2024-03-28T21:50:19Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/85022019-08-30T11:32:14Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Kruft, Volker
author
Eubel, Holger
author
Jänsch, Lothar
author
Werhahn, Wolf
author
Braun, Hans-Peter
author
2001-12
Plant Physiology 2001 127(4):1694-1710
0032-0889
1532-2548
11743114
http://hdl.handle.net/10033/8502
133574
Proteomic Approach to Identify Novel Mitochondrial Proteins in Arabidopsis1
oai:repository.helmholtz-hzi.de:10033/86702019-08-30T11:31:45Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Eubel, Holger
author
Jänsch, Lothar
author
Braun, Hans-Peter
author
2003-09
Plant Physiology 2003 133(1):274-286
0032-0889
1532-2548
12970493
10.1104/pp.103.024620
http://hdl.handle.net/10033/8670
196604
New Insights into the Respiratory Chain of Plant Mitochondria. Supercomplexes and a Unique Composition of Complex II1
oai:repository.helmholtz-hzi.de:10033/86742019-08-30T11:24:26Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Focke, Manfred
author
Gieringer, Ellen
author
Schwan, Sabine
author
Jänsch, Lothar
author
Binder, Stefan
author
Braun, Hans-Peter
author
2003-10
Plant Physiology 2003 133(2):875-884
0032-0889
1532-2548
12972648
10.1104/pp.103.027375
http://hdl.handle.net/10033/8674
219060
Fatty Acid Biosynthesis in Mitochondria of Grasses: Malonyl-Coenzyme A Is Generated by a MitochondrialLocalized Acetyl-Coenzyme A Carboxylase1
oai:repository.helmholtz-hzi.de:10033/86912019-08-30T11:25:07Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Wissing, Josef
author
Heim, Sabina
author
Wagner, Karl G.
author
1989-08
Plant Physiology 1989 90(4):1546-1551
0032-0889
1532-2548
http://hdl.handle.net/10033/8691
1061923
Diacylglycerol Kinase from Suspension Cultured Plant Cells 1
oai:repository.helmholtz-hzi.de:10033/86962019-08-30T11:30:28Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Wissing, Josef B.
author
Wagner, Karl G.
author
1992-03
Plant Physiology 1992 98(3):1148-1153
0032-0889
1532-2548
http://hdl.handle.net/10033/8696
1080320
Diacylglycerol Kinase from Suspension Cultured Plant Cells 1
oai:repository.helmholtz-hzi.de:10033/153322019-08-30T11:35:39Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Sunderhaus, Stephanie
author
Dudkina, Natalya V
author
Jänsch, Lothar
author
Klodmann, Jennifer
author
Heinemeyer, Jesco
author
Perales, Mariano
author
Zabaleta, Eduardo
author
Boekema, Egbert J
author
Braun, Hans-Peter
author
2006-03-10
Complex I of Arabidopsis includes five structurally related subunits representing gamma-type carbonic anhydrases termed CA1, CA2, CA3, CAL1, and CAL2. The position of these subunits within complex I was investigated. Direct analysis of isolated subcomplexes of complex I by liquid chromatography linked to tandem mass spectrometry allowed the assignment of the CA subunits to the membrane arm of complex I. Carbonate extraction experiments revealed that CA2 is an integral membrane protein that is protected upon protease treatment of isolated mitoplasts, indicating a location on the matrix-exposed side of the complex. A structural characterization by single particle electron microscopy of complex I from the green alga Polytomella and a previous analysis from Arabidopsis indicate a plant-specific spherical extra-domain of about 60 A in diameter, which is attached to the central part of the membrane arm of complex I on its matrix face. This spherical domain is proposed to contain a heterotrimer of three CA subunits, which are anchored with their C termini to the hydrophobic arm of complex I. Functional implications of the complex I-integrated CA subunits are discussed.
Carbonic anhydrase subunits form a matrix-exposed domain attached to the membrane arm of mitochondrial complex I in plants. 2006, 281 (10):6482-8 J. Biol. Chem.
0021-9258
16407270
10.1074/jbc.M511542200
http://hdl.handle.net/10033/15332
The Journal of biological chemistry
Carbonic anhydrase subunits form a matrix-exposed domain attached to the membrane arm of mitochondrial complex I in plants.
oai:repository.helmholtz-hzi.de:10033/153182019-08-30T11:35:39Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Thedieck, Kathrin
author
Hain, Torsten
author
Mohamed, Walid
author
Tindall, Brian J
author
Nimtz, Manfred
author
Chakraborty, Trinad
author
Wehland, Jürgen
author
Jänsch, Lothar
author
2006-12
Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the VirR-dependent virulence regulon, recently identified in Listeria monocytogenes. Lmo1695 encodes a membrane protein of 98 kDa with strong homology to the multiple peptide resistance factor (MprF) of Staphylococcus aureus. Like staphylococcal MprF, we found that Lmo1695 is involved in the synthesis of the membrane phospholipid lysylphosphatidylglycerol (L-PG). In addition, Lmo1695 is also essential for lysinylation of diphosphatidylglycerol (DPG), another phospholipid widely distributed in bacterial membranes. A Deltalmo1695 mutant lacking the lysinylated phospholipids was particularly susceptible to CAMPs of human and bacterial origin. The mutant strain infected both epithelial cells and macrophages only poorly and was attenuated for virulence when tested in a mouse model of infection. Lmo1695 is a member of a growing list of survival factors which enable growth of L. monocytogenes in different environments.
The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes. 2006, 62 (5):1325-39 Mol. Microbiol.
0950-382X
17042784
10.1111/j.1365-2958.2006.05452.x
http://hdl.handle.net/10033/15318
Molecular microbiology
The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes.
oai:repository.helmholtz-hzi.de:10033/166152019-08-30T11:36:04Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Loessner, Holger
author
Endmann, Anne
author
Leschner, Sara
author
Westphal, Kathrin
author
Rohde, Manfred
author
Miloud, Tewfik
author
Hämmerling, Günter
author
Neuhaus, Klaus
author
Weiss, Siegfried
author
2007-06
We have used Salmonella enterica serovar Typhimurium (S. typhimurium) which are able to colonize tumours besides spleen and liver. Bacteria were equipped with constructs encoding green fluorescent protein or luciferase as reporters under control of the promoter PBAD that is inducible with L-arabinose. Reporter genes could be induced in culture but also when the bacteria resided within the mouse macrophages J774A.1. More important, strong expression of reporters by the bacteria could be detected in mice after administration of L-arabinose. This was especially pronounced in bacteria colonizing tumours. Histology demonstrated that the bacteria had accumulated in and close to necrotic areas of tumours. Bacterial gene induction was observed in both regions. PBAD is tightly controlled also in vivo because gene E of bacteriophage PhiX174 could be introduced as inducible suicide gene. The possibility to deliberately induce genes in bacterial carriers within the host should render them extremely powerful tools for tumour therapy.
Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo. 2007, 9 (6):1529-37 Cell. Microbiol.
1462-5814
17298393
10.1111/j.1462-5822.2007.00890.x
http://hdl.handle.net/10033/16615
Cellular microbiology
Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo.
oai:repository.helmholtz-hzi.de:10033/195592019-08-30T11:25:11Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Baumgärtner, Maja
author
Kärst, Uwe
author
Gerstel, Birgit
author
Loessner, Martin
author
Wehland, Jürgen
author
Jänsch, Lothar
author
2007-01
Lipoprotein anchoring in bacteria is mediated by the prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the transfer of a diacylglyceryl moiety to the prospective N-terminal cysteine of the mature lipoprotein. Deletion of the lgt gene in the gram-positive pathogen Listeria monocytogenes (i) impairs intracellular growth of the bacterium in different eukaryotic cell lines and (ii) leads to increased release of lipoproteins into the culture supernatant. Comparative extracellular proteome analyses of the EGDe wild-type strain and the Delta lgt mutant provided systematic insight into the relative expression of lipoproteins. Twenty-six of the 68 predicted lipoproteins were specifically released into the extracellular proteome of the Delta lgt strain, and this proved that deletion of lgt is an excellent approach for experimental verification of listerial lipoproteins. Consequently, we generated Delta lgt Delta prfA double mutants to detect lipoproteins belonging to the main virulence regulon that is controlled by PrfA. Overall, we identified three lipoproteins whose extracellular levels are regulated and one lipoprotein that is posttranslationally modified depending on PrfA. It is noteworthy that in contrast to previous studies of Escherichia coli, we unambiguously demonstrated that lipidation by Lgt is not a prerequisite for activity of the lipoprotein-specific signal peptidase II (Lsp) in Listeria.
Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes. 2007, 189 (2):313-24 J. Bacteriol.
0021-9193
17041050
10.1128/JB.00976-06
http://hdl.handle.net/10033/19559
Journal of bacteriology
Inactivation of Lgt allows systematic characterization of lipoproteins from Listeria monocytogenes.
oai:repository.helmholtz-hzi.de:10033/197562019-08-30T11:25:11Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Wissing, Josef
author
Jänsch, Lothar
author
Nimtz, Manfred
author
Dieterich, Guido
author
Hornberger, Renate
author
Kéri, György
author
Wehland, Jürgen
author
Daub, Henrik
author
2007-03
Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis.
Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry. 2007, 6 (3):537-47 Mol. Cell Proteomics
1535-9476
17192257
10.1074/mcp.T600062-MCP200
http://hdl.handle.net/10033/19756
Molecular & cellular proteomics : MCP
Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.
oai:repository.helmholtz-hzi.de:10033/231562019-08-30T11:26:42Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Peters, Katrin
author
Dudkina, Natalya V
author
Jänsch, Lothar
author
Braun, Hans-Peter
author
Boekema, Egbert J
author
2008-01
The projection structures of complex I and the I+III2 supercomplex from the C4 plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 A. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric gamma-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the gamma-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant "type II" particle has a 15 A shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant "type I" particle. The I+III2 supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of "type I". This would mean that the "type II" particles are not involved in the supercomplex formation and, hence, could have a different physiological role.
A structural investigation of complex I and I+III2 supercomplex from Zea mays at 11-13 A resolution: assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I. 2008, 1777 (1):84-93 Biochim. Biophys. Acta
0006-3002
18047828
10.1016/j.bbabio.2007.10.012
http://hdl.handle.net/10033/23156
Biochimica et biophysica acta
A structural investigation of complex I and I+III2 supercomplex from Zea mays at 11-13 A resolution: assignment of the carbonic anhydrase domain and evidence for structural heterogeneity within complex I.
oai:repository.helmholtz-hzi.de:10033/304552019-08-30T11:26:42Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Langner, Kathrin F A
author
Darpel, Karin E
author
Denison, Eric
author
Drolet, Barbara S
author
Leibold, Wolfgang
author
Mellor, Philip S
author
Mertens, Peter P C
author
Nimtz, Manfred
author
Greiser-Wilke, Irene
author
2007-03
Salivary proteins of hematophagous Culicoides spp. are thought to play an important role in pathogen transmission and skin hypersensitivity. Analysis of these proteins, however, has been problematic due to the difficulty in obtaining adequate amounts of secreted Culicoides saliva. In the current study, a collection method for midge saliva was developed. Over a 3-d period, 3- to 5-d-old male and female Culicoides nubeculosus Meigen (Diptera: Ceratopogonidae) were repeatedly placed onto the collection system and allowed to deposit saliva into a filter. Salivary products were eluted from the filters and evaluated by gel electrophoresis and mass spectrometry as well as by intradermal testing and determination of clotting time. Gel electrophoresis revealed approximately 55 protein spots displaying relative molecular masses from 5 to 67 kDa and isoelectric points ranging from 4.5 to 9.8. The majority of molecular species analyzed by mass spectrometry showed high convergence with salivary proteins recently obtained from a cDNA library of Culicoides sonorensis Wirth & Jones, including proteins involved in sugarmeal digestion, defense, and coagulation inhibition as well as members of the D7 family and unclassified salivary proteins. In addition, the proteome analysis revealed a number of peptides that were related to proteins from insect species other than Culicoides. Intradermal injection of the saliva in human skin produced edema, vasodilatation, and pruritus. The anticoagulant activity of the saliva was demonstrated by significantly prolonged clotting times for human platelets. The potential role of the identified salivary proteins in the transmission of pathogens and the induction of allergies is discussed.
Collection and analysis of salivary proteins from the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae). 2007, 44 (2):238-48 J. Med. Entomol.
0022-2585
17427692
http://hdl.handle.net/10033/30455
Journal of medical entomology
Collection and analysis of salivary proteins from the biting midge Culicoides nubeculosus (Diptera: Ceratopogonidae).
oai:repository.helmholtz-hzi.de:10033/847582019-08-30T11:32:17Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Zelena, Kateryna
author
Zorn, Holger
author
Nimtz, Manfred
author
Berger, Ralf Günter
author
2009-05
For the heterologous expression of the msp2 gene from the edible mushroom Marasmius scorodonius in Escherichia coli the cDNA encoding the extracellular Msp2 peroxidase was cloned into the pBAD III expression plasmid. Expression of the protein with or without signal peptide was investigated in E. coli strains TOP10 and LMG194. Different PCR products were amplified for expression of the native target protein or a protein with a signal peptide. Omitting the native stop codon and adding six His-residues resulted in a fusion protein amenable to immune detection and purification by immobilised metal affinity chromatography. In E. coli the recombinant protein was produced in high yield as insoluble inclusion bodies. The influence of different parameters on MsP2 refolding was investigated. Active enzyme was obtained by glutathione-mediated oxidation in a medium containing urea, Ca(2+), and hemin.
Heterologous expression of the msp2 gene from Marasmius scorodonius. 2009, 191 (5):397-402 Arch. Microbiol.
1432-072X
19247632
10.1007/s00203-009-0462-2
http://hdl.handle.net/10033/84758
Archives of microbiology
Heterologous expression of the msp2 gene from Marasmius scorodonius.
oai:repository.helmholtz-hzi.de:10033/1240272019-08-30T11:26:42Zcom_10033_311624com_10033_6839com_10033_311308col_10033_311625col_10033_620721
00925njm 22002777a 4500
dc
Solbak, Sara M
author
Reksten, Tove R
author
Wray, Victor
author
Bruns, Karsten
author
Horvli, Ole
author
Raae, Arnt J
author
Henklein, Petra
author
Henklein, Peter
author
Röder, Rene
author
Mitzner, David
author
Schubert, Ulrich
author
Fossen, Torgils
author
2010
Cyclophilin A (CypA) represents a potential target for antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication, although the mechanism through which CypA modulates HIV-1 infectivity still remains unclear. The interaction of HIV-1 viral protein R (Vpr) with the human peptidyl prolyl isomerase CypA is known to occur in vitro and in vivo. However, the nature of the interaction of CypA with Pro-35 of N-terminal Vpr has remained undefined.
The intriguing cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding. 2010, 10:31 BMC Struct. Biol.
1472-6807
20920334
10.1186/1472-6807-10-31
http://hdl.handle.net/10033/124027
BMC structural biology
The intriguing cyclophilin A-HIV-1 Vpr interaction: prolyl cis/trans isomerisation catalysis and specific binding.
oai:repository.helmholtz-hzi.de:10033/1351052019-08-30T11:31:49Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Bals, Carola
author
Schambach, Axel
author
Meyer, Johann
author
Scheper, Thomas
author
Rinas, Ursula
author
2011-03-10
Stem cell factor (SCF) known as the c-kit ligand, plays important roles in spermatogenesis, melanogenesis and early stages of hematopoiesis. As for the latter, SCF is essential for growth and expansion of hematopoietic stem and progenitor cells. We herein describe the production of recombinant murine SCF from Escherichia coli as soluble thioredoxin-fusion protein. The formation of insoluble and inactive inclusion bodies, usually observed when SCF is expressed in E. coli, was almost entirely prevented. After purification based on membrane adsorber technology, the fusion protein was subsequently cleaved by TEV protease in order to release mature mSCF. Following dialysis and a final purification step, the target protein was isolated in high purity. Bioactivity of mSCF was proven by different tests (MTT analogous assay, long-term proliferation assay) applying a human megakaryocytic cell line. Furthermore, the biological activity of the uncleaved fusion protein was tested as well. We observed a significant activity, even though it was less than the activity displayed by the purified mSCF. In summary, avoiding inclusion body formation we present an efficient production procedure for mSCF, one of the most important stem cell cytokines.
Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner. 2011, 152 (1-2):1-8 J. Biotechnol.
