Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide
Nilanjana Bhattacharjee1, Sayanika Banerjee2, Samir K Dutta2
1Drug Development Diagnostics & Biotechnology Division, CSIR - Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India; Department of Zoology, New Alipore College, L Block, New Alipore, Kolkata 700053, India.
Abstract:
A winged bean trypsin inhibitor (WbTI-2) of molecular mass ∼20kDa, has been cloned and expressed in Escherichiacoli with full activity like the one from seed protein. It completely inhibits trypsin at an enzyme:inhibitor molar ratio of 1:2. PCR with cDNA and genomic DNA using same primers produced about 550 base pair product, which indicated it to be an intronless gene. Through site-directed mutagenesis, the Arg64 has been confirmed as the P1 residue. For the presence of five methionine residues in WbTI-2, cyanogen bromide (CNBr) digestion was carried out. Out of three fragments the one (about 65% of original size) containing the reactive site loop retained 50% activity.
