Abstract
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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Acknowledgments
Financial assistance to DKS from Department of Science and Technology (EMR/2014/000997); Department of Biotechnology and Indian Institute of Science partnership program, and DST-FIST for equipment support and a Research Fellowship to RJ from University Grants Commission is acknowledged.
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Singh, K.K., Jain, R., Ramanan, H., Saini, D.K. (2017). Expression and Purification of Matrix Metalloproteinases in Escherichia coli . In: Galea, C. (eds) Matrix Metalloproteases. Methods in Molecular Biology, vol 1579. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6863-3_1
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DOI: https://doi.org/10.1007/978-1-4939-6863-3_1
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-6863-3
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