Periplasmic chaperones used to enhance functional secretion of proteins in E. coli.
While Escherichia coli is in wide use as a host organism for preparative protein production, problems with the folding of the recombinant gene product as well as protein aggregation, i.e., formation of inclusion bodies, are frequently encountered. This is particularly true for proteins that carry structural disulfide bonds, including antibody fragments, cytokines, growth factors, and extracellular fragments of eukaryotic cell surface receptors. In these cases, secretion into the oxidizing milieu of the bacterial periplasm in principle enables disulfide bond formation, resulting in a correctly folded and soluble protein. However, this process often occurs at low efficiency, depending on the nature of the recombinant gene product. Therefore, we have developed the helper plasmid pTUM4, which effects overexpression of four established periplasmic chaperones and/or folding catalysts: the thiol-disulfide oxidoreductases DsbA and DsbC, which catalyze the formation and isomerization of disulfide bridges, and two peptidyl-prolyl cis/trans isomerases with chaperone activity, FkpA and SurA. Here, we present a detailed protocol how to use this system for the bacterial secretion of recombinant proteins, including human EGF as a new example, and we give hints on optimization of the expression procedure.
Lehrstuhl für Biologische Chemie, Technische Universität München, Freising-Weihenstephan, Germany. martin.schlapschy@wzw.tum.de
Martin
M
Schlapschy
Arne
A
Skerra
D004220:Disulfides / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones; D010084:Oxidation-Reduction; D019897:Periplasm / Q000235:genetics / Q000378:metabolism*; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
D004220:Disulfides; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D033903:Periplasmic Proteins; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D019704:Protein Disulfide-Isomerases; C526648:dsbA protein, E coli; C526649:dsbC protein, E coli