title: Expression and Purification of Matrix Metalloproteinases in Escherichia coli.
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.
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Krishna K
authors.initials: KK
authors.lastname: Singh
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Ruchi
authors.initials: R
authors.lastname: Jain
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India.
authors.forename: Harini
authors.initials: H
authors.lastname: Ramanan
authors.affiliation: Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India. deepak@mrdg.iisc.ernet.in.
authors.forename: Deepak K
authors.initials: DK
authors.lastname: Saini
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2
keywords: Affinity chromatography; Inclusion bodies; MMP2; Refolding; Zymography