Cloning, expression and characterization of Bauhinia variegata trypsin inhibitor BvTI.
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, 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/l of active protein, respectively, and showed protein bands of approximately 21.5 kDa by SDS-PAGE. Trypsin inhibition activity was comparable for rBvTI (Ki=4 nM) and r(mut)BvTI (Ki=6 nM). Our data suggest that the Leu to Pro substitution at the fifth amino-terminal residue was not crucial for proteinase inhibition.
de Souza
Adriana F
AF
Departamento de Bioquímica, UNIFESP-EPM, Rua 3 de Maio 100, 04044-020 São Paulo, SP, Brazil.
Torquato
Ricardo J S
RJ
Tanaka
Aparecida S
AS
Sampaio
Claudio A M
CA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D036481:Bauhinia; D002417:Cattle; D003001:Cloning, Molecular; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D016415:Sequence Alignment; D014361:Trypsin Inhibitors / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*
D014361:Trypsin Inhibitors