1873-4863
21262286
10.1016/j.jbiotec.2011.01.012
http://hdl.handle.net/10033/135105
Journal of biotechnology
Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.
oai:repository.helmholtz-hzi.de:10033/1355092019-08-30T11:32:17Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Tomala, Magda
author
Lavrentieva, Antonina
author
Moretti, Pierre
author
Rinas, Ursula
author
Kasper, Cornelia
author
Stahl, Frank
author
Schambach, Axel
author
Warlich, Eva
author
Martin, Ulrich
author
Cantz, Tobias
author
Scheper, Thomas
author
2010-09
Leukemia inhibitory factor (LIF) is a polyfunctional cytokine with numerous regulatory effects in vivo and in vitro. In stem cell cultures it is the essential media supplement for the maintenance of pluripotency of embryonic and induced pluripotent stem cells. With regard to large scale cultures of these cells, LIF is needed in high quality and quantity and represents the major cost determining factor (90%) of the culture media. In this report, we describe a novel production and purification process for human LIF (hLIF) from recombinant Escherichia coli cultures. hLIF was cloned into pET32b and expressed as soluble protein in fusion with thioredoxin. After purification based on membrane adsorber technology, the fusion protein was cleaved using TEV protease. Released, soluble hLIF was subsequently purified by cation exchange chromatography and successfully tested for its biological activity using suspension cultures of murine embryonic and induced pluripotent stem cells. Our novel protocol for the production of recombinant hLIF is very suitable and effective for the production of poorly soluble proteins through expression in fusion with the solubilizing partner thioredoxin.
Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner. 2010, 73 (1):51-7 Protein Expr. Purif.
1096-0279
20381622
10.1016/j.pep.2010.04.002
http://hdl.handle.net/10033/135509
Protein expression and purification
Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.
oai:repository.helmholtz-hzi.de:10033/1360892019-08-30T11:36:33Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Lorenz, Udo
author
Lorenz, Birgit
author
Schmitter, Tim
author
Streker, Karin
author
Erck, Christian
author
Wehland, Jürgen
author
Nickel, Joachim
author
Zimmermann, Bastian
author
Ohlsen, Knut
author
2011-01
Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections.
Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy. 2011, 55 (1):165-73 Antimicrob. Agents Chemother.
1098-6596
20956605
10.1128/AAC.01144-10
http://hdl.handle.net/10033/136089
Antimicrobial agents and chemotherapy
Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy.
oai:repository.helmholtz-hzi.de:10033/1399612019-08-30T11:37:24Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Gurramkonda, Chandrasekhar
author
Polez, Sulena
author
Skoko, Natasa
author
Adnan, Ahmad
author
Gäbel, Thomas
author
Chugh, Dipti
author
Swaminathan, Sathyamangalam
author
Khanna, Navin
author
Tisminetzky, Sergio
author
Rinas, Ursula
author
2010
The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries.
Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin. 2010, 9:31 Microb. Cell Fact.
1475-2859
20462406
10.1186/1475-2859-9-31
http://hdl.handle.net/10033/139961
Microbial cell factories
Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin.
oai:repository.helmholtz-hzi.de:10033/1399602019-08-30T11:36:59Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Martínez-Alonso, Mónica
author
García-Fruitós, Elena
author
Ferrer-Miralles, Neus
author
Rinas, Ursula
author
Villaverde, Antonio
author
2010
Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield. This minireview summarizes the most recent observations of chaperone-linked negative side effects, mostly focusing on DnaK and GroEL sets, when using these proteins as folding assistant agents. These events are discussed in the context of the complexity of the cell quality network and the consequent intricacy of the physiological responses triggered by protein misfolding.
Side effects of chaperone gene co-expression in recombinant protein production. 2010, 9:64 Microb. Cell Fact.
1475-2859
20813055
10.1186/1475-2859-9-64
http://hdl.handle.net/10033/139960
Microbial cell factories
Side effects of chaperone gene co-expression in recombinant protein production.
oai:repository.helmholtz-hzi.de:10033/2007372019-08-30T11:32:16Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Babiychuk, Elena
author
Vandepoele, Klaas
author
Wissing, Josef
author
Garcia-Diaz, Miguel
author
De Rycke, Riet
author
Akbari, Hana
author
Joubès, Jérôme
author
Beeckman, Tom
author
Jänsch, Lothar
author
Frentzen, Margrit
author
Van Montagu, Marc C E
author
Kushnir, Sergei
author
2011-04-19
Plastids are DNA-containing organelles unique to plant cells. In Arabidopsis, one-third of the genes required for embryo development encode plastid-localized proteins. To help understand the role of plastids in embryogenesis and postembryonic development, we characterized proteins of the mitochondrial transcription termination factor (mTERF) family, which in animal models, comprises DNA-binding regulators of mitochondrial transcription. Of 35 Arabidopsis mTERF proteins, 11 are plastid-localized. Genetic complementation shows that at least one plastidic mTERF, BELAYA SMERT' (BSM), is required for embryogenesis. The main postembryonic phenotypes of genetic mosaics with the bsm mutation are severe abnormalities in leaf development. Mutant bsm cells are albino, are compromised in growth, and suffer defects in global plastidic gene expression. The bsm phenotype could be phenocopied by inhibition of plastid translation with spectinomycin. Plastid translation is essential for cell viability in dicotyledonous species such as tobacco but not in monocotyledonous maize. Here, genetic interactions between BSM and the gene encoding plastid homomeric acetyl-CoA carboxylase ACC2 suggest that there is a functional redundancy in malonyl-CoA biosynthesis that permits bsm cell survival in Arabidopsis. Overall, our results indicate that biosynthesis of malonyl-CoA and plastid-derived systemic growth-promoting compounds are the processes that link plant development and plastid gene expression.
Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family. 2011, 108 (16):6674-9 Proc. Natl. Acad. Sci. U.S.A.
1091-6490
21464319
10.1073/pnas.1103442108
http://hdl.handle.net/10033/200737
Proceedings of the National Academy of Sciences of the United States of America
Plastid gene expression and plant development require a plastidic protein of the mitochondrial transcription termination factor family.
oai:repository.helmholtz-hzi.de:10033/2146502019-08-30T11:37:00Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Li, Zhaopeng
author
Kessler, Wolfgang
author
van den Heuvel, Joop
author
Rinas, Ursula
author
2011-08
Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH(4)(+) as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L(-1) product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-D-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.
Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems. 2011, 91 (4):1203-13 Appl. Microbiol. Biotechnol.
1432-0614
21698378
10.1007/s00253-011-3407-z
http://hdl.handle.net/10033/214650
Applied microbiology and biotechnology
Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems.
oai:repository.helmholtz-hzi.de:10033/2151432019-08-30T11:27:46Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Della Beffa, Cristina
author
Klawonn, Frank
author
Menetski, Joseph P
author
Schumacher, H Ralph
author
Pessler, Frank
author
2011
ABSTRACT:
Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation. 2011, 4:443 BMC Res Notes
1756-0500
10.1186/1756-0500-4-443
http://hdl.handle.net/10033/215143
BMC research notes
Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation.
oai:repository.helmholtz-hzi.de:10033/2168112019-08-30T11:25:43Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Lorenz, Udo
author
Lorenz, Birgit
author
Schmitter, Tim
author
Streker, Karin
author
Erck, Christian
author
Wehland, Jürgen
author
Nickel, Joachim
author
Zimmermann, Bastian
author
Ohlsen, Knut
author
2011-01
Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections.
Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy. 2011, 55 (1):165-73 Antimicrob. Agents Chemother.
1098-6596
20956605
10.1128/AAC.01144-10
http://hdl.handle.net/10033/216811
Antimicrobial agents and chemotherapy
Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy.
oai:repository.helmholtz-hzi.de:10033/2215122019-08-30T11:37:24Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Li, Zhaopeng
author
Nimtz, Manfred
author
Rinas, Ursula
author
2011-11
Generating sufficient quantities of labeled proteins represents a bottleneck in protein structure determination. A simple protocol for producing heavy isotope as well as selenomethionine (Se-Met)-labeled proteins was developed using T7-based Escherichia coli expression systems. The protocol is applicable for generation of single-, double-, and triple-labeled proteins ((15)N, (13)C, and (2)H) in shaker flask cultures. Label incorporation into the target protein reached 99% and 97% for (15)N and (13)C, respectively, and 75% of (non-exchangeable) hydrogen for (2)H labeling. The expression yields and final cell densities (OD600 ~16) were the same as for the production of non-labeled protein. This protocol is also applicable for Se-Met labeling, leading to Se-Met incorporation into the target protein of 70% or 90% using prototrophic or methionine auxotrophic E. coli strains, respectively.
Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems. 2011, 92 (4):823-33 Appl. Microbiol. Biotechnol.
1432-0614
21983707
10.1007/s00253-011-3603-x
http://hdl.handle.net/10033/221512
Applied microbiology and biotechnology
Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.
oai:repository.helmholtz-hzi.de:10033/2355752019-08-30T11:25:43Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Sans, Cristina
author
García-Fruitós, Elena
author
Ferraz, Rosa M
author
González-Montalbán, Núria
author
Rinas, Ursula
author
López-Santín, Josep
author
Villaverde, Antonio
author
Álvaro, Gregorio
author
2012-07-24
Fuculose-1-phosphate aldolase (FucA) has been produced in Escherichia coli as active inclusion bodies (IBs) in batch cultures. The activity of insoluble FucA has been modulated by a proper selection of producing strain, culture media, and process conditions. In some cases, when an optimized defined medium was used, FucA IBs were more active (in terms of specific activity) than the soluble protein version obtained in the same process with a conventional defined medium, supporting the concept that solubility and conformational quality are independent protein parameters. FucA IBs have been tested as biocatalysts, either directly or immobilized into Lentikat beads, in an aldolic reaction between DHAP and (S)-Cbz-alaninal, obtaining product yields ranging from 65 to 76%. The production of an active aldolase as IBs, the possibility of tailoring IBs properties by both genetic and process approaches, and the reusability of IBs by further entrapment in appropriate matrices fully support the principle of using self-assembled enzymatic clusters as tunable mechanically stable and functional biocatalysts.
Inclusion bodies of fuculose-1-phosphate aldolase as stable and reusable biocatalysts., 28 (2):421-7 Biotechnol. Prog.
1520-6033
22275283
10.1002/btpr.1518
http://hdl.handle.net/10033/235575
Biotechnology progress
Inclusion bodies of fuculose-1-phosphate aldolase as stable and reusable biocatalysts.
oai:repository.helmholtz-hzi.de:10033/2379132019-08-30T11:28:24Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Voss, Martin
author
Nimtz, Manfred
author
Leimkühler, Silke
author
2011
The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.
Elucidation of the dual role of Mycobacterial MoeZR in molybdenum cofactor biosynthesis and cysteine biosynthesis. 2011, 6 (11):e28170 PLoS ONE
1932-6203
22140533
10.1371/journal.pone.0028170
http://hdl.handle.net/10033/237913
PloS one
Elucidation of the dual role of Mycobacterial MoeZR in molybdenum cofactor biosynthesis and cysteine biosynthesis.
oai:repository.helmholtz-hzi.de:10033/2456722019-08-30T11:31:49Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Lehrke, Markus
author
Rump, Steffen
author
Heidenreich, Torsten
author
Wissing, Josef
author
Mendel, Ralf R
author
Bittner, Florian
author
2012-02-01
The Moco (molybdenum cofactor) sulfurase ABA3 from Arabidopsis thaliana catalyses the sulfuration of the Moco of aldehyde oxidase and xanthine oxidoreductase, which represents the final activation step of these enzymes. ABA3 consists of an N-terminal NifS-like domain that exhibits L-cysteine desulfurase activity and a C-terminal domain that binds sulfurated Moco. The strictly conserved Cys430 in the NifS-like domain binds a persulfide intermediate, which is abstracted from the substrate L-cysteine and finally needs to be transferred to the Moco of aldehyde oxidase and xanthine oxidoreductase. In addition to Cys⁴³⁰, another eight cysteine residues are located in the NifS-like domain, with two of them being highly conserved among Moco sulfurase proteins and, at the same time, being in close proximity to Cys⁴³⁰. By determination of the number of surface-exposed cysteine residues and the number of persulfide-binding cysteine residues in combination with the sequential substitution of each of the nine cysteine residues, a second persulfide-binding cysteine residue, Cys²⁰⁶, was identified. Furthermore, the active-site Cys⁴³⁰ was found to be located on top of a loop structure, formed by the two flanking residues Cys⁴²⁸ and Cys⁴³⁵, which are likely to form an intramolecular disulfide bridge. These findings are confirmed by a structural model of the NifS-like domain, which indicates that Cys⁴²⁸ and Cys⁴³⁵ are within disulfide bond distance and that a persulfide transfer from Cys⁴³⁰ to Cys²⁰⁶ is indeed possible.
Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis. 2012, 441 (3):823-32 Biochem. J.
1470-8728
22004669
10.1042/BJ20111170
http://hdl.handle.net/10033/245672
The Biochemical journal
Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis.
oai:repository.helmholtz-hzi.de:10033/2476312019-08-30T11:26:42Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
König, Sebastian
author
Probst-Kepper, Michael
author
Reinl, Tobias
author
Jeron, Andreas
author
Huehn, Jochen
author
Schraven, Burkhart
author
Jänsch, Lothar
author
2012
Regulatory T cells (Tregs) are essential for controlling peripheral tolerance by the active suppression of various immune cells including conventional T effector cells (Teffs). Downstream of the T cell receptor (TCR), more than 500 protein kinases encoded by the human genome have to be considered in signaling cascades regulating the activation of Tregs and Teffs, respectively. Following TCR engagement, Tregs posses a number of unique attributes, such as constitutive expression of Foxp3, hyporesponsiveness and poor cytokine production. Furthermore, recent studies showed that altered regulation of protein kinases is important for Treg function. These data indicate that signaling pathways in Tregs are distinctly organized and alterations at the level of protein kinases contribute to the unique Treg phenotype. However, kinase-based signaling networks in Tregs are poorly understood and necessitate further systematic characterization. In this study, we analyzed the differential expression of kinases in Tregs and Teffs by using a kinase-selective proteome strategy. In total, we revealed quantitative information on 185 kinases expressed in the human CD4(+) T cell subsets. The majority of kinases was equally abundant in both T cell subsets, but 11 kinases were differentially expressed in Tregs. Most strikingly, Tregs showed an altered expression of cell cycle kinases including CDK6. Quantitative proteomics generates first comparative insight into the kinase complements of the CD4(+) Teff and Treg subset. Treg-specific expression pattern of 11 protein kinases substantiate the current opinion that TCR-mediated signaling cascades are altered in Tregs and further suggests that Tregs exhibit significant specificities in cell-cycle control and progression.
First insight into the kinome of human regulatory T cells. 2012, 7 (7):e40896 PLoS ONE
1932-6203
22815858
10.1371/journal.pone.0040896
http://hdl.handle.net/10033/247631
PloS one
First insight into the kinome of human regulatory T cells.
oai:repository.helmholtz-hzi.de:10033/2678332019-08-30T11:29:17Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Hintzsche, Henning
author
Jastrow, Christian
author
Kleine-Ostmann, Thomas
author
Kärst, Uwe
author
Schrader, Thorsten
author
Stopper, Helga
author
2012
Terahertz electromagnetic fields are non-ionizing electromagnetic fields in the frequency range from 0.1 to 10 THz. Potential applications of these electromagnetic fields include the whole body scanners, which currently apply millimeter waves just below the terahertz range, but future scanners will use higher frequencies in the terahertz range. These and other applications will bring along human exposure to these fields. Up to now, only a limited number of investigations on biological effects of terahertz electromagnetic fields have been performed. Therefore, research is strongly needed to enable reliable risk assessment.Cells were exposed for 2 h, 8 h, and 24 h with different power intensities ranging from 0.04 mW/cm(2) to 2 mW/cm(2), representing levels below, at, and above current safety limits. Genomic damage on the chromosomal level was measured as micronucleus formation. DNA strand breaks and alkali-labile sites were quantified with the comet assay. No DNA strand breaks or alkali-labile sites were observed as a consequence of exposure to terahertz electromagnetic fields in the comet assay. The fields did not cause chromosomal damage in the form of micronucleus induction.
Terahertz electromagnetic fields (0.106 THz) do not induce manifest genomic damage in vitro. 2012, 7 (9):e46397 PLoS ONE
1932-6203
23029508
10.1371/journal.pone.0046397
http://hdl.handle.net/10033/267833
PloS one
Terahertz electromagnetic fields (0.106 THz) do not induce manifest genomic damage in vitro.
oai:repository.helmholtz-hzi.de:10033/2790602019-08-30T11:32:16Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Scheiter, Maxi
author
Bulitta, Björn
author
van Ham, Marco
author
Klawonn, Frank
author
König, Sebastian
author
Jänsch, Lothar
author
2013
Natural killer (NK) cells are part of the innate immune response and play a crucial role in the defense against tumors and virus-infected cells. Their effector functions include the specific killing of target cells, as well as the modulation of other immune cells by cytokine release. Kinases constitute a relevant part in signaling, are prime targets in drug research and the protein kinase inhibitor Dasatinib is already used for immune-modulatory therapies. In this study, we tested the effects of the kinase inhibitors CK59 and CID755673. These inhibitors are directed against calmodulin kinase II (CaMKII; CK59) and PKD family kinases (CID755673) that were previously suggested as novel components of NK activation pathways. Here, we use a multi-parameter, FACS-based assay to validate the influence of CK59 and CID755673 on the effector functions of primary NK cells. Treatment with CK59 and CID755673 indeed resulted in a significant dose-dependent reduction of NK cell degranulation markers and cytokine release in freshly isolated Peripheral blood mononuclear cell populations from healthy blood donors. These results underline the importance of CaMKII for NK cell signaling and suggest protein kinase D2 as a novel signaling component in NK cell activation. Notably, kinase inhibition studies on pure NK cell populations indicate significant donor variations.
