PRODUCTION OF ENZYMATICALLY ACTIVE-RAT PROTEIN DISULFIDE-ISOMERASE INESCHERICHIA-COLI

We report the development of a bacterial expression system allowing hi gh-level synthesis of enzymatically active rat protein disulfide isome rase (rPDI). After expression of the rpdi gene under control of the in ducible trc promoter (P-trc), a significant amount of soluble, active rPDI was detected in the periplasmic contents, which were released fro m the cells by cold osmotic shock. However, the exported molecules wer e incompletely or improperly processed, while the major amount of synt hesized rPDI was in fact detected in the soluble cellular fraction. Su bstitution of the autologous eukaryotic export signal with the nucleot ide (nt) sequence encoding the signal peptide (sOmpA) of the bacterial outer membrane protein A, and expression of the sompA::rpdi fusion ge ne under control of both the Ipp promoter (P-lpp) and the Inc promoter -operator (POlac), resulted in high-level production of rPDI. Furtherm ore, the latter was efficiently exported into the periplasmic compartm ent, from where it was recovered as a soluble, fully active form with the sOmpA precisely removed. The synthesis of a small 21-kDa peptide a ccompanying the production of rPDI was also observed. This rPDI-relate d peptide (rPDIf), which represented a C-terminal fragment of rPDI inc luding the second active site, arose by internal translation initiatio n within rpdi. Replacement of the presumed internal start codon by CTC completely eliminated the aforementioned phenomenon and resulted in t he production of a slightly mutated, enzymatically active enzyme (rPDI m).