Purification of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds using Single Step Immunoaffinity Column

Abstract

Problem statement: Winged bean is a rich source of protease inhibitors. The seeds of winged bean contain several serine protease inhibitors of which winged bean chymotrypsin-trypsin inhibitor (WbCTI) is of significant importance due to its dual inhibition of both chymotrypsin and trypsin. The purification of WbCTI has always been difficult and a time taking process that involved large number of steps and various purification columns. Approach: This study was focused on the single step method of purification of WbCTI to homogeneity from winged bean seeds using immunoaffinity column chromatography. Results: WbCTI was purified to homogeneity using antiWbCTI antibody column in a single step which gave a single band on SDS-PAGE analysis and with almost one and half times more yield of purified protein than obtained from previously mentioned purification methods. Conclusion: This method using immunoaffinity column is high yielding and time saving process of purification.

Purified winged bean protease inhibitor affects the growth ofBactrocera cucurbitae

“…The seeds of winged beans, P. tetragonolobus , were ground to make fine flour and were then defatted with acetone (1:10 w/v) and air dried four times. The flour after defatting was soaked in potassium phosphate buffer (10 mM, pH 7.2) (Roy & Dutta, 2009). The soaked material filtered through multiple layers of surgical gauze, next day was centrifuged at 10,000 rpm for a time interval of 15 min (4°C).…”

Nutritional and Antinutritional Factors of Some Pulses Seed and Their Effects on Human Health

“…Proteinase inhibitors of plants are small molecular weight proteins that are natural, defense-related proteins often present in seeds and induced in certain tissues by herbivory or wounding. Most storage organs such as seeds (Leguminosae and Graminae) contain about 1 to 10% of their total protein, which inhibit different types of enzymes [33]. Thus, their main function is thought to be in plant defense, the regulation of endogenous proteinases in addition to the prevention of unwanted proteolysis [34].…”

Genomic and cDNA Cloning, Expression, Purification, and Characterization of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds

“…Starting with 3.0 g of optimally induced E. coli cells containing pTrc-WbCTI plasmids, the recombinant protein (rWbCTI) was purified from the soluble portion of the bacterial lysate by single-step immunoaffinity chromatography using a rabbit anti-WbCTI antibody coupled sepharose column. The immunoaffinity column used for purification of the recombinant protein was prepared as described by Roy et al 32) The cells were harvested by centrifugation at 12;000 Â g for 15 min. The pellet was resuspended in ice-cold lysis buffer (50 mM Tris-Cl, pH 8.0, 1 mM EDTA, 150 mM NaCl, and protease inhibitor cocktail) and sonicated Labsonic 2000 (Germany) at 10 pulses of 30 s each.…”