2024-03-29T11:59:55Zhttp://repository.helmholtz-hzi.de/oai/requestoai:repository.helmholtz-hzi.de:10033/1351052019-08-30T11:31:49Zcom_10033_311308col_10033_620777
Expression and purification of bioactive soluble murine stem cell factor from recombinant Escherichia coli using thioredoxin as fusion partner.
Bals, Carola
Schambach, Axel
Meyer, Johann
Scheper, Thomas
Rinas, Ursula
Excellence Cluster Rebirth, Institute of Technical Chemistry-Life Science, Leibniz University of Hannover, Callinstr.5, 30167 Hannover, Germany.
Animals
Cell Line
Cell Proliferation
Electrophoresis, Polyacrylamide Gel
Escherichia coli
Genetic Vectors
Humans
Mice
Polymerase Chain Reaction
Stem Cell Factor
Thioredoxins
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.
2011-07-01T13:45:04Z
2011-07-01T13:45:04Z
2011-03-10
Article
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
en
oai:repository.helmholtz-hzi.de:10033/1355092019-08-30T11:32:17Zcom_10033_311308col_10033_620777
Preparation of bioactive soluble human leukemia inhibitory factor from recombinant Escherichia coli using thioredoxin as fusion partner.
Tomala, Magda
Lavrentieva, Antonina
Moretti, Pierre
Rinas, Ursula
Kasper, Cornelia
Stahl, Frank
Schambach, Axel
Warlich, Eva
Martin, Ulrich
Cantz, Tobias
Scheper, Thomas
Institute of Technical Chemistry, Leibniz University of Hannover, Callinstr. 5, 30167 Hannover, Germany.
Animals
Antigens, CD15
Base Sequence
Cell Line
Cell Proliferation
Cloning, Molecular
Electrophoresis, Polyacrylamide Gel
Embryonic Stem Cells
Endopeptidases
Escherichia coli
Flow Cytometry
Humans
Leukemia Inhibitory Factor
Mice
Molecular Sequence Data
Recombinant Fusion Proteins
Solubility
Thioredoxins
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.
2011-07-07T08:11:30Z
2011-07-07T08:11:30Z
2010-09
Article
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
en
oai:repository.helmholtz-hzi.de:10033/1399612019-08-30T11:37:24Zcom_10033_311308col_10033_620777
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.
Gurramkonda, Chandrasekhar
Polez, Sulena
Skoko, Natasa
Adnan, Ahmad
Gäbel, Thomas
Chugh, Dipti
Swaminathan, Sathyamangalam
Khanna, Navin
Tisminetzky, Sergio
Rinas, Ursula
Helmholtz Centre for Infection Research, Braunschweig, Germany.
Cloning, Molecular
Culture Media
Glycerol
Humans
Insulin
Methanol
Pichia
Technology, Pharmaceutical
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.
2011-08-17T13:47:12Z
2011-08-17T13:47:12Z
2010
Article
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
en
oai:repository.helmholtz-hzi.de:10033/1399602019-08-30T11:36:59Zcom_10033_311308col_10033_620777
Side effects of chaperone gene co-expression in recombinant protein production.
Martínez-Alonso, Mónica
García-Fruitós, Elena
Ferrer-Miralles, Neus
Rinas, Ursula
Villaverde, Antonio
Institute for Biotechnology and Biomedicine, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Bacterial Proteins
Chaperonin 60
Escherichia coli Proteins
HSP70 Heat-Shock Proteins
Molecular Chaperones
Protein Folding
Protein Stability
Recombinant Proteins
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.
2011-08-17T13:35:58Z
2011-08-17T13:35:58Z
2010
Article
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
en
oai:repository.helmholtz-hzi.de:10033/2146502019-08-30T11:37:00Zcom_10033_311308col_10033_620777
Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems.
Li, Zhaopeng
Kessler, Wolfgang
van den Heuvel, Joop
Rinas, Ursula
Helmholtz Centre for Infection Research (SB), Braunschweig, Germany.
Bacteriological Techniques
Culture Media
Escherichia coli
Gene Expression
Genetic Vectors
Podoviridae
Protein Engineering
Recombinant Proteins
Transcriptional Activation
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.
2012-03-07T15:27:29Z
2012-03-07T15:27:29Z
2011-08
Article
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
en
oai:repository.helmholtz-hzi.de:10033/2168112019-08-30T11:25:43Zcom_10033_311308col_10033_620777
Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy.
Lorenz, Udo
Lorenz, Birgit
Schmitter, Tim
Streker, Karin
Erck, Christian
Wehland, Jürgen
Nickel, Joachim
Zimmermann, Bastian
Ohlsen, Knut
Department of General, Visceral, Vascular and Paediatric Surgery, University Clinic of Würzburg, Wuerzburg, Germany. u.lorenz@mail.uni-wuerzburg.de
Animals
Antibodies, Bacterial
Antigens, Bacterial
Female
Fluorescent Antibody Technique, Indirect
Male
Mice
Sepsis
Staphylococcal Infections
Staphylococcus aureus
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.