Protein Kinase Inhibitors CK59 and CID755673 Alter Primary Human NK Cell Effector Functions. 2013, 4:66 Front Immunol
1664-3224
23508354
10.3389/fimmu.2013.00066
http://hdl.handle.net/10033/279060
Frontiers in immunology
Protein Kinase Inhibitors CK59 and CID755673 Alter Primary Human NK Cell Effector Functions.
oai:repository.helmholtz-hzi.de:10033/2835722019-08-30T11:30:58Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Vallejo, Luis F
author
Rinas, Ursula
author
2013-01
The kinetics of folding and dimerization of bone morphogenetic protein-2 (BMP-2), a disulfide-connected, homodimeric cystine-knot protein and a member of the transforming growth factor-β superfamily, was analyzed under a variety of different conditions. Refolding and dimerization of BMP-2 were extremely slow under all conditions studied, and could be described by consecutive first-order reactions involving at least one long-lived intermediate. The rate constants vary from ~ 0.2 × 10(-5) to ~ 3.5 × 10(-5) s(-1), and were strongly dependent on temperature, redox conditions, and the presence of stabilizing or destabilizing ions. In particular, the combined impact of ionic strength and redox conditions on the rates indicates that electrostatic interactions control thiol-disulfide exchange reactions on the path from the unfolded and reduced monomers to the disulfide-connected growth factor in a rate-determining way.
Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-β family. 2013, 280 (1):83-92 FEBS J.
1742-4658
23122408
10.1111/febs.12051
http://hdl.handle.net/10033/283572
The FEBS journal
Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-β family.
oai:repository.helmholtz-hzi.de:10033/2911422019-08-30T11:34:22Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Dahl, Jan-Ulrik
author
Radon, Christin
author
Bühning, Martin
author
Nimtz, Manfred
author
Leichert, Lars I
author
Denis, Yann
author
Jourlin-Castelli, Cécile
author
Iobbi-Nivol, Chantal
author
Méjean, Vincent
author
Leimkühler, Silke
author
2013-02-22
The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a ΔtusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the ΔtusA mutant. Characterization of the ΔtusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.
The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis. 2013, 288 (8):5426-42 J. Biol. Chem.
1083-351X
23281480
10.1074/jbc.M112.431569
http://hdl.handle.net/10033/291142
The Journal of biological chemistry
The sulfur carrier protein TusA has a pleiotropic role in Escherichia coli that also affects molybdenum cofactor biosynthesis.
oai:repository.helmholtz-hzi.de:10033/2962882019-08-30T11:28:22Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Klawonn, Frank
author
Lechner, Werner M.
author
Grigull, Lorenz
author
2013-03
10.1007/978-3-642-37899-7_12
http://hdl.handle.net/10033/296288
Case-Centred Multidimensional Scaling for Classification Visualisation in Medical Diagnosis
oai:repository.helmholtz-hzi.de:10033/2970562019-08-30T11:33:05Zcom_10033_311624com_10033_6839com_10033_311308com_10033_338554col_10033_621787col_10033_311625col_10033_620777
00925njm 22002777a 4500
dc
Khetarpal, Niyati
author
Poddar, Ankur
author
Nemani, Satish K
author
Dhar, Nisha
author
Patil, Aravind
author
Negi, Priyanka
author
Perween, Ashiya
author
Viswanathan, Ramaswamy
author
Lünsdorf, Heinrich
author
Tyagi, Poornima
author
Raut, Rajendra
author
Arora, Upasana
author
Jain, Swatantra K
author
Rinas, Ursula
author
Swaminathan, Sathyamangalam
author
Khanna, Navin
author
2013
Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate.
Dengue-specific subviral nanoparticles: design, creation and characterization. 2013, 11 (1):15 J Nanobiotechnology
1477-3155
23706089
10.1186/1477-3155-11-15
http://hdl.handle.net/10033/297056
Journal of nanobiotechnology
Dengue-specific subviral nanoparticles: design, creation and characterization.
oai:repository.helmholtz-hzi.de:10033/2972222019-08-30T11:35:13Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Meyer, Steffen
author
Lorenz, Carmen
author
Baser, Bahar
author
Wördehoff, Mona
author
Jäger, Volker
author
van den Heuvel, Joop
author
2013
Recombinant production of complex eukaryotic proteins for structural analyses typically requires a profound screening process to identify suitable constructs for the expression of ample amounts of properly folded protein. Furthermore, the evaluation of an optimal expression host has a major impact on protein yield and quality as well as on actual cost of the production process. Here we present a novel fast expression system for multiple hosts based on a single donor vector termed pFlp-Bac-to-Mam. The range of applications of pFlp-Bac-to-Mam comprises highly efficient transient transfection of HEK293-6E in serum-free suspension culture and subsequent large-scale production of challenging proteins expressing in mg per Liter level using either the baculoviral expression vector system or stable CHO production cell lines generated by Flp-mediated cassette exchange. The success of the multi-host expression vector to identify the optimal expression strategy for efficient production of high quality protein is demonstrated in a comparative expression study of three model proteins representing different protein classes: intracellular expression using a fluorescent protein, secretion of a single-chain-Fv-hIgG1Fc fusion construct and production of a large amount of highly homogeneous protein sample of the extracellular domain of a Toll-like receptor. The evaluation of the production efficiency shows that the pFlp-Bac-to-Mam system allows a fast and individual optimization of the expression strategy for each protein class.
Multi-host expression system for recombinant production of challenging proteins. 2013, 8 (7):e68674 PLoS ONE
1932-6203
23874717
10.1371/journal.pone.0068674
http://hdl.handle.net/10033/297222
PloS one
Multi-host expression system for recombinant production of challenging proteins.
oai:repository.helmholtz-hzi.de:10033/2974382019-08-30T11:34:48Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Nicke, Tristan
author
Schnitzer, Tobias
author
Münch, Karin
author
Adamczack, Julia
author
Haufschildt, Kristin
author
Buchmeier, Sabine
author
Kucklick, Martin
author
Felgenträger, Undine
author
Jänsch, Lothar
author
Riedel, Katharina
author
Layer, Gunhild
author
2013
The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV-visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.
Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF. 2013, 33 (3): Biosci. Rep.
1573-4935
23683062
10.1042/BSR20130043
http://hdl.handle.net/10033/297438
Bioscience reports
Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF.
oai:repository.helmholtz-hzi.de:10033/2993872019-08-30T11:33:56Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Bodenhofer, Ulrich
author
Krone, Martin
author
Klawonn, Frank
author
2013-08-21
Testing noisy numerical data for monotonic association 2013, 245:21 Information Sciences
200255
10.1016/j.ins.2012.11.026
http://hdl.handle.net/10033/299387
Information Sciences
Testing noisy numerical data for monotonic association
oai:repository.helmholtz-hzi.de:10033/3006682019-08-30T11:37:24Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Della Beffa, Cristina
author
Slansky, Elisabeth
author
Pommerenke, Claudia
author
Klawonn, Frank
author
Li, Jialiang
author
Dai, Lie
author
Schumacher, H Ralph
author
Pessler, Frank
author
2013
Traditionally, differences in absolute numbers of cells expressing a certain marker (e.g., positive staining cells per mm(2)) have been used in immunohistological synovial tissue classification. We have begun to evaluate the relative composition of the inflammatory infiltrates, i.e. percentages of inflammatory cell types in inflammatory infiltrates, as an alternate classification tool that may potentially improve tissue diagnostics, subgrouping in clinical trials, and understanding of pathogenesis of inflammatory and noninflammatory arthropathies.
The relative composition of the inflammatory infiltrate as an additional tool for synovial tissue classification. 2013, 8 (8):e72494 PLoS ONE
1932-6203
23951325
10.1371/journal.pone.0072494
http://hdl.handle.net/10033/300668
PloS one
The relative composition of the inflammatory infiltrate as an additional tool for synovial tissue classification.
oai:repository.helmholtz-hzi.de:10033/3018082019-08-30T11:34:48Zcom_10033_311308col_10033_620561
00925njm 22002777a 4500
dc
Hofmeyer, Thomas
author
Schmelz, Stefan
author
Degiacomi, Matteo T
author
Dal Peraro, Matteo
author
Daneschdar, Matin
author
Scrima, Andrea
author
van den Heuvel, Joop
author
Heinz, Dirk W
author
Kolmar, Harald
author
2013-04-26
The complement system as a major part of innate immunity is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune response. Additional recognition of necrotic or apoptotic cells demands a fine-tune regulation of this powerful system. C4b-binding protein (C4BP) is the major inhibitor of the classical complement and lectin pathway. The crystal structure of the human C4BP oligomerization domain in its 7α isoform and molecular simulations provide first structural insights of C4BP oligomerization. The heptameric core structure is stabilized by intermolecular disulfide bonds. In addition, thermal shift assays indicate that layers of electrostatic interactions mainly contribute to the extraordinary thermodynamic stability of the complex. These findings make C4BP a promising scaffold for multivalent ligand display with applications in immunology and biological chemistry.
Arranged sevenfold: structural insights into the C-terminal oligomerization domain of human C4b-binding protein. 2013, 425 (8):1302-17 J. Mol. Biol.
1089-8638
23274142
10.1016/j.jmb.2012.12.017
http://hdl.handle.net/10033/301808
Journal of molecular biology
Arranged sevenfold: structural insights into the C-terminal oligomerization domain of human C4b-binding protein.
oai:repository.helmholtz-hzi.de:10033/3031522019-08-30T11:26:42Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Scheiter, Maxi
author
Lau, Ulrike
author
van Ham, Marco
author
Bulitta, Björn
author
Gröbe, Lothar
author
Garritsen, Henk
author
Klawonn, Frank
author
König, Sebastian
author
Jänsch, Lothar
author
2013-05
The recent Natural Killer (NK) cell maturation model postulates that CD34(+) hematopoietic stem cells (HSC) first develop into CD56(bright) NK cells, then into CD56(dim)CD57(-) and finally into terminally maturated CD56(dim)CD57(+). The molecular mechanisms of human NK cell differentiation and maturation however are incompletely characterized. Here we present a proteome analysis of distinct developmental stages of human primary NK cells, isolated from healthy human blood donors. Peptide sequencing was used to comparatively analyze CD56(bright) NK cells versus CD56(dim) NK cells and CD56(dim)CD57(-) NK cells versus CD56(dim)CD57(+) NK cells and revealed distinct protein signatures for all of these subsets. Quantitative data for about 3400 proteins were obtained and support the current differentiation model. Furthermore, 11 donor-independently, but developmental stage specifically regulated proteins so far undescribed in NK cells were revealed, which may contribute to NK cell development and may elucidate a molecular source for NK cell effector functions. Among those proteins, S100A4 (Calvasculin) and S100A6 (Calcyclin) were selected to study their dynamic subcellular localization. Upon activation of human primary NK cells, both proteins are recruited into the immune synapse (NKIS), where they colocalize with myosin IIa.
Proteome analysis of distinct developmental stages of human natural killer (NK) cells. 2013, 12 (5):1099-114 Mol. Cell Proteomics
1535-9484
23315794
10.1074/mcp.M112.024596
http://hdl.handle.net/10033/303152
Molecular & cellular proteomics : MCP
Proteome analysis of distinct developmental stages of human natural killer (NK) cells.
oai:repository.helmholtz-hzi.de:10033/3051922019-08-30T11:30:58Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Gurramkonda, Chandrasekhar
author
Zahid, Maria
author
Nemani, Satish Kumar
author
Adnan, Ahmad
author
Gudi, Satheesh Kumar
author
Khanna, Navin
author
Ebensen, Thomas
author
Lünsdorf, Heinrich
author
Guzmán, Carlos A
author
Rinas, Ursula
author
2013-12-01
Following earlier studies on high-level intracellular production of hepatitis B surface antigen (HBsAg) using recombinant Pichia pastoris, we present here in detail an enhanced method for the purification of recombinant HBsAg virus-like particles (VLPs). We have screened various detergents for their ability to promote the solubilization of recombinant intracellular HBsAg. In addition, we have analyzed the effect of cell disruption and extraction regarding their impact on the release of HBsAg. Our results show that introduction of the mild nonionic detergent Tween 20 in the initial process of cell lysis at ∼600bars by high pressure homogenization leads to the best results. The subsequent purification steps involved polyethylene glycol precipitation of host cell contaminants, hydrophobic adsorption of HBsAg to colloidal silica followed by ion-exchange chromatography and either isopycnic density ultracentrifugation or size exclusion chromatography for the recovery of the VLPs. After final KSCN treatment and dialysis, a total yield of ∼3% with a purity of >99% was reached. The pure protein was characterized by electron microscopy, showing the presence of uniform VLPs which are the pre-requisite for immunogenicity. The intramuscular co-administration of HBsAg VLPs, with either alum or a PEGylated-derivative of the toll-like receptor 2/6 agonist MALP-2, to mice resulted in the elicitation of significantly higher HBsAg-specific IgG titers as well as a stronger cellular immune response compared to mice vaccinated with a gold standard vaccine (Engerix™). These results show that P. pastoris derived HBsAg VLPs exhibit a high potential as a superior biosimilar vaccine against hepatitis B.
Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties. 2013, 940:104-11 J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
1873-376X
24141044
10.1016/j.jchromb.2013.09.030
http://hdl.handle.net/10033/305192
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.
oai:repository.helmholtz-hzi.de:10033/3058782019-08-30T11:27:46Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Tielen, Petra
author
Rosin, Nathalie
author
Meyer, Ann-Kathrin
author
Dohnt, Katrin
author
Haddad, Isam
author
Jänsch, Lothar
author
Klein, Johannes
author
Narten, Maike
author
Pommerenke, Claudia
author
Scheer, Maurice
author
Schobert, Max
author
Schomburg, Dietmar
author
Thielen, Bernhard
author
Jahn, Dieter
author
2013
Biofilms of the Gram-negative bacterium Pseudomonas aeruginosa are one of the major causes of complicated urinary tract infections with detrimental outcome. To develop novel therapeutic strategies the molecular adaption strategies of P. aeruginosa biofilms to the conditions of the urinary tract were investigated thoroughly at the systems level using transcriptome, proteome, metabolome and enzyme activity analyses. For this purpose biofilms were grown anaerobically in artificial urine medium (AUM). Obtained data were integrated bioinformatically into gene regulatory and metabolic networks. The dominating response at the transcriptome and proteome level was the adaptation to iron limitation via the broad Fur regulon including 19 sigma factors and up to 80 regulated target genes or operons. In agreement, reduction of the iron cofactor-dependent nitrate respiratory metabolism was detected. An adaptation of the central metabolism to lactate, citrate and amino acid as carbon sources with the induction of the glyoxylate bypass was observed, while other components of AUM like urea and creatinine were not used. Amino acid utilization pathways were found induced, while fatty acid biosynthesis was reduced. The high amounts of phosphate found in AUM explain the reduction of phosphate assimilation systems. Increased quorum sensing activity with the parallel reduction of chemotaxis and flagellum assembly underscored the importance of the biofilm life style. However, reduced formation of the extracellular polysaccharide alginate, typical for P. aeruginosa biofilms in lungs, indicated a different biofilm type for urinary tract infections. Furthermore, the obtained quorum sensing response results in an increased production of virulence factors like the extracellular lipase LipA and protease LasB and AprA explaining the harmful cause of these infections.
Regulatory and metabolic networks for the adaptation of Pseudomonas aeruginosa biofilms to urinary tract-like conditions. 2013, 8 (8):e71845 PLoS ONE
1932-6203
23967252
10.1371/journal.pone.0071845
http://hdl.handle.net/10033/305878
PloS one
Regulatory and metabolic networks for the adaptation of Pseudomonas aeruginosa biofilms to urinary tract-like conditions.
oai:repository.helmholtz-hzi.de:10033/3061572019-08-30T11:34:48Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Becher, Dörte
author
Bernhardt, Jörg
author
Fuchs, Stephan
author
Riedel, Katharina
author
2013-10
Soil- and litter-borne microorganisms vitally contribute to biogeochemical cycles. However, changes in environmental parameters but also human interferences may alter species composition and elicit alterations in microbial activities. Soil and litter metaproteomics, implying the assignment of soil and litter proteins to specific phylogenetic and functional groups, has a great potential to provide essential new insights into the impact of microbial diversity on soil ecosystem functioning. This article will illuminate challenges and perspectives of current soil and litter metaproteomics research, starting with an introduction to an appropriate experimental design and state-of-the-art proteomics methodologies. This will be followed by a summary of important studies aimed at (i) the discovery of the major biotic drivers of leaf litter decomposition, (ii) metaproteomics analyses of rhizosphere-inhabiting microbes, and (iii) global approaches to study bioremediation processes. The review will be closed by a brief outlook on future developments and some concluding remarks, which should assist the reader to develop successful concepts for soil and litter metaproteomics studies.
