title: Matrix-assisted refolding, purification and activity assessment using a 'form invariant' assay for matrix metalloproteinase 2 (MMP2).
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.
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Krishna Kumar
authors.initials: KK
authors.lastname: Singh
authors.forename: Ruchi
authors.initials: R
authors.lastname: Jain
authors.forename: Harini
authors.initials: H
authors.lastname: Ramanan
authors.forename: Deepak Kumar
authors.initials: DK
authors.lastname: Saini
mesh_terms: D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding
chemical_list: D004792:Enzyme Precursors; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2