cDNA cloning, expression, and rapid purification of a Kunitz-type winged bean chymotrypsin inhibitor.
A 183-residue Kunitz-type winged bean chymotrypsin inhibitor (WbCI), inhibits its cognate protease at a molar ratio of 1:2, instead of the usual ratio of 1:1 common to other members of the family. From the cDNA pool obtained by reverse transcription of the poly(A)+ RNA of the developing winged bean seeds, the structural gene of WbCI has been amplified by PCR using primers designed to delete the 24-residue signal peptide and introduce EcoRI and SalI sites at the ends of the amplified DNA. The latter is cloned in pBluescript and the insert has been sequenced to confirm its authenticity. Subcloning it in pTrc99A, a high-expression vector for Escherichia coli has generated a chimeric plasmid, pTrc-WbCI, which has a reading frame for a recombinant protein (rWbCI), having an additional tripeptide (M-E-F) fused to the N-terminus of WbCI. The expression of rWbCI has been ascertained by immunoblot analysis using rabbit anti-WbCI immune sera and quantitated by ELISA. The optimal conditions for the induction of the protein by IPTG, avoiding complications of protein-body formation, have also been standardized. rWbCI has been purified by a simple and rapid procedure of immunoaffinity chromatography, with an overall yield of 1.3 mg/g wet cell. SDS-PAGE analysis shows the presence of a single protein band, attesting to the homogeneity of the preparation; functionally, it is indistinguishable from WbCI since they inhibit alpha-chymotrypsin in an identical manner.
Ghosh
S
S
Biochemical Engineering Division, Indian Institute of Chemical Biology, Calcutta, India.
Singh
M
M
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin / Q000037:antagonists & inhibitors; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli; D017343:Genes, Plant; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D018076:DNA, Complementary; D018744:DNA, Plant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014361:Trypsin Inhibitors; C440588:WCI protein, Psophocarpus tetragonolobus; D002918:Chymotrypsin