Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI
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2005-11-01
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Artigo
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A Bauhinia variegata trypsin inhibitor (BvTI) cDNA fragment was cloned into the pCANTAB5E phagemid. the clone pAS 1.1.3 presented a cDNA fragment of 733 bp, including the coding region for a mature BvTI protein comprising 175 amino acid residues. the deduced amino acid sequence for BvTI confirmed it as a member of the Kunitz-type plant serine proteinase inhibitor family. the BvTI cDNA fragment encoding the mature form was cloned into the expression vector, pET-14b, and expressed in E coli BL2I (DE3) pLysS in an active form. in addition, a BvTI mutant form, r(mut)BvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. the recombinant proteins, rBvTI and r(mut)BvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/I of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (K-I=4 nm) and r(mut)BvTI (K-i=6 nm). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.
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Biological Chemistry. Berlin: Walter de Gruyter & Co, v. 386, n. 11, p. 1185-1189, 2005.