Singh, Krishna K and Jain, Ruchi and Ramanan, Harini and Saini, Deepak K (2017) Expression and Purification of Matrix Metalloproteinases in Escherichia coli. [Book Chapter]
Full text not available from this repository.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.
| Item Type: | Book Chapter |
|---|---|
| Publisher: | Humana Press Inc. |
| Additional Information: | The Copyright of this article belongs to the Humana Press Inc. |
| Keywords: | Affinity chromatography; Inclusion bodies; MMP2; Refolding; Zymography; Chromatography, Affinity; Cloning, Molecular; Escherichia coli; Gene Expression; HEK293 Cells; Humans; Matrix Metalloproteinase 2; Protein Engineering; Protein Folding; Recombinant Proteins; complementary DNA; gelatinase A; gelatinase A; MMP2 protein, human; recombinant protein; affinity chromatography; bacterial strain; cell inclusion; cloning; DNA sequence; enzyme activity; enzyme purification; Escherichia coli; HEK293 cell line; nonhuman; protein expression; protein refolding; transformed cell; zymography; chemistry; Escherichia coli; gene expression; genetics; growth, development and aging; human; metabolism; molecular cloning; procedures; protein engineering; protein folding |
| Department/Centre: | Division of Biological Sciences > Molecular Reproduction, Development & Genetics |
| Date Deposited: | 27 May 2022 04:29 |
| Last Modified: | 31 May 2022 05:06 |
| URI: | https://eprints.iisc.ac.in/id/eprint/72566 |
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