Metaproteomics to unravel major microbial players in leaf litter and soil environments: challenges and perspectives. 2013, 13 (18-19):2895-909 Proteomics
1615-9861
23894095
10.1002/pmic.201300095
http://hdl.handle.net/10033/306157
Proteomics
Metaproteomics to unravel major microbial players in leaf litter and soil environments: challenges and perspectives.
oai:repository.helmholtz-hzi.de:10033/3066962019-08-30T11:28:51Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Gamrekelashvili, Jaba
author
Kapanadze, Tamar
author
Han, Miaojun
author
Wissing, Josef
author
Ma, Chi
author
Jaensch, Lothar
author
Manns, Michael P
author
Armstrong, Todd
author
Jaffee, Elizabeth
author
White, Ayla O
author
Citrin, Deborah E
author
Korangy, Firouzeh
author
Greten, Tim F
author
2013-10-08
Cross-priming of CD8+ T cells and generation of effector immune responses is pivotal for tumor immunity as well as for successful anticancer vaccination and therapy. Dead and dying cells produce signals that can influence Ag processing and presentation; however, there is conflicting evidence regarding the immunogenicity of necrotic cell death. We used a mouse model of sterile necrosis, in which mice were injected with sterile primary necrotic cells, to investigate a role of these cells in priming of CD8+ T cells. We discovered a molecular mechanism operating in Ag donor cells that regulates cross-priming of CD8+ T cells during primary sterile necrosis and thereby controls adaptive immune responses. We found that the cellular peptidases dipeptidyl peptidase 3 (DPP-3) and thimet oligopeptidase 1 (TOP-1), both of which are present in nonimmunogenic necrotic cells, eliminated proteasomal degradation products and blocked Ag cross-presentation. While sterile necrotic tumor cells failed to induce CD8+ T cell responses, their nonimmunogenicity could be reversed in vitro and in vivo by inactivation of DPP-3 and TOP-1. These results indicate that control of cross-priming and thereby immunogenicity of primary sterile necrosis relies on proteasome-dependent oligopeptide generation and functional status of peptidases in Ag donor cells.
Peptidases released by necrotic cells control CD8+ T cell cross-priming. 2013: J. Clin. Invest.
1558-8238
24216478
10.1172/JCI65698
http://hdl.handle.net/10033/306696
The Journal of clinical investigation
Peptidases released by necrotic cells control CD8+ T cell cross-priming.
oai:repository.helmholtz-hzi.de:10033/3113092019-08-30T11:33:55Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Haase-Aschoff, Paul
author
Linke, Diana
author
Nimtz, Manfred
author
Popper, Lutz
author
Berger, Ralf G.
author
2014-01-14
An enzyme from Auricularia auricula-judae combining both benzoyl and cinnamoyl esterase activity 2013, 48 (12):1872 Process Biochemistry
13595113
10.1016/j.procbio.2013.09.016
http://hdl.handle.net/10033/311309
Process Biochemistry
An enzyme from Auricularia auricula-judae combining both benzoyl and cinnamoyl esterase activity
oai:repository.helmholtz-hzi.de:10033/3251842019-08-30T11:35:39Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Bartsch, Annekathrin
author
Bunk, Boyke
author
Haddad, Isam
author
Klein, Johannes
author
Münch, Richard
author
Johl, Thorsten
author
Kärst, Uwe
author
Jänsch, Lothar
author
Jahn, Dieter
author
Retter, Ida
author
2011-04-01
GeneReporter is a web tool that reports functional information and relevant literature on a protein-coding sequence of interest. Its purpose is to support both manual genome annotation and document retrieval. PubMed references corresponding to a sequence are detected by the extraction of query words from UniProt entries of homologous sequences. Data on protein families, domains, potential cofactors, structure, function, cellular localization, metabolic contribution and corresponding DNA binding sites complement the information on a given gene product of interest.
GeneReporter--sequence-based document retrieval and annotation. 2011, 27 (7):1034-5 Bioinformatics
1367-4811
21310745
10.1093/bioinformatics/btr047
http://hdl.handle.net/10033/325184
Bioinformatics (Oxford, England)
GeneReporter--sequence-based document retrieval and annotation.
oai:repository.helmholtz-hzi.de:10033/3260092019-08-30T11:36:05Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Vit, Allegra
author
Misson, Laëtitia
author
Blankenfeldt, Wulf
author
Seebeck, Florian Peter
author
2014-05
Ergothioneine is an amino-acid betaine derivative of histidine that was discovered more than one century ago. Despite significant research pointing to a function in oxidative stress defence, the exact mechanisms of action of ergothioneine remain elusive. Although both humans and bacterial pathogens such as Mycobacterium tuberculosis seem to depend on ergothioneine, humans are devoid of the corresponding biosynthetic enzymes. Therefore, its biosynthesis may emerge as potential drug target in the development of novel therapeutics against tuberculosis. The recent identification of ergothioneine-biosynthetic genes in M. smegmatis enables a more systematic study of its biology. The pathway is initiated by EgtD, a SAM-dependent methyltransferase that catalyzes a trimethylation reaction of histidine to give N(α),N(α),N(α)-trimethylhistidine. Here, the recombinant production, purification and crystallization of EgtD are reported. Crystals of native EgtD diffracted to 2.35 Å resolution at a synchrotron beamline, whereas crystals of seleno-L-methionine-labelled protein diffracted to 1.75 Å resolution and produced a significant anomalous signal to 2.77 Å resolution at the K edge. All of the crystals belonged to space group P212121, with two EgtD monomers in the asymmetric unit.
Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD. 2014, 70 (Pt 5):676-80 Acta Crystallogr F Struct Biol Commun
2053-230X
24817736
10.1107/S2053230X1400805X
http://hdl.handle.net/10033/326009
Acta crystallographica. Section F, Structural biology communications
Crystallization and preliminary X-ray analysis of the ergothioneine-biosynthetic methyltransferase EgtD.
oai:repository.helmholtz-hzi.de:10033/3260082019-08-30T11:36:33Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Kleinboelting, Silke
author
van den Heuvel, Joop
author
Kambach, Christian
author
Weyand, Michael
author
Leipelt, Martina
author
Steegborn, Clemens
author
2014-04
The second messenger cAMP is synthesized in mammals by ten differently regulated adenylyl cyclases (AC1-10). These ACs are grouped into nucleotidyl cyclase class III based on homologies in their catalytic domains. The catalytic domain of AC10 is unique, however, in being activated through direct interaction with calcium and bicarbonate. Here, the production, crystallization and X-ray diffraction analysis of the catalytic domain of human AC10 are described as a basis for structural studies of regulator binding sites and mechanisms. The recombinant protein had high specific AC activity, and crystals of AC10 in space group P63 diffracted to ∼2.0 Å resolution on a synchrotron beamline. A complete diffraction data set revealed unit-cell parameters a = b = 99.65, c = 98.04 Å, indicating one AC10 catalytic domain per asymmetric unit, and confirmed that the obtained crystals are suitable for structure solution and mechanistic studies.
Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase. 2014, 70 (Pt 4):467-9 Acta Crystallogr F Struct Biol Commun
2053-230X
24699740
10.1107/S2053230X14004014
http://hdl.handle.net/10033/326008
Acta crystallographica. Section F, Structural biology communications
Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase.
oai:repository.helmholtz-hzi.de:10033/3261982019-08-30T11:36:05Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Li, Zhaopeng
author
Nimtz, Manfred
author
Rinas, Ursula
author
2014
The proteome reflects the available cellular machinery to deal with nutrients and environmental challenges. The most common E. coli strain BL21 growing in different, commonly employed media was evaluated using a detailed quantitative proteome analysis.
The metabolic potential of Escherichia coli BL21 in defined and rich medium. 2014, 13 (1):45 Microb. Cell Fact.
1475-2859
24656150
10.1186/1475-2859-13-45
http://hdl.handle.net/10033/326198
Microbial cell factories
The metabolic potential of Escherichia coli BL21 in defined and rich medium.
oai:repository.helmholtz-hzi.de:10033/3370282019-08-30T11:34:48Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Blankenfeldt, Wulf
author
Parsons, James F
author
2014-09-09
The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis.
The structural biology of phenazine biosynthesis. 2014, 29C:26-33 Curr. Opin. Struct. Biol.
1879-033X
25215885
10.1016/j.sbi.2014.08.013
http://hdl.handle.net/10033/337028
Current opinion in structural biology
The structural biology of phenazine biosynthesis.
oai:repository.helmholtz-hzi.de:10033/3387232019-08-30T11:34:48Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Halbedel, Sven
author
Reiss, Swantje
author
Hahn, Birgit
author
Albrecht, Dirk
author
Mannala, Gopala Krishna
author
Chakraborty, Trinad
author
Hain, Torsten
author
Engelmann, Susanne
author
Flieger, Antje
author
2014-11
Listeria monocytogenes is a firmicute bacterium causing serious infections in humans upon consumption of contaminated food. Most of its virulence factors are secretory proteins either released to the medium or attached to the bacterial surface. L. monocytogenes encodes at least six different protein secretion pathways. Although great efforts have been made in the past to predict secretory proteins and their secretion routes using bioinformatics, experimental evidence is lacking for most secretion systems. Therefore, we constructed mutants in the main housekeeping protein secretion systems, which are the Sec-dependent transport, the YidC membrane insertases SpoIIIJ and YqjG, as well as the twin-arginine pathway, and analyzed their secretion and virulence defects. Our results demonstrate that Sec-dependent secretion and membrane insertion of proteins via YidC proteins are essential for viability of L. monocytogenes. Depletion of SecA or YidC activity severely affected protein secretion, whereas loss of the Tat-pathway was without any effect on secretion, viability, and virulence. Two-dimensional gel electrophoresis combined with protein identification by mass spectrometry revealed that secretion of many virulence factors and of enzymes synthesizing and degrading the cell wall depends on the SecA route. This finding was confirmed by SecA inhibition experiments using sodium azide. Analysis of secretion of substrates typically dependent on the accessory SecA2 ATPase in wild type and azide resistant mutants of L. monocytogenes revealed for the first time that SecA2-dependent protein secretion also requires the ATPase activity of the house-keeping SecA protein.
A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems. 2014, 13 (11):3063-81 Mol. Cell Proteomics
1535-9484
25056936
10.1074/mcp.M114.041327
http://hdl.handle.net/10033/338723
Molecular & cellular proteomics : MCP
A systematic proteomic analysis of Listeria monocytogenes house-keeping protein secretion systems.
oai:repository.helmholtz-hzi.de:10033/3453762019-08-30T11:28:51Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Shevchuk, Olga
author
Pägelow, Dennis
author
Rasch, Janine
author
Döhrmann, Simon
author
Günther, Gabriele
author
Hoppe, Julia
author
Ünal, Can Murat
author
Bronietzki, Marc
author
Gutierrez, Maximiliano Gabriel
author
Steinert, Michael
author
2014-11
L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.
Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection. 2014, 304 (8):1169-81 Int. J. Med. Microbiol.
1618-0607
25218702
10.1016/j.ijmm.2014.08.010
http://hdl.handle.net/10033/345376
International journal of medical microbiology : IJMM
Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.
oai:repository.helmholtz-hzi.de:10033/3465282019-08-30T11:37:23Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Kuhle, Katja
author
Krausze, Joern
author
Curth, Ute
author
Rössle, Manfred
author
Heuner, Klaus
author
Lang, Christina
author
Flieger, Antje
author
2014-07-04
The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 μm where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.
Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity. 2014, 289 (27):18657-66 J. Biol. Chem.
1083-351X
24811180
10.1074/jbc.M114.573196
http://hdl.handle.net/10033/346528
The Journal of biological chemistry
Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity.
oai:repository.helmholtz-hzi.de:10033/5526802019-08-30T11:30:52Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Büssow, Konrad
author
2015-03-21
The mammalian cell lines HEK293 and CHO have become important expression hosts in structural biology. Generating stable mammalian cell lines remains essential for studying the function and structure of recombinant proteins, despite the emergence of highly efficient transient transfection protocols. Production with stable cell lines can be scaled up easily and high volumetric product yield can be achieved. Protein structure reports of the past two years that used stable cell lines were surveyed for this review. Well-established techniques and novel approaches for generating stable cell lines and stable cell pools are presented, including cell sorting, site-specific recombination, transposons, the Lentivirus system and phage integrases. Host cell line optimization by endoglycosidase overexpression and sequence-specific genome engineering is highlighted.
Stable mammalian producer cell lines for structural biology. 2015, 32:81-90 Curr. Opin. Struct. Biol.
1879-033X
25804355
10.1016/j.sbi.2015.03.002
http://hdl.handle.net/10033/552680
Current opinion in structural biology
Stable mammalian producer cell lines for structural biology.
oai:repository.helmholtz-hzi.de:10033/5530692019-08-30T11:27:46Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Worthmann, Hans
author
Tryc, Anita B
author
Dirks, Meike
author
Schuppner, Ramona
author
Brand, Korbinian
author
Klawonn, Frank
author
Lichtinghagen, Ralf
author
Weissenborn, Karin
author
2015
Ischemic stroke patients are prone to infection by stroke-induced immunodepression. We hypothesized that levels of lipopolysaccharide binding protein (LBP), interleukin-10 (IL-10), IL-6 and C-reactive protein (CRP) are early predictors for the development of stroke-associated infection.
Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection. 2015, 12 (1):13 J Neuroinflammation
1742-2094
25613713
10.1186/s12974-014-0231-2
http://hdl.handle.net/10033/553069
Journal of neuroinflammation
Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection.
oai:repository.helmholtz-hzi.de:10033/5566622019-08-30T11:26:42Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Seele, Jana
author
Beineke, Andreas
author
Hillermann, Lena-Maria
author
Jaschok-Kentner, Beate
author
von Pawel-Rammingen, Ulrich
author
Valentin-Weigand, Peter
author
Baums, Christoph Georg
author
2015
Streptococcus (S.) suis is one of the most important pathogens in pigs causing meningitis, arthritis, endocarditis and serositis. Furthermore, it is also an emerging zoonotic agent. In our previous work we identified a highly specific IgM protease in S. suis, designated Ide Ssuis . The objective of this study was to characterize the function of Ide Ssuis in the host-pathogen interaction. Edman-sequencing revealed that Ide Ssuis cleaves the heavy chain of the IgM molecule between constant domain 2 and 3. As the C1q binding motif is located in the C3 domain, we hypothesized that Ide Ssuis is involved in complement evasion. Complement-mediated hemolysis induced by porcine hyperimmune sera containing erythrocyte-specific IgM was abrogated by treatment of these sera with recombinant Ide Ssuis . Furthermore, expression of Ide Ssuis reduced IgM-triggered complement deposition on the bacterial surface. An infection experiment of prime-vaccinated growing piglets suggested attenuation in the virulence of the mutant 10Δide Ssuis . Bactericidal assays confirmed a positive effect of Ide Ssuis expression on bacterial survival in porcine blood in the presence of high titers of specific IgM. In conclusion, this study demonstrates that Ide Ssuis is a novel complement evasion factor, which is important for bacterial survival in porcine blood during the early adaptive (IgM-dominated) immune response.
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis , is involved in complement evasion. 2015, 46 (1):45 Vet. Res.
1297-9716
25928761
10.1186/s13567-015-0171-6
http://hdl.handle.net/10033/556662
Veterinary research
The immunoglobulin M-degrading enzyme of Streptococcus suis, Ide Ssuis , is involved in complement evasion.
oai:repository.helmholtz-hzi.de:10033/5591542019-08-30T11:32:17Zcom_10033_311308col_10033_621053
00925njm 22002777a 4500
dc
Simon, Bernd
author
Masiewicz, Pawel
author
Ephrussi, Anne
author
Carlomagno, Teresa
author
2015-06-18
mRNA localization by active transport is a regulated process that requires association of mRNPs with protein motors for transport along either the microtubule or the actin cytoskeleton. oskar mRNA localization at the posterior pole of the Drosophila oocyte requires a specific mRNA sequence, termed the SOLE, which comprises nucleotides of both exon 1 and exon 2 and is assembled upon splicing. The SOLE folds into a stem-loop structure. Both SOLE RNA and the exon junction complex (EJC) are required for oskar mRNA transport along the microtubules by kinesin. The SOLE RNA likely constitutes a recognition element for a yet unknown protein, which either belongs to the EJC or functions as a bridge between the EJC and the mRNA. Here, we determine the solution structure of the SOLE RNA by Nuclear Magnetic Resonance spectroscopy. We show that the SOLE forms a continuous helical structure, including a few noncanonical base pairs, capped by a pentanucleotide loop. The helix displays a widened major groove, which could accommodate a protein partner. In addition, the apical helical segment undergoes complex dynamics, with potential functional significance.
