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

• Published in: Russian journal of bioorganic chemistry Vol. 34; no. 3; pp. 310 - 317
• Main Authors: Valueva, T. A., Speranskaya, A. S., Revina, T. A., Shevelev, A. B.
• Format: Journal Article
• Language: English
• Published: Dordrecht SP MAIK Nauka/Interperiodica 01-05-2008
• Subjects: Biochemistry; Biomedical and Life Sciences; Biomedicine; Bioorganic Chemistry; Escherichia coli; Life Sciences; Organic Chemistry; Solanum tuberosum; genes; proteases; Kunitz type protease inhibitors
• ISSN: 1068-1620; 1608-330X
• DOI: 10.1134/S1068162008030102

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.