2012-03-28T09:37:24Z
2012-03-28T09:37:24Z
2011-01
Article
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
en
Archived with thanks to Antimicrobial agents and chemotherapy
oai:repository.helmholtz-hzi.de:10033/2215122019-08-30T11:37:24Zcom_10033_311308col_10033_620777
Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems.
Li, Zhaopeng
Nimtz, Manfred
Rinas, Ursula
Helmholtz Centre for Infection Research (SB), Braunschweig, Germany.
Carbon Isotopes
Deuterium
Escherichia coli
Gene Expression
Genetic Vectors
Isotope Labeling
Nitrogen Isotopes
Proteins
Selenomethionine
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.
2012-05-02T09:54:29Z
2012-05-02T09:54:29Z
2011-11
Article
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
en
Archived with thanks to Applied microbiology and biotechnology
oai:repository.helmholtz-hzi.de:10033/2355752019-08-30T11:25:43Zcom_10033_311308col_10033_620777
Inclusion bodies of fuculose-1-phosphate aldolase as stable and reusable biocatalysts.
Sans, Cristina
García-Fruitós, Elena
Ferraz, Rosa M
González-Montalbán, Núria
Rinas, Ursula
López-Santín, Josep
Villaverde, Antonio
Álvaro, Gregorio
Dept. d'Enginyeria Química, Escola d'Enginyeria, Unitat de Biocatàlisi Aplicada Associada al IQAC (CSIC), Universitat Autònoma de Barcelona, Edifici Q, 08193 Bellaterra, Spain.
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.
2012-07-24T14:43:28Z
2012-07-24T14:43:28Z
2012-07-24
Article
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
en
Archived with thanks to Biotechnology progress
oai:repository.helmholtz-hzi.de:10033/2835722019-08-30T11:30:58Zcom_10033_311308col_10033_620777
Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-β family.
Vallejo, Luis F
Rinas, Ursula
Helmholtz Centre for Infection Research, Braunschweig, Germany.
Bone Morphogenetic Protein 2
Buffers
Glutathione Disulfide
Guanidine
Humans
Kinetics
Models, Biological
Oxidation-Reduction
Protein Denaturation
Protein Folding
Protein Multimerization
Protein Stability
Sodium Chloride
TGF-beta Superfamily Proteins
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.
2013-04-22T14:40:11Z
2013-04-22T14:40:11Z
2013-01
Article
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
en
Archived with thanks to The FEBS journal
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
Dengue-specific subviral nanoparticles: design, creation and characterization.
Khetarpal, Niyati
Poddar, Ankur
Nemani, Satish K
Dhar, Nisha
Patil, Aravind
Negi, Priyanka
Perween, Ashiya
Viswanathan, Ramaswamy
Lünsdorf, Heinrich
Tyagi, Poornima
Raut, Rajendra
Arora, Upasana
Jain, Swatantra K
Rinas, Ursula
Swaminathan, Sathyamangalam
Khanna, Navin
Recombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110067, India. swami@icgeb.res.in.
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.
2013-07-26T14:14:02Z
2013-07-26T14:14:02Z
2013
Article
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
en
Archived with thanks to Journal of nanobiotechnology
oai:repository.helmholtz-hzi.de:10033/2972222019-08-30T11:35:13Zcom_10033_311308col_10033_620777
Multi-host expression system for recombinant production of challenging proteins.
Meyer, Steffen
Lorenz, Carmen
Baser, Bahar
Wördehoff, Mona
Jäger, Volker
van den Heuvel, Joop
Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany.
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.
2013-08-01T10:49:35Z
2013-08-01T10:49:35Z
2013
Article
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
en
Archived with thanks to PloS one
oai:repository.helmholtz-hzi.de:10033/3051922019-08-30T11:30:58Zcom_10033_311308col_10033_620777
Purification of hepatitis B surface antigen virus-like particles from recombinant Pichia pastoris and in vivo analysis of their immunogenic properties.
Gurramkonda, Chandrasekhar
Zahid, Maria
Nemani, Satish Kumar
Adnan, Ahmad
Gudi, Satheesh Kumar
Khanna, Navin
Ebensen, Thomas
Lünsdorf, Heinrich
Guzmán, Carlos A
Rinas, Ursula
Department of Structural Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; International Centre for Genetic Engineering and Biotechnology, New Delhi, India; Technology Research Centre, Department of Chemical, Biochemical and Environmental Engineering, University of Maryland Baltimore County, Baltimore, USA. Electronic address: chskrg@umbc.edu.
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.
2013-11-11T13:54:47Z
2013-11-11T13:54:47Z
2013-12-01
Article
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
en
Archived with thanks to Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
oai:repository.helmholtz-hzi.de:10033/3260082019-08-30T11:36:33Zcom_10033_311308col_10033_620777
Expression, purification, crystallization and preliminary X-ray diffraction analysis of a mammalian type 10 adenylyl cyclase.
Kleinboelting, Silke
van den Heuvel, Joop
Kambach, Christian
Weyand, Michael
Leipelt, Martina
Steegborn, Clemens
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.
2014-09-09T13:41:53Z
2014-09-09T13:41:53Z
2014-04
Article
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
en
Archived with thanks to Acta crystallographica. Section F, Structural biology communications
oai:repository.helmholtz-hzi.de:10033/3261982019-08-30T11:36:05Zcom_10033_311308col_10033_620777
The metabolic potential of Escherichia coli BL21 in defined and rich medium.
