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 anti-WbCTI 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.
Highlights
Protease inhibitors are ubiquitous in nature and are found to be involved in various important biological functions like digestion of proteins, control of blood clotting, apoptosis, signaling receptors interaction in animals and in plant defense against insect attack[11]
The present study describes about a rapid single step purification method of winged bean chymotrypsin-trypsin inhibitor (WbCTI) using an immunoaffinity column chromatography with the high yield of purified biologically active protein
Western blot analysis using this antibody against other inhibitors purified from winged bean showed that there was no immunological crossreactivity between anti-WbCTI antibody and other protease inhibitors present in winged bean seeds (Fig. 1A)
Summary
Protease inhibitors are ubiquitous in nature and are found to be involved in various important biological functions like digestion of proteins, control of blood clotting, apoptosis, signaling receptors interaction in animals and in plant defense against insect attack[11]. Shibata et al.[12] purified seven inhibitors including their isoinhibitors from winged bean using a large number of conventional ionexchange and gel filtration columns All these inhibitors were Kunitz type having molecular weights of around 20 kDa, including four half cysteine residues. Three different protease inhibitors from winged bean have been purified, namely a chymotrypsin inhibitor (WbCI), a trypsin inhibitor (WbTI) and a dual chymotrypsin-trypsin inhibitor (WbCTI) using the conventional trypsin/chymotrypsin sepharose columns and FPLC gel filtration columns[3] Despite their presence in large amounts in seeds, isolation of individual inhibitors free from each other using affinity chromatography has always posed difficulties. The present study describes about a rapid single step purification method of WbCTI using an immunoaffinity column chromatography with the high yield of purified biologically active protein. WbCTI, a dual inhibitor of 18.6 kDa molecular weight, inhibits both trypsin and chymotrypsin in 1:1 molar ratio but does not form a ternary complex with the cognate proteases[3]
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