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.
The MMP (matrix metalloproteinases) family of endopeptidases are involved in cleavage induced remodelling of the extracellular matrix including collagen, fibrinogen, elastin, and gelatin. Owing to their proteolytic activity which can cleave and degrade multiple intracellular substrates, the overexpression and purification of these proteins tends to be toxic. Here we describe a novel "matrix assisted refolding" protocol to overcome the technical challenges associated with overexpression and purification of full-length MMPs. The toxicity issue associated with MMP expression, is circumvented by expressing the recombinant protein in Escherichia coli in an inactive insoluble form. The methodology used for obtaining full-length MMP2 protein from these inclusion bodies, by its subsequent purification and refolding using affinity chromatography, through a single-step matrix based refolding protocol is presented here. The protocol described yields high concentrations of pure full-length and active MMP2 protein useful for downstream applications.
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