Li, Zhaopeng
Nimtz, Manfred
Rinas, Ursula
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.
2014-09-17T14:49:26Z
2014-09-17T14:49:26Z
2014
Article
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
en
Archived with thanks to Microbial cell factories
oai:repository.helmholtz-hzi.de:10033/5526802019-08-30T11:30:52Zcom_10033_311308col_10033_620777
Stable mammalian producer cell lines for structural biology.
Büssow, Konrad
Helmholtz Centre for Infection Research, Structure and Function of Proteins, Inhoffenstr. 7, 38124 Braunschweig, Germany.
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.
2015-05-12T14:03:28Z
2015-05-12T14:03:28Z
2015-03-21
Article
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
ENG
oai:repository.helmholtz-hzi.de:10033/5611762019-08-30T11:27:46Zcom_10033_311308col_10033_620777
Cryo-EM structure of Hepatitis C virus IRES bound to the human ribosome at 3.9-Å resolution.
Quade, Nick
Boehringer, Daniel
Leibundgut, Marc
van den Heuvel, Joop
Ban, Nenad
Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany.
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.
2015-07-28T10:41:32Z
2015-07-28T10:41:32Z
2015
Article
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
en
info:eu-repo/grantAgreement/EC/FP7/250071
openAccess
oai:repository.helmholtz-hzi.de:10033/5662532019-08-30T11:34:22Zcom_10033_311308col_10033_620777
Assessing stability and assembly of the hepatitis B surface antigen into virus-like particles during down-stream processing.
Zahid, Maria
Lünsdorf, Heinrich
Rinas, Ursula
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
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.
2015-08-13T10:57:33Z
2015-08-13T10:57:33Z
2015-07-17
Article
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
en
oai:repository.helmholtz-hzi.de:10033/6207902019-08-30T11:25:43Zcom_10033_311308col_10033_620777col_10033_620777
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
Gurramkonda, Chandrasekhar
Adnan, Ahmad
Gäbel, Thomas
Lünsdorf, Heinrich
Ross, Anton
Nemani, Satish K
Swaminathan, Sathyamangalam
Khanna, Navin
Rinas, Ursula
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.
2017-01-27T11:53:19Z
2017-01-27T11:53:19Z
2009-02-10
2015-09-04T08:25:50Z
Journal Article
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
en
Gurramkonda et al.
oai:repository.helmholtz-hzi.de:10033/6207762018-06-12T21:27:46Zcom_10033_311308col_10033_620777
Dengue-specific subviral nanoparticles: design, creation and characterization
Khetarpal, Niyati
Poddar, Ankur
Nemani, Satish K
Dhar, Nisha
Patil, Aravind
Negi, Priyanka
Perween, Ashiya
Viswanathan, Ramaswamy
Lünsdorf, Heinrich
Tyagi, Poornima
Raut, Rajendra
Arora, Upasana
Jain, Swatantra K
Rinas, Ursula
Swaminathan, Sathyamangalam
Khanna, Navin
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.
2017-01-27T10:30:39Z
2017-01-27T10:30:39Z
2013-05-25
2015-09-04T08:27:52Z
Journal Article
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
en
Khetarpal et al.; licensee BioMed Central Ltd.
oai:repository.helmholtz-hzi.de:10033/6207672018-06-13T15:13:41Zcom_10033_311308col_10033_620777
Protein folding and conformational stress in microbial cells producing recombinant proteins: a host comparative overview
Gasser, Brigitte
Saloheimo, Markku
Rinas, Ursula
Dragosits, Martin
Rodríguez-Carmona, Escarlata
Baumann, Kristin
Giuliani, Maria
Parrilli, Ermenegilda
Branduardi, Paola
Lang, Christine
Porro, Danilo
Ferrer, Pau
Luisa Tutino, Maria
Mattanovich, Diethard
Villaverde, Antonio
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.
2017-01-27T09:46:39Z
2017-01-27T09:46:39Z
2008-04-04
2015-09-04T08:28:24Z
Journal Article
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
en
Gasser et al.
oai:repository.helmholtz-hzi.de:10033/6207572019-08-30T11:31:23Zcom_10033_311308col_10033_620777
Packaging protein drugs as bacterial inclusion bodies for therapeutic applications
Villaverde, Antonio
García-Fruitós, Elena
Rinas, Ursula
Seras-Franzoso, Joaquin
Kosoy, Ana
Corchero, José L
Vazquez, Esther
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.
2017-01-27T08:36:00Z
2017-01-27T08:36:00Z
2012-06-11
2015-09-04T08:29:13Z
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
en
Villaverde et al.; licensee BioMed Central Ltd.
oai:repository.helmholtz-hzi.de:10033/6207542019-08-30T11:31:23Zcom_10033_311308col_10033_620777
Smart sustainable bottle (SSB) system for E. coli based recombinant protein production
Li, Zhaopeng
Carstensen, Bettina
Rinas, Ursula
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.
