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Functional Expression of Horsegram (Dolichos biflorus) Trypsin Inhibitor Using pTwin1 System

Rooparani, G.N. (2010) Functional Expression of Horsegram (Dolichos biflorus) Trypsin Inhibitor Using pTwin1 System. [Student Project Report]

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Abstract

This Dissertation / Report is the outcome of investigation carried out by the creator(s) / author(s) at the department/division of Central Food Technological Research Institute (CFTRI), Mysore mentioned below in this page.

Item Type: Student Project Report
Additional Information: Proteinase inhibitors are essential in nature to regulate the proteinases which catalyze many physiological and pathophysiological processes. Bowman–Birk Inhibitor (BBI) family is a group of small serine proteinase inhibitors found mainly in seeds of legumes. Much evidence shows that many different proteinase inhibitors in their pure forms suppress carcinogenesis. In the present study, we have used an intein-based expression system to express BBI of major horsegram-III (HGI-III) inhibitor. Genomic DNA was extracted from horsegram seed fine powder and the open reading frame of 228 bp BBI gene amplified. SapI and Pst I restriction site were engineered into the gene and cloned using directional cloning strategy into pTwin1 vector. The putative clone was analyzed by gel shift assay and PCR amplification. The DNA sequence of pTwin-HGI-III was determined by cycle sequencing using Big Dye Terminator chemistry .The pTwin1-HGI-III was expressed in E.coli B ER2566 strain. The fusion protein expressed as a soluble protein when induced with 0.4 mM IPTG when grown at 16°C. SDS-PAGE and densitometric analysis shows high level expression (50% of total protein) of the fusion protein. The trypsin inhibitory activity of the crude extract was 356 TIU/mg. The trypsin inhibition was further confirmed by gelatin embedded PAGE. The cleavage of HGI-III from its fusion partner was optimised at pH 6.5 The expressed HGI-III was purified by trypsin sepharose affinity chromatography. The specific activity of the purified protein was 3.3×10 5 trypsin inhibitor units /mg protein. Preliminary kinetic analysis indicated that rHGI-III was a potent competitive inhibitor of bovine trypsin. The Ki for trypsin inhibition was 2.66 x 10-9M.
Uncontrolled Keywords: horsegram-III; expression system; Protein proteinase inhibitor
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 29 Protein Chemistry
600 Technology > 08 Food technology > 22 Legumes-Pulses
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 14 Jul 2010 10:32
Last Modified: 28 Dec 2011 10:16
URI: http://ir.cftri.com/id/eprint/9557

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