Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI

Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI

Author Souza, A. F. de Google Scholar
Torquato, RJS Google Scholar
Tanaka, A. S. Google Scholar
Sampaio, CAM Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract 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.
Keywords Bauhinia variegata
cloning
expression
Kunitz-type serine proteinase inhibitor
Leguminosae
trypsin inhibitor
Language English
Date 2005-11-01
Published in Biological Chemistry. Berlin: Walter de Gruyter & Co, v. 386, n. 11, p. 1185-1189, 2005.
ISSN 1431-6730 (Sherpa/Romeo, impact factor)
Publisher Walter de Gruyter & Co
Extent 1185-1189
Origin http://dx.doi.org/10.1515/BC.2005.135
Access rights Closed access
Type Article
Web of Science ID WOS:000233703800012
URI http://repositorio.unifesp.br/handle/11600/28546

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