Matrix-Assisted Refolding, Purification and Activity Assessment Using a `Form Invariant' Assay for Matrix Metalloproteinase 2 (MMP2)

Singh, Krishna Kumar and Jain, Ruchi and Ramanan, Harini and Saini, Deepak Kumar (2014) Matrix-Assisted Refolding, Purification and Activity Assessment Using a `Form Invariant' Assay for Matrix Metalloproteinase 2 (MMP2). In: MOLECULAR BIOTECHNOLOGY, 56 (12). pp. 1121-1132.

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Abstract

Matrix metalloproteinases expression is used as biomarker for various cancers and associated malignancies. Since these proteinases can cleave many intracellular proteins, overexpression tends to be toxic; hence, a challenge to purify them. To overcome these limitations, we designed a protocol where full length pro-MMP2 enzyme was overexpressed in E. coli as inclusion bodies and purified using 6xHis affinity chromatography under denaturing conditions. In one step, the enzyme was purified and refolded directly on the affinity matrix under redox conditions to obtain a bioactive protein. The pro-MMP2 protein was characterized by mass spectrometry, CD spectroscopy, zymography and activity analysis using a simple in-house developed `form invariant' assay, which reports the total MMP2 activity independent of its various forms. The methodology yielded higher yields of bioactive protein compared to other strategies reported till date, and we anticipate that using the protocol, other toxic proteins can also be overexpressed and purified from E. coli and subsequently refolded into active form using a one step renaturation protocol.

Item Type:	Journal Article
Publication:	MOLECULAR BIOTECHNOLOGY
Publisher:	HUMANA PRESS INC
Additional Information:	Copyright for this article belongs to the HUMANA PRESS INC, 999 RIVERVIEW DRIVE SUITE 208, TOTOWA, NJ 07512 USA
Keywords:	MMP (matrix metalloproteinase); Refolding; Zymography; Activity assay
Department/Centre:	Division of Biological Sciences > Molecular Reproduction, Development & Genetics
Date Deposited:	19 Dec 2014 07:40
Last Modified:	19 Dec 2014 07:40
URI:	http://eprints.iisc.ac.in/id/eprint/50461

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