2017-01-27T08:30:06Z
2017-01-27T08:30:06Z
2014-11-05
2015-09-04T08:29:17Z
Journal Article
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
en
Li et al.; licensee BioMed Central Ltd.
oai:repository.helmholtz-hzi.de:10033/6207042019-08-30T11:37:44Zcom_10033_311308col_10033_620777
The intra- and extracellular proteome of Aspergillus niger growing on defined medium with xylose or maltose as carbon substrate
Lu, Xin
Sun, Jibin
Nimtz, Manfred
Wissing, Josef
Zeng, An-Ping
Rinas, Ursula
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.
2017-01-16T15:28:35Z
2017-01-16T15:28:35Z
2010-04-20
2015-09-04T08:30:22Z
Journal Article
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
en
Lu et al.
oai:repository.helmholtz-hzi.de:10033/6206892018-06-12T22:52:32Zcom_10033_311308col_10033_620777
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
Gurramkonda, Chandrasekhar
Polez, Sulena
Skoko, Natasa
Adnan, Ahmad
Gäbel, Thomas
Chugh, Dipti
Swaminathan, Sathyamangalam
Khanna, Navin
Tisminetzky, Sergio
Rinas, Ursula
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.
2017-01-10T11:49:36Z
2017-01-10T11:49:36Z
2010-05-12
2015-09-04T08:31:28Z
Journal Article
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
en
Gurramkonda et al.
oai:repository.helmholtz-hzi.de:10033/5788812019-08-30T11:33:30Zcom_10033_6832com_10033_311308col_10033_620777col_10033_6833
Metabolic peculiarities of Aspergillus niger disclosed by comparative metabolic genomics.
Sun, Jibin
Lu, Xin
Rinas, Ursula
Zeng, An Ping
Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
Aspergillus niger
Biotechnology
Citrate (si)-Synthase
Citric Acid
Fungal Proteins
Gene Expression Regulation, Fungal
Genes, Fungal
Genome
Genomics
Metabolism
Mitochondrial Proteins
Models, Biological
Models, Genetic
Models, Theoretical
Oxidoreductases
Phylogeny
Plant Proteins
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.
2015-09-29T11:44:49Z
2015-09-29T11:44:49Z
2007
Article
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
en
oai:repository.helmholtz-hzi.de:10033/5797162019-08-30T11:24:31Zcom_10033_311308col_10033_620777
Decrease of UPR- and ERAD-related proteins in Pichia pastoris during methanol-induced secretory insulin precursor production in controlled fed-batch cultures.
Vanz, Ana Letícia
Nimtz, Manfred
Rinas, Ursula
Batch Cell Culture Techniques
Endoplasmic Reticulum-Associated Degradation
Glycerol
Methanol
Pichia
Proinsulin
Proteome
Recombinant Proteins
Secretory Pathway
Unfolded Protein Response
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.
2015-10-15T08:01:43Z
2015-10-15T08:01:43Z
2014
Article
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
en
oai:repository.helmholtz-hzi.de:10033/5944172019-08-30T11:37:00Zcom_10033_311308col_10033_620777
Generation of anti-TLR2 intrabody mediating inhibition of macrophage surface TLR2 expression and TLR2-driven cell activation.
Kirschning, Carsten J
Dreher, Stefan
Maass, Björn
Fichte, Sylvia
Schade, Jutta
Köster, Mario
Noack, Andreas
Lindenmaier, Werner
Wagner, Hermann
Böldicke, Thomas
Helmholtz Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany.
Adenoviridae
Amino Acid Sequence
Animals
Antibodies, Monoclonal
Antibody Specificity
Base Sequence
Cell Line
Endoplasmic Reticulum
Genetic Vectors
Humans
Interleukin-6
Macrophages
Mice
Mice, Inbred C57BL
Molecular Sequence Data
Signal Transduction
Single-Chain Antibodies
Toll-Like Receptor 2
Transfection
Tumor Necrosis Factor-alpha
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.
2016-01-20T14:17:07Z
2016-01-20T14:17:07Z
2010
Article
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
en
oai:repository.helmholtz-hzi.de:10033/6009822019-08-30T11:37:44Zcom_10033_311308col_10033_620777
Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.
Bleckmann, Maren
Schürig, Margitta
Chen, Fang-Fang
Yen, Zen-Zen
Lindemann, Nils
Meyer, Steffen
Spehr, Johannes
van den Heuvel, Joop
Helmholtz Centre for infection research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany.
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.
2016-03-09T08:18:20Z
2016-03-09T08:18:20Z
2016
Article
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
en
oai:repository.helmholtz-hzi.de:10033/6039232019-08-30T11:31:23Zcom_10033_311308col_10033_620777
Genomic Analysis and Isolation of RNA Polymerase II Dependent Promoters from Spodoptera frugiperda.
Bleckmann, Maren
Fritz, Markus H-Y
Bhuju, Sabin
Jarek, Michael
Schürig, Margitta
Geffers, Robert
Benes, Vladimir
Besir, Hüseyin
van den Heuvel, Joop
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
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.
2016-03-29T14:34:28Z
2016-03-29T14:34:28Z
2015
Article
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
en
oai:repository.helmholtz-hzi.de:10033/6039962019-08-30T11:35:14Zcom_10033_311308col_10033_620777
Fast plasmid based protein expression analysis in insect cells using an automated SplitGFP screen.
