Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion.

Nilanjana Bhattacharjee, Sayanika Banerjee, Samir K Dutta

Protein expression and purification 2014 Apr

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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. Copyright © 2014 Elsevier Inc. All rights reserved.

Citation

Nilanjana Bhattacharjee, Sayanika Banerjee, Samir K Dutta. Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion. Protein expression and purification. 2014 Apr;96:26-31