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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.
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