Bleckmann, Maren
Schmelz, Stefan
Schinkowski, Christian
Scrima, Andrea
van den Heuvel, Joop
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
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.
2016-03-30T09:42:36Z
2016-03-30T09:42:36Z
2016-02-23
Article
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
ENG
oai:repository.helmholtz-hzi.de:10033/6159992019-08-30T11:31:23Zcom_10033_311308col_10033_620777
High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells.
Jäger, Volker
Büssow, Konrad
Wagner, Andreas
Weber, Susanne
Hust, Michael
Frenzel, André
Schirrmann, Thomas
Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Cloning, Molecular
Genetic Vectors
HEK293 Cells
Humans
Immunoglobulin Fc Fragments
Peptide Library
Recombinant Fusion Proteins
Ribonucleases
Single-Chain Antibodies
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.
2016-07-13T13:14:38Z
2016-07-13T13:14:38Z
2013
Article
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
en
oai:repository.helmholtz-hzi.de:10033/6200302019-08-30T11:28:23Zcom_10033_311308col_10033_620777
Specific in vivo knockdown of protein function by intrabodies.
Marschall, Andrea L J
Dübel, Stefan
Böldicke, Thomas
Helmholtz Centre for infection researchz, Inhoffenstr. 7, 38124 Braunschweig.
Animals
Antibodies
Cell Nucleus
Cytoplasm
Endoplasmic Reticulum
Gene Knockdown Techniques
Humans
Intracellular Space
Models, Immunological
Proteome
Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed.
2016-09-09T14:04:03Z
2016-09-09T14:04:03Z
2015
Article
Specific in vivo knockdown of protein function by intrabodies. 2015, 7 (6):1010-35 MAbs
1942-0870
26252565
10.1080/19420862.2015.1076601
http://hdl.handle.net/10033/620030
mAbs
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6209042019-08-30T11:35:39Zcom_10033_311308col_10033_620777
Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.
Kleinboelting, Silke
Ramos-Espiritu, Lavoisier
Buck, Hannes
Colis, Laureen
van den Heuvel, Joop
Glickman, J Fraser
Levin, Lonny R
Buck, Jochen
Steegborn, Clemens
Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Adenosine Triphosphate
Adenylyl Cyclases
Allosteric Regulation
Bicarbonates
Bithionol
Catalytic Domain
Crystallography, X-Ray
Humans
The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs.
2017-05-03T13:27:51Z
2017-05-03T13:27:51Z
2016-04-29
Article
Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site. 2016, 291 (18):9776-84 J. Biol. Chem.
1083-351X
26961873
10.1074/jbc.M115.708255
http://hdl.handle.net/10033/620904
The Journal of biological chemistry
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6213022019-08-30T11:36:33Zcom_10033_311308col_10033_620777
Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase.
Ramos-Espiritu, Lavoisier
Kleinboelting, Silke
Navarrete, Felipe A
Alvau, Antonio
Visconti, Pablo E
Valsecchi, Federica
Starkov, Anatoly
Manfredi, Giovanni
Buck, Hannes
Adura, Carolina
Zippin, Jonathan H
van den Heuvel, Joop
Glickman, J Fraser
Steegborn, Clemens
Levin, Lonny R
Buck, Jochen
Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Adenylyl Cyclase Inhibitors
Adenylyl Cyclases
Allosteric Regulation
Dose-Response Relationship, Drug
Humans
Models, Molecular
Molecular Structure
Pyrimidines
Solubility
Structure-Activity Relationship
Thiophenes
The prototypical second messenger cAMP regulates a wide variety of physiological processes. It can simultaneously mediate diverse functions by acting locally in independently regulated microdomains. In mammalian cells, two types of adenylyl cyclase generate cAMP: G-protein-regulated transmembrane adenylyl cyclases and bicarbonate-, calcium- and ATP-regulated soluble adenylyl cyclase (sAC). Because each type of cyclase regulates distinct microdomains, methods to distinguish between them are needed to understand cAMP signaling. We developed a mass-spectrometry-based adenylyl cyclase assay, which we used to identify a new sAC-specific inhibitor, LRE1. LRE1 bound to the bicarbonate activator binding site and inhibited sAC via a unique allosteric mechanism. LRE1 prevented sAC-dependent processes in cellular and physiological systems, and it will facilitate exploration of the therapeutic potential of sAC inhibition.
2018-03-02T15:30:30Z
2018-03-02T15:30:30Z
2016
Article
Discovery of LRE1 as a specific and allosteric inhibitor of soluble adenylyl cyclase. 2016, 12 (10):838-44 Nat. Chem. Biol.
1552-4469
27547922
10.1038/nchembio.2151
http://hdl.handle.net/10033/621302
Nature chemical biology
en
http://creativecommons.org/licenses/by-nc-sa/4.0/
oai:repository.helmholtz-hzi.de:10033/6216342019-08-30T11:31:21Zcom_10033_620533com_10033_311308col_10033_620534col_10033_620777col_10033_559591
Investigations on the mode of action of gephyronic acid, an inhibitor of eukaryotic protein translation from myxobacteria.
