Overproduction of bacterial protein disulfide isomerase (DsbC) and its modulator (DsbD) markedly enhances periplasmic production of human nerve growth factor in Escherichia coli.
Production of eukaryotic proteins with multiple disulfide bonds in the Escherichia coli periplasm often encounters difficulty in obtaining soluble products with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms insoluble aggregates when expressed in E. coli. We now report that overexpression of Dsb proteins known to catalyze formation and isomerization of disulfide bonds can substantially enhance periplasmic production of NGF. A set of pACYC184-based plasmids that permit dsb expression under the araB promoter were introduced into cells carrying a compatible plasmid that expresses NGF. The efficiency of periplasmic production of NGF fused to the OmpT signal peptide was strikingly improved by coexpression of DsbCD or DsbABCD proteins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly effective, whereas that of DsbAC increased the total yield but not the periplasmic expression. These results suggest synergistic roles of DsbC and DsbD in disulfide isomerization that appear to become limiting upon NGF production. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 cells.
HSP Research Institute, Kyoto Research Park, Kyoto 600-8813, Japan.
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Y
Kurokawa
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Yanagi
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T
Yura
D000818:Animals; D001089:Arabinose / Q000378:metabolism; D002384:Catalysis; D002455:Cell Division; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D008957:Models, Genetic; D020932:Nerve Growth Factor / Q000096:biosynthesis*; D016716:PC12 Cells; D019897:Periplasm / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D019704:Protein Disulfide-Isomerases / Q000096:biosynthesis*; D021382:Protein Sorting Signals; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors
D004220:Disulfides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D020932:Nerve Growth Factor; D001089:Arabinose; D019704:Protein Disulfide-Isomerases