Vegetos- An International Journal of Plant Research

  • Year: 2012
  • Volume: 25
  • Issue: 2

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

  • Author:
  • Mukesh Kumar, Rekha Kansal1, Vinay Kumar3, P S Srivastava2, K R Koundal1
  • Total Page Count: 7
  • DOI:
  • Page Number: 158 to 164

Department of Biotechnology, S V P University of Agriculture and Technology, Meerut-250 110, UP, India

1National Research Centre on Plant Biotechnology, IARI, New Delhi, India

3Department of Agricultural Biotechnology, Anand Agricultural University, Anand, Gujarat

2Department of Biotechnology, Jamia Hamdard, New Delhi, India

Abstract

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.

Keywords

Pigeonpea, Protease Inhibitor, Ammonium, DEAE-cellulose, Gel filtration Alkaline