Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI

• Published in: Biological chemistry Vol. 386; no. 11; pp. 1185 - 1189
• Main Authors: de Souza, Adriana F., Torquato, Ricardo J.S., Tanaka, Aparecida S., Sampaio, Claudio A.M.
• Format: Journal Article
• Language: English
• Published: Germany Walter de Gruyter 01.11.2005
• Subjects: Amino Acid Sequence; Animals; Base Sequence; Bauhinia; Bauhinia variegata; Cattle; cloning; Cloning, Molecular; expression; Gene Expression; Humans; Kunitz-type serine proteinase inhibitor; Leguminosae; Molecular Sequence Data; Plants, Medicinal; Sequence Alignment; trypsin inhibitor; Trypsin Inhibitors - biosynthesis; Trypsin Inhibitors - chemistry; Trypsin Inhibitors - genetics
• ISSN: 1431-6730; 1437-4315
• DOI: 10.1515/BC.2005.135

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 ex-pressed in E. coli BL21 (DE3) pLysS in an active form. In addition, a BvTI mutant form, rmutBvTI, with a Pro residue as the fifth amino acid in place of Leu, was produced. The recombinant proteins, rBvTI and rmutBvTI, were purified on a trypsin-Sepharose column, yielding 29 and 1.44 mg/l 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 rmutBvTI (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.