Synthesis of proteins with disulfide bonds in E-coli using defined culture media

摘要

Synthesis of eukaryotic proteins containing disulfide bonds in E. coli normally produces misfolded products because the low redox potential of the bacterial cytosol is not appropriated for the formation of the disulfide bonds of these polypeptides. Although in many cases those denatured proteins can be refolded to a native conformation, the yield of this process is usually very low and the product too heterogeneous for high-resolution structural studies. Several eukaryotic cell-based systems have been described as alternatives to the E. coli-based ones. Nevertheless, those systems are considerably more cumbersome and more expensive. Moreover, with eukaryotic systems, it is difficult to obtain a homogeneously labeled protein, either with seleno-methionine for crystallographic analyses or with 15N and 13C for NMR. Therefore, the development of expression systems that allow the synthesis in native conformation of eukaryotic disulfide bondcontaining proteins in E. coli grown in defined media is still an attractive biotechnological goal. Several systems for synthesizing this type of protein in E. coli (in native conformation) have been delineated that have worked successfully in some cases. Such is the case of the E. coli strains in which the thioredoxin and glutaredoxin reduction pathways have been knocked out to raise the thiol-disulfide redox potential of the cytoplasm (2). Other approaches, such as the plN-III-ompA vector-based systems (9), couple protein synthesis to its secretion to the more oxidizing environment of the periplasm, where the disulfide bond-forming and isomerizing enzymes are present. However, labeling requires the culture to be grown in minimal medium, a condition in which protein synthesis using plN-III-ompA vectors becomes quite inefficient (C.F.-T., personal communication). Accordingly, polypeptide synthesis of medium- or large-scale quantities using ompA vectors usually requires culture media enriched with either casamino acids or yeast extracts (5,11).