Isolation and Characterization of Protease Inhibitor Protein from Pigeon pea (Cajanus cajan L.)

Protease Inhibitor (PI) protein was isolated from four varieties of Pigeonpea viz. Pusa-855, Pusa33, Pusa-987 ND Pusa-84 by shaking defatted flour of mature seeds with 0.1 M sodium phosphate buffer (pH 7.6). The crude extracts were shacked for four hours at RT in a shaker. The homogenates were heat denatured and centrifuged. The supernatants subjected to 0–20%, 2040%, 40–60%, 60–80% and 80–100% ammonium sulphate fractions and the precipitates obtained were dialyzed extensively with same buffer. The dialyzed sample of Pusa-33, which had higher PI activity in 40–80% fraction, was used for further purification. The dialyzed protein sample loaded on pretreated DEAE-cellulose (anion exchanger) column to purify the protein followed by gel filtration. The fraction showing PI activity after ion exchange and gel filtration was pooled and resolved on SDS-PAGE and Native-PAGE. It showed approx 26kD band on SDS-PAGE but on gel filtration the molecular weight was found 24.95 kD. The purified PI showed stability 92–94% activity alkaline pH and also retains 85% activity at 90°C but become inactive on boiling and autoclaving.