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 Esch erichia coli periplasm often encounters difficulty in obtaining soluble pro ducts with native structure. Human nerve growth factor beta (NGF) contains three disulfide bonds between nonconsecutive cysteine residues and forms in soluble aggregates when expressed in E. coli. We now report that overexpres sion of Dsb proteins known to catalyze formation and isomerization of disul fide bonds can substantially enhance periplasmic production of NGF. A set o f pACYC184-based plasmids that permit dsb expression under the araB promote r were introduced into cells carrying a compatible plasmid that expresses N GF. The efficiency of periplasmic production of NGF fused to the OmpT signa l peptide was strikingly improved by coexpression of DsbCD or DsbABCD prote ins (up to 80% of total NGF produced). Coexpression of DsbAB was hardly eff ective, whereas that of DsbAC increased the total yield but not the peripla smic expression. These results suggest synergistic roles of DsbC and DsbD i n disulfide isomerization that appear to become limiting upon NGF productio n. Furthermore, recombinant NGF produced with excess DsbCD (or DsbABCD) was biologically active judged by the neurite outgrowth assay using rat PC12 c ells.