The structure of the SOLE element of oskar mRNA. 2015: RNA
1469-9001
26089324
10.1261/rna.049601.115
http://hdl.handle.net/10033/559154
RNA (New York, N.Y.)
The structure of the SOLE element of oskar mRNA.
oai:repository.helmholtz-hzi.de:10033/5608732019-08-30T11:34:22Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Stentzel, Sebastian
author
Sundaramoorthy, Nandakumar
author
Michalik, Stephan
author
Nordengrün, Maria
author
Schulz, Sarah
author
Kolata, Julia
author
Kloppot, Peggy
author
Engelmann, Susanne
author
Steil, Leif
author
Hecker, Michael
author
Schmidt, Frank
author
Völker, Uwe
author
Roghmann, Mary-Claire
author
Bröker, Barbara M
author
2015-07-05
Although Staphylococcus aureus is a prominent cause of infections, no vaccine is currently available. Active vaccination relies on immune memory, a core competence of the adaptive immune system. To elucidate whether adaptive immunity can provide protection from serious complications of S. aureus infection, a prospective observational study of 44 patients with S. aureus infection complicated by bacteremia was conducted. At diagnosis, serum IgG binding to S. aureus extracellular proteins was quantified on immunoblots and with Luminex-based FLEXMAP 3D™ assays comprising 64 recombinant S. aureus proteins. Results were correlated with the course of the infection with sepsis as the main outcome variable. S. aureus-specific serum IgG levels at diagnosis of S. aureus infection were lower in patients developing sepsis than in patients without sepsis (P<0.05). The pattern of IgG binding to eight selected S. aureus proteins correctly predicted the disease course in 75% of patients. Robust immune memory of S. aureus was associated with protection from serious complications of bacterial invasion. Serum IgG binding to eight conserved S. aureus proteins enabled stratification of patients with high and low risk of sepsis early in the course of S. aureus infections complicated by bacteremia.
Specific serum IgG at diagnosis of Staphylococcus aureus bloodstream invasion is correlated with disease progression. 2015, 128:1-7 J Proteomics
1876-7737
26155744
10.1016/j.jprot.2015.06.018
http://hdl.handle.net/10033/560873
Journal of proteomics
Specific serum IgG at diagnosis of Staphylococcus aureus bloodstream invasion is correlated with disease progression.
oai:repository.helmholtz-hzi.de:10033/5611762019-08-30T11:27:46Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Quade, Nick
author
Boehringer, Daniel
author
Leibundgut, Marc
author
van den Heuvel, Joop
author
Ban, Nenad
author
2015
Hepatitis C virus (HCV), a widespread human pathogen, is dependent on a highly structured 5'-untranslated region of its mRNA, referred to as internal ribosome entry site (IRES), for the translation of all of its proteins. The HCV IRES initiates translation by directly binding to the small ribosomal subunit (40S), circumventing the need for many eukaryotic translation initiation factors required for mRNA scanning. Here we present the cryo-EM structure of the human 40S ribosomal subunit in complex with the HCV IRES at 3.9 Å resolution, determined by focused refinement of an 80S ribosome-HCV IRES complex. The structure reveals the molecular details of the interactions between the IRES and the 40S, showing that expansion segment 7 (ES7) of the 18S rRNA acts as a central anchor point for the HCV IRES. The structural data rationalizes previous biochemical and genetic evidence regarding the initiation mechanism of the HCV and other related IRESs.
Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution. 2015, 6:7646 Nat Commun
2041-1723
26155016
10.1038/ncomms8646
http://hdl.handle.net/10033/561176
Nature communications
Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution.
oai:repository.helmholtz-hzi.de:10033/5613172019-08-30T11:25:43Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Hellert, Jan
author
Krausze, Joern
author
Schulz, Thomas F
author
Lührs, Thorsten
author
2014-11
The latency-associated nuclear antigen (LANA) is the latent origin-binding protein and chromatin anchor of the Kaposi's sarcoma herpesvirus (KSHV/HHV-8) genome. Its C-terminal domain (CTD) binds sequence-specifically to the viral origin of replication, whereas the N-terminal domain links it to nucleosomes of cellular chromatin for long-term persistence in dividing host cells. Here, the crystallization and X-ray data acquisition of a mutant LANA CTD in complex with its wild-type target DNA LBS1 is described. This report describes the rational protein engineering for successful co-crystallization with DNA and X-ray diffraction data collection at room temperature on the high-brilliance third-generation synchrotron PETRA III at DESY, Germany.
Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA. 2014, 70 (Pt 11):1570-4 Acta Crystallogr F Struct Biol Commun
2053-230X
25372834
10.1107/S2053230X14019906
http://hdl.handle.net/10033/561317
Acta crystallographica. Section F, Structural biology communications
Crystallization, room-temperature X-ray diffraction and preliminary analysis of Kaposi's sarcoma herpesvirus LANA bound to DNA.
oai:repository.helmholtz-hzi.de:10033/5662532019-08-30T11:34:22Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Zahid, Maria
author
Lünsdorf, Heinrich
author
Rinas, Ursula
author
2015-07-17
The hepatitis B surface antigen (HBsAg) is a recombinant protein-based vaccine being able to form virus-like particles (VLPs). HBsAg is mainly produced using yeast-based expression systems, however, recent results strongly suggest that VLPs are not formed within the yeast cells during the cultivation but are formed in a gradual manner during the following down-stream procedures. VLPs are also not detectable during the first down-stream steps including mechanical and EDTA/detergent-assisted cell destruction. Moreover, VLPs are not detectable in the cell lysate treated with polyethylene glycol and colloidal silica. The first VLP resembling structures appear after elution of HBsAg from colloidal silica to which it binds through hydrophobic interaction. These first VLP resembling structures are non-symmetrical as well as heterodisperse and exhibit a high tendency toward cluster formation presumably because of surface exposed hydrophobic patches. More symmetrical and monodisperse VLPs appear after the following ion-exchange and size-exclusion chromatography most likely as the result of buffer changes during these purification steps (toward more neutral pH and less salt). Final treatment of the VLPs with the denaturant KSCN at moderate concentrations with following KSCN removal by dialysis does not cause unfolding and VLP disassembly but results in a re- and fine-structuring of the VLP surface topology.
Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing. 2015, 33 (31):3739-45 Vaccine
1873-2518
26079614
10.1016/j.vaccine.2015.05.066
http://hdl.handle.net/10033/566253
Vaccine
Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing.
oai:repository.helmholtz-hzi.de:10033/6210292018-06-13T09:23:33Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Bauer, Isabel
author
Günther, Juliane
author
Wheeler, Thomas T
author
Engelmann, Susanne
author
Seyfert, Hans-Martin
author
2015-07-30
Abstract Background Considerably divergent data have been published from attempts to model the E. coli vs. S. aureus specific immune reaction of the udder using primary cultures of bovine mammary epithelial cells from cows (pbMEC). Some groups reported a swift, strong and transient inflammatory response against challenges with E. coli and only a weak and retarded response against S. aureus, in agreement with the respective reaction of the udder. Others found almost the reverse. Presence or absence of fetal calf serum distinguished the experimental setting between both groups. We examined here if this causes the divergent reaction of the pbMEC towards both pathogen species. We challenged pbMEC with proteins from heat killed E. coli or S. aureus pathogens or purified TLR2 and TLR4 ligands. The stimuli were applied in normal growth medium with (SM10) or without (SM0) 10 % fetal calf serum, or in the basal medium supplemented with 10 mg/ml milk proteins (SM Milk). Results Withdrawal of FCS slowed down and decreased the extent by which E. coli or LPS enhanced the expression of cyto- and chemokine encoding genes through impaired TLR4 signalling but enforced their expression during stimulation with S. aureus. SM Milk strongly quenched the induction of those genes. S. aureus strain specific differences in the reaction of the pbMEC could only be recorded in SM0. NF-κB factors were activated by E. coli in all stimulation media, but only to a small extent by S. aureus, solely in SM0. Purified ligands for TLR2 stimulated expression of those genes and activated NF-κB equally well in SM10 and SM0. The mRNA destabilizing factor tristetraproline was only induced by E. coli in SM10 and by purified PAMPs. Conclusions Our data cross validate the correctness of previously published divergent data on the pathogen-specific induction of key immune genes in pbMEC. The differences are due to the presence of FCS, modulating signalling through TLR4 and TLR-unrelated pathogen receptors. S. aureus does not substantially activate any TLR signalling in MEC. Rather, receptors distinct from TLRs perceive the presence of S. aureus and control the immune response against this pathogen in MEC.
BMC Veterinary Research. 2015 Jul 30;11(1):172
http://dx.doi.org/10.1186/s12917-015-0489-3
http://hdl.handle.net/10033/621029
Extracellular milieu grossly alters pathogen-specific immune response of mammary epithelial cells
oai:repository.helmholtz-hzi.de:10033/6208332019-08-30T11:25:43Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Solbak, Sara M
author
Wray, Victor
author
Horvli, Ole
author
Raae, Arnt J
author
Flydal, Marte I
author
Henklein, Petra
author
Henklein, Peter
author
Nimtz, Manfred
author
Schubert, Ulrich
author
Fossen, Torgils
author
2011-12-20
Abstract Background Cyclophilin A (CypA) represents a potential key molecule in future antiretroviral therapy since inhibition of CypA suppresses human immunodeficiency virus type 1 (HIV-1) replication. CypA interacts with the virus proteins Capsid (CA) and Vpr, however, the mechanism through which CypA influences HIV-1 infectivity still remains unclear. Results Here the interaction of full-length HIV-1 Vpr with the host cellular factor CypA has been characterized and quantified by surface plasmon resonance spectroscopy. A C-terminal region of Vpr, comprising the 16 residues 75GCRHSRIGVTRQRRAR90, with high binding affinity for CypA has been identified. This region of Vpr does not contain any proline residues but binds much more strongly to CypA than the previously characterized N-terminal binding domain of Vpr, and is thus the first protein binding domain to CypA described involving no proline residues. The fact that the mutant peptide Vpr75-90 R80A binds more weakly to CypA than the wild-type peptide confirms that Arg-80 is a key residue in the C-terminal binding domain. The N- and C-terminal binding regions of full-length Vpr bind cooperatively to CypA and have allowed a model of the complex to be created. The dissociation constant of full-length Vpr to CypA was determined to be approximately 320 nM, indicating that the binding may be stronger than that of the well characterized interaction of HIV-1 CA with CypA. Conclusions For the first time the interaction of full-length Vpr and CypA has been characterized and quantified. A non-proline-containing 16-residue region of C-terminal Vpr which binds specifically to CypA with similar high affinity as full-length Vpr has been identified. The fact that this is the first non-proline containing binding motif of any protein found to bind to CypA, changes the view on how CypA is able to interact with other proteins. It is interesting to note that several previously reported key functions of HIV-1 Vpr are associated with the identified N- and C-terminal binding domains of the protein to CypA.
BMC Structural Biology. 2011 Dec 20;11(1):49
http://dx.doi.org/10.1186/1472-6807-11-49
http://hdl.handle.net/10033/620833
The Host-Pathogen interaction of human cyclophilin A and HIV-1 Vpr requires specific N-terminal and novel C-terminal domains
oai:repository.helmholtz-hzi.de:10033/6207902019-08-30T11:25:43Zcom_10033_311308col_10033_620777col_10033_620777
00925njm 22002777a 4500
dc
Gurramkonda, Chandrasekhar
author
Adnan, Ahmad
author
Gäbel, Thomas
author
Lünsdorf, Heinrich
author
Ross, Anton
author
Nemani, Satish K
author
Swaminathan, Sathyamangalam
author
Khanna, Navin
author
Rinas, Ursula
author
2009-02-10
Abstract Background Hepatitis B is a serious global public health concern. Though a safe and efficacious recombinant vaccine is available, its use in several resource-poor countries is limited by cost. We have investigated the production of Hepatitis B virus surface antigen (HBsAg) using the yeast Pichia pastoris GS115 by inserting the HBsAg gene into the alcohol oxidase 1 locus. Results Large-scale production was optimized by developing a simple fed-batch process leading to enhanced product titers. Cells were first grown rapidly to high-cell density in a batch process using a simple defined medium with low salt and high glycerol concentrations. Induction of recombinant product synthesis was carried out using rather drastic conditions, namely through the addition of methanol to a final concentration of 6 g L-1. This methanol concentration was kept constant for the remainder of the cultivation through continuous methanol feeding based on the on-line signal of a flame ionization detector employed as methanol analyzer in the off-gas stream. Using this robust feeding protocol, maximum concentrations of ~7 grams HBsAg per liter culture broth were obtained. The amount of soluble HBsAg, competent for assembly into characteristic virus-like particles (VLPs), an attribute critical to its immunogenicity and efficacy as a hepatitis B vaccine, reached 2.3 grams per liter of culture broth. Conclusion In comparison to the highest yields reported so far, our simple cultivation process resulted in an ~7 fold enhancement in total HBsAg production with more than 30% of soluble protein competent for assembly into VLPs. This work opens up the possibility of significantly reducing the cost of vaccine production with implications for expanding hepatitis B vaccination in resource-poor countries.
Microbial Cell Factories. 2009 Feb 10;8(1):13
http://dx.doi.org/10.1186/1475-2859-8-13
http://hdl.handle.net/10033/620790
Simple high-cell density fed-batch technique for high-level recombinant protein production with Pichia pastoris: Application to intracellular production of Hepatitis B surface antigen
oai:repository.helmholtz-hzi.de:10033/6207882019-08-30T11:36:32Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Martínez-Alonso, Mónica
author
García-Fruitós, Elena
author
Ferrer-Miralles, Neus
author
Rinas, Ursula
author
Villaverde, Antonio
author
2010-09-02
Abstract Insufficient availability of molecular chaperones is observed as a major bottleneck for proper protein folding in recombinant protein production. Therefore, co-production of selected sets of cell chaperones along with foreign polypeptides is a common approach to increase the yield of properly folded, recombinant proteins in bacterial cell factories. However, unbalanced amounts of folding modulators handling folding-reluctant protein species might instead trigger undesired proteolytic activities, detrimental regarding recombinant protein stability, quality and yield. This minireview summarizes the most recent observations of chaperone-linked negative side effects, mostly focusing on DnaK and GroEL sets, when using these proteins as folding assistant agents. These events are discussed in the context of the complexity of the cell quality network and the consequent intricacy of the physiological responses triggered by protein misfolding.
Microbial Cell Factories. 2010 Sep 02;9(1):64
http://dx.doi.org/10.1186/1475-2859-9-64
http://hdl.handle.net/10033/620788
Side effects of chaperone gene co-expression in recombinant protein production
oai:repository.helmholtz-hzi.de:10033/6207852019-08-30T11:37:44Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Jäger, Volker
author
Büssow, Konrad
author
Wagner, Andreas
author
Weber, Susanne
author
Hust, Michael
author
Frenzel, André
author
Schirrmann, Thomas
author
2013-06-26
Abstract Background The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. Results In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. Conclusion Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.
BMC Biotechnology. 2013 Jun 26;13(1):52
http://dx.doi.org/10.1186/1472-6750-13-52
http://hdl.handle.net/10033/620785
High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells
oai:repository.helmholtz-hzi.de:10033/6207832018-06-13T00:40:48Zcom_10033_311308col_10033_620561
00925njm 22002777a 4500
dc
He, Feng Q
author
Sauermann, Ulrike
author
Beer, Christiane
author
Winkelmann, Silke
author
Yu, Zheng
author
Sopper, Sieghart
author
Zeng, An-Ping
author
Wirth, Manfred
author
2014-08-27
Abstract Background The deciphering of cellular networks to determine susceptibility to infection by HIV or the related simian immunodeficiency virus (SIV) is a major challenge in infection biology. Results Here, we have compared gene expression profiles of a human CD4+ T cell line at 24 h after infection with a cell line of the same origin permanently releasing SIVmac. A new knowledge-based-network approach (Inter-Chain-Finder, ICF) has been used to identify sub-networks associated with cell survival of a chronically SIV-infected T cell line. Notably, the method can identify not only differentially expressed key hub genes but also non-differentially expressed, critical, ‘hidden’ regulators. Six out of the 13 predicted major hidden key regulators were among the landscape of proteins known to interact with HIV. Several sub-networks were dysregulated upon chronic infection with SIV. Most prominently, factors reported to be engaged in early stages of acute viral infection were affected, e.g. entry, integration and provirus transcription and other cellular responses such as apoptosis and proliferation were modulated. For experimental validation of the gene expression analyses and computational predictions, individual pathways/sub-networks and significantly altered key regulators were investigated further. We showed that the expression of caveolin-1 (Cav-1), the top hub in the affected protein-protein interaction network, was significantly upregulated in chronically SIV-infected CD4+ T cells. Cav-1 is the main determinant of caveolae and a central component of several signal transduction pathways. Furthermore, CD4 downregulation and modulation of the expression of alternate and co-receptors as well as pathways associated with viral integration into the genome were also observed in these cells. Putatively, these modifications interfere with re-infection and the early replication cycle and inhibit cell death provoked by syncytia formation and bystander apoptosis. Conclusions Thus, by using the novel approach for network analysis, ICF, we predict that in the T cell line chronically infected with SIV, cellular processes that are known to be crucial for early phases of HIV/SIV replication are altered and cellular responses that result in cell death are modulated. These modifications presumably contribute to cell survival despite chronic infection.