Muthukumar, Yazh
Münkemer, Johanna
Mathieu, Daniel
Richter, Christian
Schwalbe, Harald
Steinmetz, Heinrich
Kessler, Wolfgang
Reichelt, Joachim
Beutling, Ulrike
Frank, Ronald
Büssow, Konrad
van den Heuvel, Joop
Brönstrup, Mark
Taylor, Richard E
LASCHAT, SABINE
Sasse, Florenz
The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.
2019-01-07T14:43:20Z
2019-01-07T14:43:20Z
2018-01-01
Article
PLoS One. 2018 Jul 31;13(7):e0201605. doi: 10.1371/journal.pone.0201605 eCollection 2018.
1932-6203
30063768
10.1371/journal.pone.0201605
http://hdl.handle.net/10033/621634
PLOSOne
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
PLOS
PloS one
oai:repository.helmholtz-hzi.de:10033/6217752019-08-30T11:30:26Zcom_10033_620589com_10033_620652com_10033_311308col_10033_620672col_10033_620777col_10033_620608col_10033_559591
ER intrabody-mediated inhibition of interferon α secretion by mouse macrophages and dendritic cells.
Büssow, Konrad
Themann, Philipp
Luu, Sabine
Pentrowski, Paul
Harting, Claudia
Majewski, Mira
Vollmer, Veith
Köster, Mario
Grashoff, Martina
Zawatzky, Rainer
van den Heuvel, Joop
Kröger, Andrea
Böldicke, Thomas
HZI, Helmholtz Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany.
Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNβ. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNβ-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.
2019-05-15T07:43:02Z
2019-05-15T07:43:02Z
2019-01-01
Article
PLoS One. 2019 Apr 16;14(4):e0215062. doi: 10.1371/journal.pone.0215062. eCollection 2019.
1932-6203
30990863
10.1371/journal.pone.0215062
http://hdl.handle.net/10033/621775
PLOS ONE
en
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
Plos
PloS one
oai:repository.helmholtz-hzi.de:10033/6218472019-08-30T11:24:27Zcom_10033_311308col_10033_620777
Identifying parameters to improve the reproducibility of transient gene expression in High Five cells.
Bleckmann, Maren
Schürig, Margitta
Endres, Michelle
Samuels, Anke
Gebauer, Daniela
Konisch, Nadine
van den Heuvel, Joop
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Virus-free, transient gene expression (TGE) in High Five cells was recently
presented as an efficient protein production method. However, published TGE
protocols have not been standardized to a general protocol. Therefore,
reproducibility and implementation of the method in other labs remains difficult.
The aim of this study is to analyse the parameters determining the
reproducibility of the TGE in insect cells. Here, we identified that using linear
40 kDa PEI instead of 25 kDa PEI was one of the most important aspects to improve
TGE. Furthermore, DNA amount, DNA:PEI ratio, growth phase of the cells before
transfection, passage number, the origin of the High-Five cell isolates and the
type of cultivation medium were considered. Interestingly, a correlation of the
passage number to the DNA content of single cells (ploidy) and to the
transfection efficacy could be shown. The optimal conditions for critical
parameters were used to establish a robust TGE method. Finally, we compared the
achieved product yields in High Five cells using our improved TGE method with
both the baculoviral expression system and TGE in the mammalian HEK293-6E cell
line. In conclusion, the presented robust TGE protocol in High Five cells is easy
to establish and produces ample amounts of high-quality recombinant protein,
bridging the gap in expression level of this method to the well-established
mammalian TGE in HEK293 cells as well as to the baculoviral expression vector
system (BEVS).
2019-07-04T13:30:10Z
2019-07-04T13:30:10Z
2019-01-01
Article
PLoS One. 2019 Jun 6;14(6):e0217878. doi: 10.1371/journal.pone.0217878. eCollection 2019.
1932-6203
31170233
10.1371/journal.pone.0217878
http://hdl.handle.net/10033/621847
PLOS ONE
en
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
PLOS
PloS one
oai:repository.helmholtz-hzi.de:10033/6225622020-11-07T01:46:52Zcom_10033_620533com_10033_311308com_10033_620618com_10033_620656col_10033_620534col_10033_620777col_10033_620657col_10033_620619
Synthetic studies of cystobactamids as antibiotics and bacterial imaging carriers lead to compounds with high: In vivo efficacy
Testolin, Giambattista
Cirnski, Katarina
Rox, Katharina
Prochnow, Hans
Fetz, Verena
Grandclaudon, Charlotte
Mollner, Tim
Baiyoumy, Alain
Ritter, Antje
Leitner, Christian
Krull, Jana
van den Heuvel, Joop
Vassort, Aurelie
Sordello, Sylvie
Hamed, Mostafa M.
Elgaher, Walid A.M.
Herrmann, Jennifer
Hartmann, Rolf W.
Müller, Rolf
Brönstrup, Mark
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany.
