期刊
PHYTOCHEMISTRY
卷 70, 期 4, 页码 465-472出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.phytochem.2009.02.001
关键词
Propapain; Cysteine protease; High yield expression; Inclusion body; Refolding; Activation
资金
- Council for Scientific and Industrial Research [21/0653/06/EMR-II]
- Department of Science and Technology [SR/WOS-A/LS-161/2004]
- Department of Biotechnology, Government of India [BT/PRO0139/RD/15/11/96]
Papain (EC 3.4.22.2), the archetypal cysteine protease of C1 family, is of considerable commercial significance. In order to obtain substantial quantities of active papain, the DNA coding for propapain, the papain precursor, has been cloned and expressed at a high level in Escherichia coli BL21(DE3) transformed with two T7 promoter based pET expression vectors - pET30 Ek/LIC and pET28a(+) each containing the propapain gene. In both cases, recombinant propapain was expressed as an insoluble Histagged fusion protein, which was solubilized, and purified by nickel chelation affinity chromatography under denaturing conditions. By systematic variation of parameters influencing the folding, disulfide bond formation and prevention of aggregate formation, a straightforward refolding procedure, based on dilution method, has been designed. This refolded protein was subjected to size exclusion chromatography to remove impurities and around 400 mg of properly refolded propapain was obtained from 1 L of bacterial culture. The expressed protein was further verified by Western blot analysis by crossreacting it with a polyclonal anti-papain antibody and the proteolytic activity was confirmed by gelatin SDS-PAGE. This refolded propapain could be converted to mature active papain by autocatalytic processing at low pH and the recombinant papain so obtained has a specific activity closely similar to the native papain. This is a simple and efficient expression and purification procedure to obtain a yield of active papain, which is the highest reported so far for any recombinant plant cysteine protease. (C) 2009 Elsevier Ltd. All rights reserved.
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