Virology Journal. 2014 Aug 27;11(1):152
http://dx.doi.org/10.1186/1743-422X-11-152
http://hdl.handle.net/10033/620783
Identification of molecular sub-networks associated with cell survival in a chronically SIVmac-infected human CD4+ T cell line
oai:repository.helmholtz-hzi.de:10033/6207792019-08-30T11:25:43Zcom_10033_311308col_10033_559591
00925njm 22002777a 4500
dc
Wilke, Sonja
author
Krausze, Joern
author
Büssow, Konrad
author
2012-07-19
Abstract Background The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. Results The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel β-prism fold formed by two β-sheets bent by β-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. Conclusion The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown β-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.
BMC Biology. 2012 Jul 19;10(1):62
http://dx.doi.org/10.1186/1741-7007-10-62
http://hdl.handle.net/10033/620779
Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx
oai:repository.helmholtz-hzi.de:10033/6207762018-06-12T21:27:46Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Khetarpal, Niyati
author
Poddar, Ankur
author
Nemani, Satish K
author
Dhar, Nisha
author
Patil, Aravind
author
Negi, Priyanka
author
Perween, Ashiya
author
Viswanathan, Ramaswamy
author
Lünsdorf, Heinrich
author
Tyagi, Poornima
author
Raut, Rajendra
author
Arora, Upasana
author
Jain, Swatantra K
author
Rinas, Ursula
author
Swaminathan, Sathyamangalam
author
Khanna, Navin
author
2013-05-25
Abstract Background Dengue is today the most significant of arboviral diseases. Novel tools are necessary to effectively address the problem of dengue. Virus-like particles (VLP) offer a versatile nanoscale platform for developing tools with potential biomedical applications. From the perspective of a potentially useful dengue-specific tool, the dengue virus envelope protein domain III (EDIII), endowed with serotype-specificity, host receptor recognition and the capacity to elicit virus-neutralizing antibodies, is an attractive candidate. Methods We have developed a strategy to co-express and co-purify Hepatitis B virus surface (S) antigen in two forms: independently and as a fusion with EDIII. We characterized these physically and functionally. Results The two forms of the S antigen associate into VLPs. The ability of these to display EDIII in a functionally accessible manner is dependent upon the relative levels of the two forms of the S antigen. Mosaic VLPs containing the fused and un-fused components in 1:4 ratio displayed maximal functional competence. Conclusions VLPs armed with EDIII may be potentially useful in diagnostic, therapeutic and prophylactic applications.
Journal of Nanobiotechnology. 2013 May 25;11(1):15
http://dx.doi.org/10.1186/1477-3155-11-15
http://hdl.handle.net/10033/620776
Dengue-specific subviral nanoparticles: design, creation and characterization
oai:repository.helmholtz-hzi.de:10033/6207702019-08-30T11:25:43Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Vanz, Ana L
author
Lünsdorf, Heinrich
author
Adnan, Ahmad
author
Nimtz, Manfred
author
Gurramkonda, Chandrasekhar
author
Khanna, Navin
author
Rinas, Ursula
author
2012-08-08
Abstract Background Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. Conclusions In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.
Microbial Cell Factories. 2012 Aug 08;11(1):103
http://dx.doi.org/10.1186/1475-2859-11-103
http://hdl.handle.net/10033/620770
Physiological response of Pichia pastoris GS115 to methanol-induced high level production of the Hepatitis B surface antigen: catabolic adaptation, stress responses, and autophagic processes
oai:repository.helmholtz-hzi.de:10033/6207692018-06-13T00:14:16Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Worthmann, Hans
author
Tryc, Anita B
author
Dirks, Meike
author
Schuppner, Ramona
author
Brand, Korbinian
author
Klawonn, Frank
author
Lichtinghagen, Ralf
author
Weissenborn, Karin
author
2015-01-23
Abstract Background Ischemic stroke patients are prone to infection by stroke-induced immunodepression. We hypothesized that levels of lipopolysaccharide binding protein (LBP), interleukin-10 (IL-10), IL-6 and C-reactive protein (CRP) are early predictors for the development of stroke-associated infection. Methods Fifty-six patients with ischemic stroke (n = 51) and transient ischemic attack (TIA) (n = 5) who presented within 6 hours after symptom onset and who were free of detectable infection on admission were included in the study. Of these, 20 developed early infections during the first week. Blood samples were taken at 6, 12, and 24 hours and at 3 and 7 days after stroke onset. Levels of LBP, Il-10, IL-6 and CRP, as well as S100B, were measured as markers of inflammation and brain damage by commercially available immunometric tests. Results In the univariate analysis, levels of LBP, IL-10, IL-6 and CRP significantly differed between patients who developed an infection and those who did not. In the binary logistic regression analysis, which was adjusted for National Institutes of Health Stroke Scale (NIHSS) on admission, stroke subtype and S100B peak levels, as indicator of the extent of brain damage, IL-10 at 6 hours, CRP at 6 hours and NIHSS on admission were identified as independent predictors of infection (IL-10: P = 0.009; CRP: P = 0.018; NIHSS: P = 0.041). The area under the curve (AUC) of the receiver operating characteristic (ROC) curves in relation to the dichotomized status of the infection (infection versus no infection) was 0.74 (95% confidence interval: 0.59 to 0.88) for CRP at 6 hours, 0.76 (0.61 to 0.9) for IL-10 at 6 hours, 0.83 (0.71 to 0.94) for NIHSS on admission and 0.94 (0.88 to 1) for the combination of CRP, IL-10 and NIHSS. In a subanalysis, 16 patients with early infections were matched with 16 patients without infection according to S100B peak levels. Here, the temporal pattern of LBP, IL-10, IL-6 and CRP significantly differed between the patient groups. Conclusions Our data show that blood levels of inflammation markers may be used as early predictors of stroke-associated infection. We propose prospective studies to investigate if the calculated cut-offs of CRP, IL-10 and NIHSS might help to identify patients who should receive early preventive antibiotic treatment.
Journal of Neuroinflammation. 2015 Jan 23;12(1):13
http://dx.doi.org/10.1186/s12974-014-0231-2
http://hdl.handle.net/10033/620769
Lipopolysaccharide binding protein, interleukin-10, interleukin-6 and C-reactive protein blood levels in acute ischemic stroke patients with post-stroke infection
oai:repository.helmholtz-hzi.de:10033/6207672018-06-13T15:13:41Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Gasser, Brigitte
author
Saloheimo, Markku
author
Rinas, Ursula
author
Dragosits, Martin
author
Rodríguez-Carmona, Escarlata
author
Baumann, Kristin
author
Giuliani, Maria
author
Parrilli, Ermenegilda
author
Branduardi, Paola
author
Lang, Christine
author
Porro, Danilo
author
Ferrer, Pau
author
Luisa Tutino, Maria
author
Mattanovich, Diethard
author
Villaverde, Antonio
author
2008-04-04
Abstract Different species of microorganisms including yeasts, filamentous fungi and bacteria have been used in the past 25 years for the controlled production of foreign proteins of scientific, pharmacological or industrial interest. A major obstacle for protein production processes and a limit to overall success has been the abundance of misfolded polypeptides, which fail to reach their native conformation. The presence of misfolded or folding-reluctant protein species causes considerable stress in host cells. The characterization of such adverse conditions and the elicited cell responses have permitted to better understand the physiology and molecular biology of conformational stress. Therefore, microbial cell factories for recombinant protein production are depicted here as a source of knowledge that has considerably helped to picture the extremely rich landscape of in vivo protein folding, and the main cellular players of this complex process are described for the most important cell factories used for biotechnological purposes.
Microbial Cell Factories. 2008 Apr 04;7(1):11
http://dx.doi.org/10.1186/1475-2859-7-11
http://hdl.handle.net/10033/620767
Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview
oai:repository.helmholtz-hzi.de:10033/6207652019-08-30T11:30:58Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Coldewey, Sina M
author
Hartmann, Maike
author
Schmidt, Dorothea S
author
Engelking, Uta
author
Ukena, Sya N
author
Gunzer, Florian
author
2007-03-26
Abstract Background Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σS, encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. Methods Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. Results First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. Conclusion The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype.
BMC Microbiology. 2007 Mar 26;7(1):21
http://dx.doi.org/10.1186/1471-2180-7-21
http://hdl.handle.net/10033/620765
Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli
oai:repository.helmholtz-hzi.de:10033/6207572019-08-30T11:31:23Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Villaverde, Antonio
author
García-Fruitós, Elena
author
Rinas, Ursula
author
Seras-Franzoso, Joaquin
author
Kosoy, Ana
author
Corchero, José L
author
Vazquez, Esther
author
2012-06-11
Abstract A growing number of insights on the biology of bacterial inclusion bodies (IBs) have revealed intriguing utilities of these protein particles. Since they combine mechanical stability and protein functionality, IBs have been already exploited in biocatalysis and explored for bottom-up topographical modification in tissue engineering. Being fully biocompatible and with tuneable bio-physical properties, IBs are currently emerging as agents for protein delivery into mammalian cells in protein-replacement cell therapies. So far, IBs formed by chaperones (heat shock protein 70, Hsp70), enzymes (catalase and dihydrofolate reductase), grow factors (leukemia inhibitory factor, LIF) and structural proteins (the cytoskeleton keratin 14) have been shown to rescue exposed cells from a spectrum of stresses and restore cell functions in absence of cytotoxicity. The natural penetrability of IBs into mammalian cells (reaching both cytoplasm and nucleus) empowers them as an unexpected platform for the controlled delivery of essentially any therapeutic polypeptide. Production of protein drugs by biopharma has been traditionally challenged by IB formation. However, a time might have arrived in which recombinant bacteria are to be engineered for the controlled packaging of therapeutic proteins as nanoparticulate materials (nanopills), for their extra- or intra-cellular release in medicine and cosmetics.
Microbial Cell Factories. 2012 Jun 11;11(1):76
http://dx.doi.org/10.1186/1475-2859-11-76
http://hdl.handle.net/10033/620757
Packaging protein drugs as bacterial inclusion bodies for therapeutic applications
oai:repository.helmholtz-hzi.de:10033/6207542019-08-30T11:31:23Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Li, Zhaopeng
author
Carstensen, Bettina
author
Rinas, Ursula
author
2014-11-05
Abstract Background Recombinant proteins are usually required in laboratories interested in the protein but not in the production process itself. Thus, technical equipment which is easy to handle and straight forward protein production procedures are of great benefit to those laboratories. Companies selling single use cultivation bags and bioreactors are trying to satisfy at least part of these needs. However, single-use systems can contribute to major costs which might be acceptable when “good manufacturing practices” are required but not acceptable for most laboratories facing tight funding. Results The assembly and application of a simple self-made “smart sustainable bottle” (SSB) system for E. coli based protein production is presented. The core of the SSB system is a 2-L glass bottle which is operated at constant temperature, air flow, and stirrer speed without measurement and control of pH and dissolved oxygen. Oxygen transfer capacities are in the range as in conventional bioreactors operated at intermediate aeration rates and by far exceed those found in conventional shaking flasks and disposable bioreactors. The SSB system was applied for the production of various recombinant proteins using T7-based expression systems and a defined autoinduction medium. The production performance regarding amount and solubility of proteins with robust and delicate properties was as good as in state-of-the-art stirred tank commercial bioreactors. Conclusions The SSB system represents a low cost protein production device applicable for easy, effective, and reproducible recombinant protein production.
Microbial Cell Factories. 2014 Nov 05;13(1):153
http://dx.doi.org/10.1186/s12934-014-0153-9
http://hdl.handle.net/10033/620754
Smart sustainable bottle (SSB) system for E. coli based recombinant protein production
oai:repository.helmholtz-hzi.de:10033/6207122019-08-30T11:37:00Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Della Beffa, Cristina
author
Klawonn, Frank
author
Menetski, Joseph P
author
Schumacher, H R
author
Pessler, Frank
author
2011-10-25
Abstract Background The murine air pouch membrane represents an easily accessible tissue for studies on gene regulation in acute inflammation. Considering that acute inflammation may affect expression of molecular reference genes, we evaluated the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prolylpeptidyl isomerase A (PPIA) in the air pouch membrane during a complete time course of urate crystal inflammation and correlated the results with expression of interleukin (IL)-1β and hypoxia inducible factor (HIF)-1α. In addition, we aimed to identify alternate potential reference genes. Methods Using custom microfluidic real-time PCR arrays, the expression of 96 genes including GAPDH, PPIA, IL-1β, and HIF-1α was determined in dissected air pouch membranes 1, 4, 9, 18, 27, and 50 hours (h) after injecting monosodium urate (MSU) crystals into the pouch. One-way ANOVA was used to detect differential gene expression throughout the time course. Using the genes on these arrays as a convenience sample, alternate candidate reference genes were sought (1) with a biostatistical approach and (2) using the geNorm software tool. Results Pouch leukocytes peaked at t = 9h and declined toward t = 50h. PPIA expression was not differentially regulated (p = 0.52, ANOVA). In contrast, GAPDH mRNA increased steadily after crystal injection, reaching a maximal 2.8-fold increase at t = 18h (p = 0.0006, t test), which followed a marked induction of IL-1β (max., 208-fold at t = 4h, p = 8.4 × 10-5, t test) and HIF-1α (max., 6.6-fold at t = 4h, p = 0.00025, t test). Fifteen genes were artifactually identified as "significantly regulated" when Ct values were normalized against GAPDH expression. The biostatistical approach and the geNorm analysis identified overlapping sets of candidate reference genes. Both ranked PPIA as the best candidate, followed by defender against cell death 1 (DAD1) and high-mobility group B1 (HMGB1). Conclusions GAPDH mRNA expression is up-regulated in urate crystal inflammation, possibly due to inflammation-associated hypoxia. Using GAPDH mRNA for molecular normalization resulted in significant artifacts in the calculated expression of the target mRNAs. PPIA and other stably expressed genes promise to be more appropriate reference genes in this model.
BMC Research Notes. 2011 Oct 25;4(1):443
http://dx.doi.org/10.1186/1756-0500-4-443
http://hdl.handle.net/10033/620712
Evaluation of glyceraldehyde-3-phosphate, prolylpeptidyl isomerase A, and a set of stably expressed genes as reference mRNAs in urate crystal inflammation
oai:repository.helmholtz-hzi.de:10033/6206042019-08-30T11:31:23Zcom_10033_311308col_10033_620561
00925njm 22002777a 4500
dc
Moes, Lorin
author
Wirth, Manfred
author
2007-11-22
Bovine viral diarrhea virus (BVDV) is the prototype representative of the pestivirus genus in the Flaviviridae family. It has been shown that the initiation of translation of BVDV RNA occurs by an internal ribosome entry mechanism mediated by the 5' untranslated region of the viral RNA 1. The 5' and 3' boundaries of the IRES of the cytopathic BVDV NADL have been mapped and it has been suggested that the IRES extends into the coding of the BVDV polyprotein 2. A putative pseudoknot structure has been recognized in the BVDV 5'UTR in close proximity to the AUG start codon. A pseudoknot structure is characteristic for flavivirus IRESes and in the case of the closely related classical swine fever virus (CSFV) and the more distantly related Hepatitis C virus (HCV) pseudoknot function in translation has been demonstrated.
To characterize the BVDV IRESes in detail, we studied the BVDV translational initiation by transfection of dicistronic expression plasmids into mammalian cells. A region coding for the amino terminus of the BVDV SD-1 polyprotein contributes considerably to efficient initiation of translation. The translation efficiency mediated by the IRES of BVDV strains NADL and SD-1 approximates the poliovirus type I IRES directed translation in BHK cells. Compared to the poliovirus IRES increased expression levels are mediated by the BVDV IRES of strain SD-1 in murine cell lines, while lower levels are observed in human cell lines. Site directed mutagenesis revealed that a RNA pseudoknot upstream of the initiator AUG is an important structural element for IRES function. Mutants with impaired ability to base pair in stem I or II lost their translational activity. In mutants with repaired base pairing either in stem 1 or in stem 2 full translational activity was restored. Thus, the BVDV IRES translation is dependent on the pseudoknot integrity. These features of the pestivirus IRES are reminiscent of those of the classical swine fever virus, a pestivirus, and the hepatitis C viruses, another genus of the Flaviviridae.
