Production of enzymatically active rat protein disulfide isomerase in Escherichia coli.
We report the development of a bacterial expression system allowing high-level synthesis of enzymatically active rat protein disulfide isomerase (rPDI). After expression of the rpdi gene under control of the inducible trc promoter (Ptrc), a significant amount of soluble, active rPDI was detected in the periplasmic contents, which were released from the cells by cold osmotic shock. However, the exported molecules were incompletely or improperly processed, while the major amount of synthesized rPDI was in fact detected in the soluble cellular fraction. Substitution of the autologous eukaryotic export signal with the nucleotide (nt) sequence encoding the signal peptide (sOmpA) of the bacterial outer membrane protein A, and expression of the sompA::rpdi fusion gene under control of both the lpp promoter (Plpp) and the lac promoter-operator (POlac), resulted in high-level production of rPDI. Furthermore, the latter was efficiently exported into the periplasmic compartment, from where it was recovered as a soluble, fully active form with the sOmpA precisely removed. The synthesis of a small 21-kDa peptide accompanying the production of rPDI was also observed. This rPDI-related peptide (rPDIf), which represented a C-terminal fragment of rPDI including the second active site, arose by internal translation initiation within rpdi. Replacement of the presumed internal start codon by CTC completely eliminated the aforementioned phenomenon and resulted in the production of a slightly mutated, enzymatically active enzyme (rPDIm).
De Sutter
K
K
Laboratory of Molecular Biology, University, Gent, Belgium.
Hostens
K
K
Vandekerckhove
J
J
Fiers
W
W
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003062:Codon; D004926:Escherichia coli / Q000235:genetics; D007535:Isomerases / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases; D021382:Protein Sorting Signals; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis
D003062:Codon; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D007535:Isomerases; D019704:Protein Disulfide-Isomerases