Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion.
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.
Drug 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.
Nilanjana
N
Bhattacharjee
Drug Development Diagnostics & Biotechnology Division, CSIR - Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India.
Sayanika
S
Banerjee
Drug Development Diagnostics & Biotechnology Division, CSIR - Indian Institute of Chemical Biology, 4, Raja S.C. Mullick Road, Kolkata 700032, India. Electronic address: samirkdutta@iicb.res.in.
Samir K
SK
Dutta
D001483:Base Sequence; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D007887:Fabaceae / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016415:Sequence Alignment; D014361:Trypsin Inhibitors / Q000096:biosynthesis / Q000235:genetics
D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C067097:trypsin inhibitor, Kunitz winged-bean seed; D003488:Cyanogen Bromide
CNBr digestion; Cloning; Expression; Psophocarpus tetragonolobus; Site-directed mutagenesis; Trypsin inhibitor