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

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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.

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Acknowledgments

Financial assistance to DKS from Department of Science and Technology (DST); Junior Research Fellowship to RJ from UGC; Assistance from proteomics facility, IISc for mass spectrometry and Gajendra, Department of Biochemistry, IISc for CD spectroscopy is gratefully acknowledged.

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Authors and Affiliations

  1. Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India

    Krishna Kumar Singh, Ruchi Jain, Harini Ramanan & Deepak Kumar Saini

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  1. Krishna Kumar Singh
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  2. Ruchi Jain
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  3. Harini Ramanan
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  4. Deepak Kumar Saini
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Correspondence to Deepak Kumar Saini.

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Krishna Kumar Singh, Ruchi Jain, Harini Ramanan have contributed equally to this work.

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Singh, K.K., Jain, R., Ramanan, H. et al. Matrix-Assisted Refolding, Purification and Activity Assessment Using a ‘Form Invariant’ Assay for Matrix Metalloproteinase 2 (MMP2). Mol Biotechnol 56, 1121–1132 (2014). https://doi.org/10.1007/s12033-014-9792-7

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