There is an alarming scarcity of novel chemical matter with bioactivity against multidrug-resistant Gram-negative bacterial pathogens. Cystobactamids, recently discovered natural products from myxobacteria, are an exception to this trend. Their unusual chemical structure, composed of oligomeric para-aminobenzoic acid moieties, is associated with a high antibiotic activity through the inhibition of gyrase. In this study, structural determinants of cystobactamid's antibacterial potency were defined at five positions, which were varied using three different synthetic routes to the cystobactamid scaffold. The potency against Acinetobacter baumannii could be increased ten-fold to an MIC (minimum inhibitory concentration) of 0.06 μg mL−1, and the previously identified spectrum gap of Klebsiella pneumoniae could be closed compared to the natural products (MIC of 0.5 μg mL−1). Proteolytic degradation of cystobactamids by the resistance factor AlbD was prevented by an amide-triazole replacement. Conjugation of cystobactamid's N-terminal tetrapeptide to a Bodipy moiety induced the selective localization of the fluorophore for bacterial imaging purposes. Finally, a first in vivo proof of concept was obtained in an E. coli infection mouse model, where derivative 22 led to the reduction of bacterial loads (cfu, colony-forming units) in muscle, lung and kidneys by five orders of magnitude compared to vehicle-treated mice. These findings qualify cystobactamids as highly promising lead structures against infections caused by Gram-positive and Gram-negative bacterial pathogens.
Deutsches Zentrum für Infektionsforschung
2020-11-06T14:53:02Z
2020-11-06T14:53:02Z
2020-01-01
Article
Chem. Sci (2020);11(5);DOI:10.1039/C9SC04769G.
20416520
10.1039/c9sc04769g
http://hdl.handle.net/10033/622562
20416539
Chemical Science
2-s2.0-85079237979
SCOPUS_ID:85079237979
en
Attribution-NonCommercial-ShareAlike 4.0 International
http://creativecommons.org/licenses/by-nc-sa/4.0/
RSC
11
5
1316
1334
Chemical Science
oai:repository.helmholtz-hzi.de:10033/6226582021-01-05T01:41:19Zcom_10033_311308col_10033_620777
Baculovirus-free insect cell expression system for high yield antibody and antigen production.
Korn, Janin
Schäckermann, Dorina
Kirmann, Toni
Bertoglio, Federico
Steinke, Stephan
Heisig, Janyn
Ruschig, Maximilian
Rojas, Gertrudis
Langreder, Nora
Wenzel, Esther Veronika
Roth, Kristian Daniel Ralph
Becker, Marlies
Meier, Doris
van den Heuvel, Joop
Hust, Michael
Dübel, Stefan
Schubert, Maren
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Mammalian cells are the most commonly used production system for therapeutic antibodies. Protocols for the expression of recombinant antibodies in HEK293-6E cells in different antibody formats are described in detail. As model, antibodies against Kallikrein-related peptidase 7 (KLK7) were used. KLK7 is a key player in skin homeostasis and represents an emerging target for pharmacological interventions. Potent inhibitors can not only help to elucidate physiological and pathophysiological functions but also serve as a new archetype for the treatment of inflammatory skin disorders. Phage display-derived affinity-matured human anti-KLK7 antibodies were converted to scFv-Fc, IgG, and Fab formats and transiently produced in the mammalian HEK293-6E system. For the production of the corresponding antigen-KLK7-the baculovirus expression vector system (BEVS) and virus-free expression in Hi5 insect cells were used in a comparative approach. The target proteins were isolated by various chromatographic methods in a one- or multistep purification strategy. Ultimately, the interaction between anti-KLK7 and KLK7 was characterized using biolayer interferometry. Here, protocols for the expression of recombinant antibodies in different formats are presented and compared for their specific features. Furthermore, biolayer interferometry (BLI), a fast and high-throughput biophysical analytical technique to evaluate the kinetic binding constant and affinity constant of the different anti-KLK7 antibody formats against Kallikrein-related peptidase 7 is presented.
2021-01-04T13:43:47Z
2021-01-04T13:43:47Z
2020-12-07
Article
Other
Sci Rep. 2020 Dec 7;10(1):21393. doi: 10.1038/s41598-020-78425-9.
33288836
10.1038/s41598-020-78425-9
http://hdl.handle.net/10033/622658
2045-2322
Scientific reports
en
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
Nature research
10
1
21393
Scientific reports
England
oai:repository.helmholtz-hzi.de:10033/6229142021-07-01T01:43:54Zcom_10033_311308col_10033_620777col_10033_559591
Reproducible and Easy Production of Mammalian Proteins by Transient Gene Expression in High Five Insect Cells.
Schubert, Maren
Nimtz, Manfred
Bertoglio, Federico
Schmelz, Stefan
Lukat, Peer
van den Heuvel, Joop
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Expression vector
High five
Insect cells
TGE
Transient gene expression
he expression of mammalian recombinant proteins in insect cell lines using transient-plasmid-based gene expression enables the production of high-quality protein samples. Here, the procedure for virus-free transient gene expression (TGE) in High Five insect cells is described in detail. The parameters that determine the efficiency and reproducibility of the method are presented in a robust protocol for easy implementation and set-up of the method. The applicability of the TGE method in High Five cells for proteomic, structural, and functional analysis of the expressed proteins is shown.
