Molecular cloning and expression of genes of Kunitz-type C protease inhibitors from potato

摘要

We cloned the products of polymerase chain reaction of the genome DNA of potato (Solanum tuberosum L., Istrinskii cultivar) and isolated 35 clones, which represent copies of eight genes encoding Kunitz type C proteases. Their nucleotide sequences were established. All the genes were found for the first time and designated as PKPI-C1-PKPI-C8. The gene PKPI-C2, which we had earlier presumed to encode the subtilisin PKSI inhibitor, was cloned into pQE30 vector and then expressed in Escherichia coli cells. The recombinant protein PKPI-C2 underwent spontaneous folding and transformation into a soluble state. We purified it to homogeneity by affinity chromatography. The PKPI-C2 protein efficiently inhibited subtilisin Carlsberg activity and did not act on trypsin, chymotrypsin, or papain.