http://hdl.handle.net/10033/128389 Tue, 18 Jun 2013 22:33:42 GMT 2013-06-18T22:33:42Z http://hdl.handle.net/10033/283572 Title: Folding and dimerization kinetics of bone morphogenetic protein-2, a member of the transforming growth factor-β family. Authors: Vallejo, Luis F; Rinas, Ursula Abstract: 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. Tue, 01 Jan 2013 00:00:00 GMT http://hdl.handle.net/10033/283572 2013-01-01T00:00:00Z http://hdl.handle.net/10033/236989 Title: More than just a metabolic regulator--elucidation and validation of new targets of PdhR in Escherichia coli. Authors: Göhler, Anna-Katharina; Kökpinar, Öznur; Schmidt-Heck, Wolfgang; Geffers, Robert; Guthke, Reinhard; Rinas, Ursula; Schuster, Stefan; Jahreis, Knut; Kaleta, Christoph Abstract: The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach. Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10033/236989 2011-01-01T00:00:00Z http://hdl.handle.net/10033/235575 Title: Inclusion bodies of fuculose-1-phosphate aldolase as stable and reusable biocatalysts. Authors: Sans, Cristina; García-Fruitós, Elena; Ferraz, Rosa M; González-Montalbán, Núria; Rinas, Ursula; López-Santín, Josep; Villaverde, Antonio; Álvaro, Gregorio Abstract: 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. Tue, 24 Jul 2012 00:00:00 GMT http://hdl.handle.net/10033/235575 2012-07-24T00:00:00Z http://hdl.handle.net/10033/221512 Title: Optimized procedure to generate heavy isotope and selenomethionine-labeled proteins for structure determination using Escherichia coli-based expression systems. Authors: Li, Zhaopeng; Nimtz, Manfred; Rinas, Ursula Abstract: 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. Tue, 01 Nov 2011 00:00:00 GMT http://hdl.handle.net/10033/221512 2011-11-01T00:00:00Z