2021-06-30T15:31:55Z
2021-06-30T15:31:55Z
2021-05-21
Article
Methods Mol Biol. 2021;2305:129-140. doi: 10.1007/978-1-0716-1406-8_6.
33950387
10.1007/978-1-0716-1406-8_6
http://hdl.handle.net/10033/622914
1940-6029
Methods in molecular biology (Clifton, N.J.)
en
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
Springer
2305
129
140
Methods in molecular biology (Clifton, N.J.)
United States
oai:repository.helmholtz-hzi.de:10033/6230102021-09-02T02:41:32Zcom_10033_311308col_10033_620777
Discovery of TDI-10229: A Potent and Orally Bioavailable Inhibitor of Soluble Adenylyl Cyclase (sAC, ADCY10).
Fushimi, Makoto
Buck, Hannes
Balbach, Melanie
Gorovyy, Anna
Ferreira, Jacob
Rossetti, Thomas
Kaur, Navpreet
Levin, Lonny R
Buck, Jochen
Quast, Jonathan
van den Heuvel, Joop
Steegborn, Clemens
Finkin-Groner, Efrat
Kargman, Stacia
Michino, Mayako
Foley, Michael A
Miller, Michael
Liverton, Nigel J
Huggins, David J
Meinke, Peter T
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Soluble adenylyl cyclase (sAC) has gained attention as a potential therapeutic target given the role of this enzyme in intracellular signaling. We describe successful efforts to design improved sAC inhibitors amenable for in vivo interrogation of sAC inhibition to assess its potential therapeutic applications. This work culminated in the identification of TDI-10229 (12), which displays nanomolar inhibition of sAC in both biochemical and cellular assays and exhibits mouse pharmacokinetic properties sufficient to warrant its use as an in vivo tool compound.
2021-09-01T08:21:19Z
2021-09-01T08:21:19Z
2021-07-14
Article
ACS Med Chem Lett. 2021 Jul 14;12(8):1283-1287. doi: 10.1021/acsmedchemlett.1c00273.
1948-5875
34413957
10.1021/acsmedchemlett.1c00273
http://hdl.handle.net/10033/623010
ACS medicinal chemistry letters
en
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
ACS
12
8
1283
1287
ACS medicinal chemistry letters
United States
United States
oai:repository.helmholtz-hzi.de:10033/6230302021-09-15T01:55:10Zcom_10033_620652com_10033_311308com_10033_622921col_10033_622922col_10033_620666col_10033_620777
A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.
Bertoglio, Federico
Fühner, Viola
Ruschig, Maximilian
Heine, Philip Alexander
Abassi, Leila
Klünemann, Thomas
Rand, Ulfert
Meier, Doris
Langreder, Nora
Steinke, Stephan
Ballmann, Rico
Schneider, Kai-Thomas
Roth, Kristian Daniel Ralph
Kuhn, Philipp
Riese, Peggy
Schäckermann, Dorina
Korn, Janin
Koch, Allan
Chaudhry, M Zeeshan
Eschke, Kathrin
Kim, Yeonsu
Zock-Emmenthal, Susanne
Becker, Marlies
Scholz, Margitta
Moreira, Gustavo Marçal Schmidt Garcia
Wenzel, Esther Veronika
Russo, Giulio
Garritsen, Hendrikus S P
Casu, Sebastian
Gerstner, Andreas
Roth, Günter
Adler, Julia
Trimpert, Jakob
Hermann, Andreas
Schirrmann, Thomas
Dübel, Stefan
Frenzel, André
van den Heuvel, Joop
Čičin-Šain, Luka
Schubert, Maren
Hust, Michael
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
RBD
SARS-CoV-2
SARS-CoV-2 variants
coronavirus
immune library
in vivo neutralization
neutralizing antibody
phage display
recombinant antibody
spike protein
The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.
2021-09-14T12:11:25Z
2021-09-14T12:11:25Z
2021-07-07
Article
Cell Rep. 2021 Jul 27;36(4):109433. doi: 10.1016/j.celrep.2021.109433. Epub 2021 Jul 7.
34273271
10.1016/j.celrep.2021.109433
http://hdl.handle.net/10033/623030
2211-1247
Cell reports
en
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
Cell Press
36
4
109433
Cell reports
United States
oai:repository.helmholtz-hzi.de:10033/6230632021-10-07T01:51:22Zcom_10033_311308col_10033_620777
Recombinant protein production provoked accumulation of ATP, fructose-1,6-bisphosphate and pyruvate in E. coli K12 strain TG1.
Weber, Jan
Li, Zhaopeng
Rinas, Ursula
HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.
Escherichia coli
Metabolic burden
Recombinant protein production
Background: Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden.
Results: Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate.
Conclusions: Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated "metabolic burden". Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.
2021-10-06T11:14:37Z
2021-10-06T11:14:37Z
2021-08-26
Article
Microb Cell Fact. 2021 Aug 26;20(1):169. doi: 10.1186/s12934-021-01661-9. PMID: 34446023.
34446023
10.1186/s12934-021-01661-9
http://hdl.handle.net/10033/623063
1475-2859
Microbial cell factories
en
Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
BMC
20
1
169
Microbial cell factories
England