The IRES of the non-cytopathic BVDV SD-1 strain displays features known from other pestivirus IRESes. The predicted pseudoknot in the 5'UTR of BVDV SD-1 virus represents an important structural element in BVDV translation.
The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon. 2007, 4:124 Virol. J.
1743-422X
18034871
10.1186/1743-422X-4-124
http://hdl.handle.net/10033/620604
Virology journal
The internal initiation of translation in bovine viral diarrhea virus RNA depends on the presence of an RNA pseudoknot upstream of the initiation codon.
oai:repository.helmholtz-hzi.de:10033/6207052019-08-30T11:25:43Zcom_10033_620626com_10033_620601com_10033_311308col_10033_620721col_10033_620627col_10033_620603
00925njm 22002777a 4500
dc
Preusse, Matthias
author
Tantawy, Mohamed A
author
Klawonn, Frank
author
Schughart, Klaus
author
Pessler, Frank
author
2013-12-17
Abstract Background Investigating the host response in the early stage of influenza A virus (IAV) infection is of considerable interest. However, it is conceivable that effects due to the anesthesia and/or intranasal infection procedure might introduce artifacts. We therefore aimed to evaluate the effects of anesthesia and/or intranasal infection on transcription of selected pulmonary mRNAs in two inbred mouse strains with differential susceptibility to IAV infection. Results DBA/2J and C57BL/6J mice were evaluated in a time course experiment in which lung tissue was sampled after 6, 12, 18, 24, 48 and 120 h. After anesthesia with ketamine and xylazine, a suspension of mouse-adapted IAV strain PR8_Mun in 20 μl sterile buffer, or 20 μl sterile buffer only, was instilled intranasally. The mice receiving anesthesia and PBS only were designated the “mock treatment” group. Pulmonary expression of 10 host mRNAs (Fos, Retnla, Irg1, Il6, Il1b, Cxcl10, Stat1, Ifng, Ifnl2, and Mx1) and viral hemagglutinin (HA) mRNA were determined at the designated time points. As expected, weight loss and viral replication were greater in the DBA/2J strain (which is more susceptible to IAV infection). Four mRNAs (Retnla, Irg1, Il6, and Cxcl10) were procedure-dependently regulated in DBA/2J mice between 6 and 24 h, and two (Retnla and Il6) in C57BL/6J mice, although to a lesser extent. All 10 mRNAs rose after infection, but one (Fos) only in DBA/2J mice. These infection-dependent effects could be separated from procedure-dependent effects beginning around 12 h in DBA/2J and 18 h in C57BL/6J mice. The interferon-related mRNAs Stat1, Ifng, Infl2, and Mx1 were unaffected by mock treatment in either mouse strain. Mx1 and Infl2 correlated best with HA mRNA expression (r = 0.97 and 0.93, respectively, in DBA/2J). Conclusions These results demonstrate effects of the anesthesia and/or intranasal infection procedure on pulmonary gene expression, which are detectable between approximately 6 and 24 h post procedure and vary in intensity and temporal evolution depending on the mouse strain used. Mock infection controls should be included in all studies on pulmonary gene expression in the early phase of infection with IAV and, likely, other respiratory pathogens.
BMC Microbiology. 2013 Dec 17;13(1):293
http://dx.doi.org/10.1186/1471-2180-13-293
http://hdl.handle.net/10033/620705
Infection- and procedure-dependent effects on pulmonary gene expression in the early phase of influenza A virus infection in mice
oai:repository.helmholtz-hzi.de:10033/6207042019-08-30T11:37:44Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Lu, Xin
author
Sun, Jibin
author
Nimtz, Manfred
author
Wissing, Josef
author
Zeng, An-Ping
author
Rinas, Ursula
author
2010-04-20
Abstract Background The filamentous fungus Aspergillus niger is well-known as a producer of primary metabolites and extracellular proteins. For example, glucoamylase is the most efficiently secreted protein of Aspergillus niger, thus the homologous glucoamylase (glaA) promoter as well as the glaA signal sequence are widely used for heterologous protein production. Xylose is known to strongly repress glaA expression while maltose is a potent inducer of glaA promoter controlled genes. For a more profound understanding of A. niger physiology, a comprehensive analysis of the intra- and extracellular proteome of Aspergillus niger AB1.13 growing on defined medium with xylose or maltose as carbon substrate was carried out using 2-D gel electrophoresis/Maldi-ToF and nano-HPLC MS/MS. Results The intracellular proteome of A. niger growing either on xylose or maltose in well-aerated controlled bioreactor cultures revealed striking similarities. In both cultures the most abundant intracellular protein was the TCA cycle enzyme malate-dehydrogenase. Moreover, the glycolytic enzymes fructose-bis-phosphate aldolase and glyceraldehyde-3-phosphate-dehydrogenase and the flavohemoglobin FhbA were identified as major proteins in both cultures. On the other hand, enzymes involved in the removal of reactive oxygen species, such as superoxide dismutase and peroxiredoxin, were present at elevated levels in the culture growing on maltose but only in minor amounts in the xylose culture. The composition of the extracellular proteome differed considerably depending on the carbon substrate. In the secretome of the xylose-grown culture, a variety of plant cell wall degrading enzymes were identified, mostly under the control of the xylanolytic transcriptional activator XlnR, with xylanase B and ferulic acid esterase as the most abundant ones. The secretome of the maltose-grown culture did not contain xylanolytic enzymes, instead high levels of catalases were found and glucoamylase (multiple spots) was identified as the most abundant extracellular protein. Surprisingly, the intracellular proteome of A. niger growing on xylose in bioreactor cultures differed more from a culture growing in shake flasks using the same medium than from the bioreactor culture growing on maltose. For example, in shake flask cultures with xylose as carbon source the most abundant intracellular proteins were not the glycolytic and the TCA cycle enzymes and the flavohemoglobin, but CipC, a protein of yet unknown function, superoxide dismutase and an NADPH dependent aldehyde reductase. Moreover, vacuolar proteases accumulated to higher and ER-resident chaperones and foldases to lower levels in shake flask compared to the bioreactor cultures. Conclusions The utilization of xylose or maltose was strongly affecting the composition of the secretome but of minor influence on the composition of the intracellular proteome. On the other hand, differences in culture conditions (pH control versus no pH control, aeration versus no aeration and stirring versus shaking) have a profound effect on the intracellular proteome. For example, lower levels of ER-resident chaperones and foldases and higher levels of vacuolar proteases render shake flask conditions less favorable for protein production compared to controlled bioreactor cultures.
Microbial Cell Factories. 2010 Apr 20;9(1):23
http://dx.doi.org/10.1186/1475-2859-9-23
http://hdl.handle.net/10033/620704
The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate
oai:repository.helmholtz-hzi.de:10033/6206972019-08-30T11:36:32Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Hain, Torsten
author
Ghai, Rohit
author
Billion, André
author
Kuenne, Carsten T
author
Steinweg, Christiane
author
Izar, Benjamin
author
Mohamed, Walid
author
Mraheil, Mobarak A
author
Domann, Eugen
author
Schaffrath, Silke
author
Kärst, Uwe
author
Goesmann, Alexander
author
Oehm, Sebastian
author
Pühler, Alfred
author
Merkl, Rainer
author
Vorwerk, Sonja
author
Glaser, Philippe
author
Garrido, Patricia
author
Rusniok, Christophe
author
Buchrieser, Carmen
author
Goebel, Werner
author
Chakraborty, Trinad
author
2012-04-24
Abstract Background Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. Results The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. Conclusion Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.
BMC Genomics. 2012 Apr 24;13(1):144
http://dx.doi.org/10.1186/1471-2164-13-144
http://hdl.handle.net/10033/620697
Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes
oai:repository.helmholtz-hzi.de:10033/6206892018-06-12T22:52:32Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Gurramkonda, Chandrasekhar
author
Polez, Sulena
author
Skoko, Natasa
author
Adnan, Ahmad
author
Gäbel, Thomas
author
Chugh, Dipti
author
Swaminathan, Sathyamangalam
author
Khanna, Navin
author
Tisminetzky, Sergio
author
Rinas, Ursula
author
2010-05-12
Abstract Background The prevalence of diabetes is predicted to rise significantly in the coming decades. A recent analysis projects that by the year 2030 there will be ~366 million diabetics around the world, leading to an increased demand for inexpensive insulin to make this life-saving drug also affordable for resource poor countries. Results A synthetic insulin precursor (IP)-encoding gene, codon-optimized for expression in P. pastoris, was cloned in frame with the Saccharomyces cerevisiae α-factor secretory signal and integrated into the genome of P. pastoris strain X-33. The strain was grown to high-cell density in a batch procedure using a defined medium with low salt and high glycerol concentrations. Following batch growth, production of IP was carried out at methanol concentrations of 2 g L-1, which were kept constant throughout the remaining production phase. This robust feeding strategy led to the secretion of ~3 gram IP per liter of culture broth (corresponding to almost 4 gram IP per liter of cell-free culture supernatant). Using immobilized metal ion affinity chromatography (IMAC) as a novel approach for IP purification, 95% of the secreted product was recovered with a purity of 96% from the clarified culture supernatant. Finally, the purified IP was trypsin digested, transpeptidated, deprotected and further purified leading to ~1.5 g of 99% pure recombinant human insulin per liter of culture broth. Conclusions A simple two-phase cultivation process composed of a glycerol batch and a constant methanol fed-batch phase recently developed for the intracellular production of the Hepatitis B surface antigen was adapted to secretory IP production. Compared to the highest previously reported value, this approach resulted in an ~2 fold enhancement of IP production using Pichia based expression systems, thus significantly increasing the efficiency of insulin manufacture.
Microbial Cell Factories. 2010 May 12;9(1):31
http://dx.doi.org/10.1186/1475-2859-9-31
http://hdl.handle.net/10033/620689
Application of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin
oai:repository.helmholtz-hzi.de:10033/5788812019-08-30T11:33:30Zcom_10033_6832com_10033_311308col_10033_620777col_10033_6833
00925njm 22002777a 4500
dc
Sun, Jibin
author
Lu, Xin
author
Rinas, Ursula
author
Zeng, An Ping
author
2007
Aspergillus niger is an important industrial microorganism for the production of both metabolites, such as citric acid, and proteins, such as fungal enzymes or heterologous proteins. Despite its extensive industrial applications, the genetic inventory of this fungus is only partially understood. The recently released genome sequence opens a new horizon for both scientific studies and biotechnological applications.
Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics. 2007, 8 (9):R182 Genome Biol.
1474-760X
17784953
10.1186/gb-2007-8-9-r182
http://hdl.handle.net/10033/578881
Genome biology
Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics.
oai:repository.helmholtz-hzi.de:10033/5797162019-08-30T11:24:31Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Vanz, Ana Letícia
author
Nimtz, Manfred
author
Rinas, Ursula
author
2014
Pichia pastoris is a popular yeast preferably employed for secretory protein production. Secretion is not always efficient and endoplasmic retention of proteins with aberrant folding properties, or when produced at exaggerated rates, can occur. In these cases production usually leads to an unfolded protein response (UPR) and the induction of the endoplasmic reticulum associated degradation (ERAD). P. pastoris is nowadays also an established host for secretory insulin precursor (IP) production, though little is known about the impact of IP production on the host cell physiology, in particular under industrially relevant production conditions. Here, we evaluate the cellular response to aox1 promoter-controlled, secretory IP production in controlled fed-batch processes using a proteome profiling approach.
Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures. 2014, 13 (1):23 Microb. Cell Fact.
1475-2859
24521445
10.1186/1475-2859-13-23
http://hdl.handle.net/10033/579716
Microbial cell factories
Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures.
oai:repository.helmholtz-hzi.de:10033/5800702019-08-30T11:36:33Zcom_10033_620626com_10033_620601com_10033_311308col_10033_620721col_10033_620627col_10033_620603
00925njm 22002777a 4500
dc
Preusse, Matthias
author
Schughart, Klaus
author
Wilk, Esther
author
Klawonn, Frank
author
Pessler, Frank
author
2015
Hematological parameters have not received much attention in small animal models of infection, particularly at very early time points. We therefore studied changes in leukocyte and thrombocyte numbers in a mouse model of influenza A virus (IAV) infection, including measurements within the first 24 h after infection, and also assessing effects, if any, of the infection/anesthesia procedure on these parameters.
Hematological parameters in the early phase of influenza A virus infection in differentially susceptible inbred mouse strains. 2015, 8:225 BMC Res Notes
1756-0500
26047817
10.1186/s13104-015-1195-8
http://hdl.handle.net/10033/580070
BMC research notes
Hematological parameters in the early phase of influenza A virus infection in differentially susceptible inbred mouse strains.
oai:repository.helmholtz-hzi.de:10033/5801142019-08-30T11:33:30Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Bauer, Isabel
author
Günther, Juliane
author
Wheeler, Thomas T
author
Engelmann, Susanne
author
Seyfert, Hans-Martin
author
2015
Considerably divergent data have been published from attempts to model the E. coli vs. S. aureus specific immune reaction of the udder using primary cultures of bovine mammary epithelial cells from cows (pbMEC). Some groups reported a swift, strong and transient inflammatory response against challenges with E. coli and only a weak and retarded response against S. aureus, in agreement with the respective reaction of the udder. Others found almost the reverse. Presence or absence of fetal calf serum distinguished the experimental setting between both groups. We examined here if this causes the divergent reaction of the pbMEC towards both pathogen species. We challenged pbMEC with proteins from heat killed E. coli or S. aureus pathogens or purified TLR2 and TLR4 ligands. The stimuli were applied in normal growth medium with (SM10) or without (SM0) 10% fetal calf serum, or in the basal medium supplemented with 10 mg/ml milk proteins (SM Milk).
Extracellular milieu grossly alters pathogen-specific immune response of mammary epithelial cells. 2015, 11:172 BMC Vet. Res.
1746-6148
26219462
10.1186/s12917-015-0489-3
http://hdl.handle.net/10033/580114
BMC veterinary research
Extracellular milieu grossly alters pathogen-specific immune response of mammary epithelial cells.
oai:repository.helmholtz-hzi.de:10033/5839162019-08-30T11:34:17Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Klawonn, Frank
author
Kruse, Rudolf
author
Winkler, Roland
author
2015-12
Fuzzy clustering: More than just fuzzification 2015, 281:272 Fuzzy Sets and Systems
1650114
10.1016/j.fss.2015.06.024
http://hdl.handle.net/10033/583916
Fuzzy Sets and Systems
Fuzzy clustering: More than just fuzzification
oai:repository.helmholtz-hzi.de:10033/5944172019-08-30T11:37:00Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Kirschning, Carsten J
author
Dreher, Stefan
author
Maass, Björn
author
Fichte, Sylvia
author
Schade, Jutta
author
Köster, Mario
author
Noack, Andreas
author
Lindenmaier, Werner
author
Wagner, Hermann
author
Böldicke, Thomas
author
2010
Toll-like receptor (TLR) 2 is a component of the innate immune system and senses specific pathogen associated molecular patterns (PAMPs) of both microbial and viral origin. Cell activation via TLR2 and other pattern recognition receptors (PRRs) contributes to sepsis pathology and chronic inflammation both relying on overamplification of an immune response. Intracellular antibodies expressed and retained inside the endoplasmatic reticulum (ER-intrabodies) are applied to block translocation of secreted and cell surface molecules from the ER to the cell surface resulting in functional inhibition of the target protein. Here we describe generation and application of a functional anti-TLR2 ER intrabody (alphaT2ib) which was generated from an antagonistic monoclonal antibody (mAb) towards human and murine TLR2 (T2.5) to inhibit the function of TLR2. alphaT2ib is a scFv fragment comprising the variable domain of the heavy chain and the variable domain of the light chain of mAb T2.5 linked together by a synthetic (Gly4Ser)3 amino acid sequence.
Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation. 2010, 10:31 BMC Biotechnol.
1472-6750
20388199
10.1186/1472-6750-10-31
http://hdl.handle.net/10033/594417
BMC biotechnology
Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation.
oai:repository.helmholtz-hzi.de:10033/6009822019-08-30T11:37:44Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Bleckmann, Maren
author
Schürig, Margitta
author
Chen, Fang-Fang
author
Yen, Zen-Zen
author
Lindemann, Nils
author
Meyer, Steffen
author
Spehr, Johannes
author
van den Heuvel, Joop
author
2016
The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.
Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells. 2016, 11 (3):e0149424 PLoS ONE
1932-6203
26934632
10.1371/journal.pone.0149424
http://hdl.handle.net/10033/600982
PloS one
Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.
oai:repository.helmholtz-hzi.de:10033/6039232019-08-30T11:31:23Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Bleckmann, Maren
author
Fritz, Markus H-Y
author
Bhuju, Sabin
author
Jarek, Michael
author
Schürig, Margitta
author
Geffers, Robert
author
Benes, Vladimir
author
Besir, Hüseyin
author
van den Heuvel, Joop
author
2015
The Baculoviral Expression Vector System (BEVS) is the most commonly used method for high expression of recombinant protein in insect cells. Nevertheless, expression of some target proteins--especially those entering the secretory pathway--provides a severe challenge for the baculovirus infected insect cells, due to the reorganisation of intracellular compounds upon viral infection. Therefore, alternative strategies for recombinant protein production in insect cells like transient plasmid-based expression or stable expression cell lines are becoming more popular. However, the major bottleneck of these systems is the lack of strong endogenous polymerase II dependent promoters, as the strong baculoviral p10 and polH promoters used in BEVS are only functional in presence of the viral transcription machinery during the late phase of infection. In this work we present a draft genome and a transcriptome analysis of Sf21 cells for the identification of the first known endogenous Spodoptera frugiperda promoters. Therefore, putative promoter sequences were identified and selected because of high mRNA level or in analogy to other strong promoters in other eukaryotic organism. The chosen endogenous Sf21 promoters were compared to early viral promoters for their efficiency to trigger eGFP expression using transient plasmid based transfection in a BioLector Microfermentation system. Furthermore, promoter activity was not only shown in Sf21 cells but also in Hi5 cells. The novel endogenous Sf21 promoters were ranked according to their activity and expand the small pool of available promoters for stable insect cell line development and transient plasmid expression in insect cells. The best promoter was used to improve plasmid based transient transfection in insect cells substantially.
Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda. 2015, 10 (8):e0132898 PLoS ONE
1932-6203
26263512
10.1371/journal.pone.0132898
http://hdl.handle.net/10033/603923
PloS one
Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.
oai:repository.helmholtz-hzi.de:10033/6039962019-08-30T11:35:14Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Bleckmann, Maren
author
Schmelz, Stefan
author
Schinkowski, Christian
author
Scrima, Andrea
author
van den Heuvel, Joop
author
2016-02-23
Recombinant protein expression often presents a bottleneck for the production of proteins for use in many areas of animal-cell biotechnology. Difficult-to-express proteins require the generation of numerous expression constructs, where popular prokaryotic screening systems often fail to identify expression of multi domain or full-length protein constructs. Post-translational modified mammalian proteins require an alternative host system such as insect cells using the Baculovirus Expression Vector System (BEVS). Unfortunately this is time-, labor- and cost-intensive. It is clearly desirable to find an automated and miniaturized fast multi-sample screening method for protein expression in such systems. With this in mind, in this paper a high-throughput initial expression screening method is described using an automated Microcultivation system in conjunction with fast plasmid based transient transfection in insect cells for the efficient generation of protein constructs. The applicability of the system is demonstrated for the difficult to express Nucleotide-binding Oligomerization Domain-containing protein 2 (NOD2). To enable detection of proper protein expression the rather weak plasmid based expression has been improved by a sensitive inline detection system. Here we present the functionality and application of the sensitive SplitGFP (split green fluorescent protein) detection system in insect cells. The successful expression of constructs is monitored by direct measurement of the fluorescence in the BioLector Microcultivation system. Additionally, we show that the results obtained with our plasmid based SplitGFP protein expression screen correlate directly to the level of soluble protein produced in BEVS. In conclusion our automated SplitGFP screen outlines a sensitive, fast and reliable method reducing the time and costs required for identifying the optimal expression construct prior to large scale protein production in baculovirus infected insect cells. This article is protected by copyright. All rights reserved.
Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen. 2016: Biotechnol. Bioeng.
1097-0290
26913471
10.1002/bit.25956
http://hdl.handle.net/10033/603996
Biotechnology and bioengineering
Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.
oai:repository.helmholtz-hzi.de:10033/6047282019-08-30T11:27:46Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Thumfart, Jörg Oliver
author
Abidi, Nada
author
Mindt, Sonani
author
Costani, Victor
author
Hofheinz, Ralf
author
Klawonn, Frank
author
Findeisen, Peter
author
2015-05-04
Preanalytical variations have major impact on most biological assays. Specifically MS-based multiparametric proteomics analyses of blood specimens are seriously affected by limited protein stability due to high intrinsic proteolytic activity of serum and plasma. However, the direct analysis of sample quality (DASQ) for serum specimens is not readily available. Here we propose the mass spectrometry based monitoring of peptide patterns that are ex vivo changing in a time dependent manner to alleviate these constrains.
LC/MS Based Monitoring of Endogenous Decay Markers for Quality Assessment of Serum Specimens 2015, 08 (05) Journal of Proteomics & Bioinformatics
0974276X
10.4172/jpb.1000356
http://hdl.handle.net/10033/604728
Journal of Proteomics & Bioinformatics
LC/MS Based Monitoring of Endogenous Decay Markers for Quality Assessment of Serum Specimens
oai:repository.helmholtz-hzi.de:10033/6092722019-08-30T11:33:30Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Selle, Martina
author
Hertlein, Tobias
author
Oesterreich, Babett
author
Klemm, Theresa
author
Kloppot, Peggy
author
Müller, Elke
author
Ehricht, Ralf
author
Stentzel, Sebastian
author
Bröker, Barbara M
author
Engelmann, Susanne
author
Ohlsen, Knut
author
2016
The pathogen Staphylococcus aureus causes a broad range of severe diseases and is feared for its ability to rapidly develop resistance to antibiotic substances. The increasing number of highly resistant S. aureus infections has accelerated the search for alternative treatment options to close the widening gap in anti-S. aureus therapy. This study analyses the humoral immune response to vaccination of Balb/c mice with sublethal doses of live S. aureus. The elicited antibody pattern in the sera of intravenously and intramuscularly vaccinated mice was determined using of a recently developed protein array. We observed a specific antibody response against a broad set of S. aureus antigens which was stronger following i.v. than i.m. vaccination. Intravenous but not intramuscular vaccination protected mice against an intramuscular challenge infection with a high bacterial dose. Vaccine protection was correlated with the strength of the anti-S. aureus antibody response. This study identified novel vaccine candidates by using protein microarrays as an effective tool and showed that successful vaccination against S. aureus relies on the optimal route of administration.
Global antibody response to Staphylococcus aureus live-cell vaccination. 2016, 6:24754 Sci Rep
2045-2322
27103319
10.1038/srep24754
http://hdl.handle.net/10033/609272
Scientific reports
Global antibody response to Staphylococcus aureus live-cell vaccination.
oai:repository.helmholtz-hzi.de:10033/6096032019-08-30T11:29:47Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Grigull, Lorenz
author
Lechner, Werner
author
Petri, Susanne
author
Kollewe, Katja
author
Dengler, Reinhard
author
Mehmecke, Sandra
author
Schumacher, Ulrike
author
Lücke, Thomas
author
Schneider-Gold, Christiane
author
Köhler, Cornelia
author
Güttsches, Anne-Katrin
author
Kortum, Xiaowei
author
Klawonn, Frank
author
2016
Diagnosis of neuromuscular diseases in primary care is often challenging. Rare diseases such as Pompe disease are easily overlooked by the general practitioner. We therefore aimed to develop a diagnostic support tool using patient-oriented questions and combined data mining algorithms recognizing answer patterns in individuals with selected neuromuscular diseases. A multicenter prospective study for the proof of concept was conducted thereafter.
Diagnostic support for selected neuromuscular diseases using answer-pattern recognition and data mining techniques: a proof of concept multicenter prospective trial. 2016, 16:31 BMC Med Inform Decis Mak
1472-6947
26957320
10.1186/s12911-016-0268-5
http://hdl.handle.net/10033/609603
BMC medical informatics and decision making
Diagnostic support for selected neuromuscular diseases using answer-pattern recognition and data mining techniques: a proof of concept multicenter prospective trial.
oai:repository.helmholtz-hzi.de:10033/6121922019-08-30T11:34:21Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Gangula, Sheetal
author
Nimtz, Manfred
author
Mischnick, Petra
author
2016-05
Relative ion intensities of maltooligosaccharide ethers in electrospray ionization ion trap mass spectrometry: A quantitative evaluation 2016, 402:57 International Journal of Mass Spectrometry
13873806
10.1016/j.ijms.2016.01.009
http://hdl.handle.net/10033/612192
International Journal of Mass Spectrometry
Relative ion intensities of maltooligosaccharide ethers in electrospray ionization ion trap mass spectrometry: A quantitative evaluation
oai:repository.helmholtz-hzi.de:10033/6123012019-08-30T11:34:22Zcom_10033_311308col_10033_621053
00925njm 22002777a 4500
dc
Marchanka, Alexander
author
Simon, Bernd
author
Althoff-Ospelt, Gerhard
author
Carlomagno, Teresa
author
2015
Knowledge of the RNA three-dimensional structure, either in isolation or as part of RNP complexes, is fundamental to understand the mechanism of numerous cellular processes. Because of its flexibility, RNA represents a challenge for crystallization, while the large size of cellular complexes brings solution-state NMR to its limits. Here, we demonstrate an alternative approach on the basis of solid-state NMR spectroscopy. We develop a suite of experiments and RNA labeling schemes and demonstrate for the first time that ssNMR can yield a RNA structure at high-resolution. This methodology allows structural analysis of segmentally labelled RNA stretches in high-molecular weight cellular machines—independent of their ability to crystallize—and opens the way to mechanistic studies of currently difficult-to-access RNA-protein assemblies.
RNA structure determination by solid-state NMR spectroscopy. 2015, 6:7024 Nat Commun
2041-1723
25960310
10.1038/ncomms8024
http://hdl.handle.net/10033/612301
Nature communications
RNA structure determination by solid-state NMR spectroscopy.
oai:repository.helmholtz-hzi.de:10033/6138592019-08-30T11:33:05Zcom_10033_311308col_10033_621053
00925njm 22002777a 4500
dc
Bowman, Andrew
author
Lercher, Lukas
author
Singh, Hari R
author
Zinne, Daria
author
Timinszky, Gyula
author
Carlomagno, Teresa
author
Ladurner, Andreas G
author
2016-04-20
Eukaryotic chromatin is a complex yet dynamic structure, which is regulated in part by the assembly and disassembly of nucleosomes. Key to this process is a group of proteins termed histone chaperones that guide the thermodynamic assembly of nucleosomes by interacting with soluble histones. Here we investigate the interaction between the histone chaperone sNASP and its histone H3 substrate. We find that sNASP binds with nanomolar affinity to a conserved heptapeptide motif in the globular domain of H3, close to the C-terminus. Through functional analysis of sNASP homologues we identified point mutations in surface residues within the TPR domain of sNASP that disrupt H3 peptide interaction, but do not completely disrupt binding to full length H3 in cells, suggesting that sNASP interacts with H3 through additional contacts. Furthermore, chemical shift perturbations from(1)H-(15)N HSQC experiments show that H3 peptide binding maps to the helical groove formed by the stacked TPR motifs of sNASP. Our findings reveal a new mode of interaction between a TPR repeat domain and an evolutionarily conserved peptide motif found in canonical H3 and in all histone H3 variants, including CenpA and have implications for the mechanism of histone chaperoning within the cell.
The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats. 2016, 44 (7):3105-17 Nucleic Acids Res.
1362-4962
26673727
10.1093/nar/gkv1372
http://hdl.handle.net/10033/613859
Nucleic acids research
The histone chaperone sNASP binds a conserved peptide motif within the globular core of histone H3 through its TPR repeats.
oai:repository.helmholtz-hzi.de:10033/6147992019-08-30T11:28:23Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Seeger, Bettina
author
Klawonn, Frank
author
Nguema Bekale, Boris
author
Steinberg, Pablo
author
2016
Consumers of fruits and vegetables are frequently exposed to small amounts of hormonally active pesticides, some of them sharing a common mode of action such as the activation of the human estrogen receptor α (hERα) or β (hERβ). Therefore, it is of particular importance to evaluate risks emanating from chemical mixtures, in which the individual pesticides are present at human-relevant concentrations, below their corresponding maximum residue levels. Binary and ternary iso-effective mixtures of estrogenic pesticides at effect concentrations eliciting a 1 or 10% effect in the presence or absence of 17β-estradiol were tested experimentally at the hERα in the yeast-based estrogen screen (YES) assay as well as in the human U2-OS cell-based ERα chemical-activated luciferase gene expression (ERα CALUX) assay and at the hERβ in the ERβ CALUX assay. The outcome was then compared to predictions calculated by means of concentration addition. In most cases, additive effects were observed with the tested combinations in all three test systems, an observation that supports the need to expand the risk assessment of pesticides and consider cumulative risk assessment. An additional testing of mixture effects at the hERβ showed that most test substances being active at the hERα could also elicit additive effects at the hERβ, but the hERβ was less sensitive. In conclusion, effects of the same ligands at the hERα and the hERβ could influence the estrogenic outcome under physiological conditions.
Mixture Effects of Estrogenic Pesticides at the Human Estrogen Receptor α and β. 2016, 11 (1):e0147490 PLoS ONE
1932-6203
26812056
10.1371/journal.pone.0147490
http://hdl.handle.net/10033/614799
PloS one
Mixture Effects of Estrogenic Pesticides at the Human Estrogen Receptor α and β.
oai:repository.helmholtz-hzi.de:10033/6159832019-08-30T11:31:23Zcom_10033_311308col_10033_620721
00925njm 22002777a 4500
dc
Rother, Ann-Katrin
author
Schwerk, Nicolaus
author
Brinkmann, Folke
author
Klawonn, Frank
author
Lechner, Werner
author
Grigull, Lorenz
author
2015
Clinical symptoms in children with pulmonary diseases are frequently non-specific. Rare diseases such as primary ciliary dyskinesia (PCD), cystic fibrosis (CF) or protracted bacterial bronchitis (PBB) can be easily missed at the general practitioner (GP).
Diagnostic Support for Selected Paediatric Pulmonary Diseases Using Answer-Pattern Recognition in Questionnaires Based on Combined Data Mining Applications--A Monocentric Observational Pilot Study. 2015, 10 (8):e0135180 PLoS ONE
1932-6203
26267801
10.1371/journal.pone.0135180
http://hdl.handle.net/10033/615983
PloS one
Diagnostic Support for Selected Paediatric Pulmonary Diseases Using Answer-Pattern Recognition in Questionnaires Based on Combined Data Mining Applications--A Monocentric Observational Pilot Study.
oai:repository.helmholtz-hzi.de:10033/6159992019-08-30T11:31:23Zcom_10033_311308col_10033_620777
00925njm 22002777a 4500
dc
Jäger, Volker
author
Büssow, Konrad
author
Wagner, Andreas
author
Weber, Susanne
author
Hust, Michael
author
Frenzel, André
author
Schirrmann, Thomas
author
2013
The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility.
High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells. 2013, 13:52 BMC Biotechnol.
1472-6750
23802841
10.1186/1472-6750-13-52
http://hdl.handle.net/10033/615999
BMC biotechnology
High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.
oai:repository.helmholtz-hzi.de:10033/6172662019-08-30T11:28:24Zcom_10033_311308col_10033_620560
00925njm 22002777a 4500
dc
Zühlke, Daniela
author
Dörries, Kirsten
author
Bernhardt, Jörg
author
Maaß, Sandra
author
Muntel, Jan
author
Liebscher, Volkmar
author
Pané-Farré, Jan
author
Riedel, Katharina
author
Lalk, Michael
author
Völker, Uwe
author
Engelmann, Susanne
author
Becher, Dörte
author
Fuchs, Stephan
author
Hecker, Michael
author
2016
Absolute protein quantification was applied to follow the dynamics of the cytoplasmic proteome of Staphylococcus aureus in response to long-term oxygen starvation. For 1,168 proteins, the majority of all expressed proteins, molecule numbers per cell have been determined to monitor the cellular investments in single branches of bacterial life for the first time. In the presence of glucose the anaerobic protein pattern is characterized by increased amounts of glycolytic and fermentative enzymes such as Eno, GapA1, Ldh1, and PflB. Interestingly, the ferritin-like protein FtnA belongs to the most abundant proteins during anaerobic growth. Depletion of glucose finally leads to an accumulation of different enzymes such as ArcB1, ArcB2, and ArcC2 involved in arginine deiminase pathway. Concentrations of 29 exo- and 78 endometabolites were comparatively assessed and have been integrated to the metabolic networks. Here we provide an almost complete picture on the response to oxygen starvation, from signal transduction pathways to gene expression pattern, from metabolic reorganization after oxygen depletion to beginning cell death and lysis after glucose exhaustion. This experimental approach can be considered as a proof of principle how to combine cell physiology with quantitative proteomics for a new dimension in understanding simple life processes as an entity.
Costs of life - Dynamics of the protein inventory of Staphylococcus aureus during anaerobiosis. 2016, 6:28172 Sci Rep
2045-2322
27344979
10.1038/srep28172
http://hdl.handle.net/10033/617266
Scientific reports
Costs of life - Dynamics of the protein inventory of Staphylococcus aureus during anaerobiosis.
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