Overproduction of Bacterial Protein Disulfide Isomerase (DsbC) and Its Modulator (DsbD) Markedly Enhances Periplasmic Production of Human Nerve Growth Factor in Escherichia coli

Abstract

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 ␤ (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.

Production of Brain-Derived Neurotrophic Factor inEscherichia coliby Coexpression of Dsb Proteins

“…The e‹ciency of periplasmic production of NGF fused to an OmpT signal peptide was improved by coexpression of DsbCD or DsbABCD pro-teins. 12) These results revealed signiˆcant eŠects of Dsb proteins on recombinant protein production in the E. coli periplasm.…”

“…15,16) In addition to PDI, overexpression of Dsb proteins has been shown to increase the yield of various eukaryotic proteins in bacteria. [8][9][10][11][12] Soluble BDNF production was estimated from the biological activity for neurite outgrowth of embryonic chick DRG neurons. Soluble BDNF fractions (Fig.…”

Soluble Expression of Recombinant Nerve Growth Factor in Cytoplasm of Escherichia coli

“…To the best of our knowledge, hNGF expression in E. coli cytoplasm has led to aggregated proteins (10)(11)(12)(13)(14)(15)(16)21). Thus, the soluble expression of hNGF in E. coli has been considered a challenging task.…”

“…In reported studies bacterial expression of human NGF demonstrated the feasibility of overproduction but also limitations in the use of E. coli as an expression host for a protein with three intra-chain disulfide bonds (10)(11)(12)(13)(14)(15)(16). Isolation and purification of hNGF from inclusion bodies require solubilization followed by refolding.…”

Role of Dimerization in the Catalytic Properties of the Escherichia coli Disulfide Isomerase DsbC

“…In E. coli, disulfide bond isomerization is the limiting step in the oxidative folding of many heterologous proteins that contain multiple cysteines. Overexpression of DsbC has been shown to enhance the yield of proteins such as human nerve growth factor, human tissue plasminogen activator (tPA) 2 and immunoglobulins (17)(18)(19). DsbC is topologically analogous to the eukaryotic proteindisulfide isomerase (PDI).…”