EXPRESSION, PURIFICATION AND CHARACTERIZATION OF CHITINASE CHIC PRECURSOR FROM Streptomyces peucetius
Journal Title: Journal of Experimental Biology and Agricultural Sciences - Year 2018, Vol 6, Issue 4
Abstract
Chitinase (ChiC) of Streptomyces peucetius produced as a precursor protein undergoes proteolytic cleavage to yield a 42 kDa protein. Specific activity and cellular location of the proteolytic cleavage is however unknown. In order to decipher the functional characteristics of the precursor, we amplified the coding region of chiC using polymerase chain reaction and cloned the chiC gene with its signal peptide in an E. coli expression vector pQE-30. Upon induction with IPTG, a protein of 66 kDa could be seen and this matched the theoretical molecular weight of full length ChiC chitinase precursor. The expressed protein was found localized in inclusion bodies, thus purification was performed under denaturing conditions using Ni-affinity chromatography. 66 kDa ChiC precursor protein purified under denaturing conditions was refolded to yield an active enzyme. Enzymatic activities of the precursor and secreted form were compared. Western analysis of the S. peucetius intracellular and secreted proteins identified the location of proteolytic cleavage.
Authors and Affiliations
Amrathlal Rabbind Singh, Chitra Thangavel, Kuppamuthu Dharmalingam
Keywords
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- EP ID EP592808
- DOI 10.18006/2018.6(4).668.676
- Views 98
- Downloads 0
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