Biomaterial Database

Expression and Purification of Matrix Metalloproteinases in Escherichia coli. 2017

Krishna K Singh, and Ruchi Jain, and Harini Ramanan, and Deepak K Saini

Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, 560012, India.

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.

UI	MeSH Term	Description	Entries
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002846	Chromatography, Affinity	A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed)	Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006801	Humans	Members of the species Homo sapiens.	Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D015202	Protein Engineering	Procedures by which protein structure and function are changed or created in vitro by altering existing or synthesizing new structural genes that direct the synthesis of proteins with sought-after properties. Such procedures may include the design of MOLECULAR MODELS of proteins using COMPUTER GRAPHICS or other molecular modeling techniques; site-specific mutagenesis (MUTAGENESIS, SITE-SPECIFIC) of existing genes; and DIRECTED MOLECULAR EVOLUTION techniques to create new genes.	Genetic Engineering of Proteins,Genetic Engineering, Protein,Proteins, Genetic Engineering,Engineering, Protein,Engineering, Protein Genetic,Protein Genetic Engineering
D015870	Gene Expression	The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION.	Expression, Gene,Expressions, Gene,Gene Expressions
D017510	Protein Folding	Processes involved in the formation of TERTIARY PROTEIN STRUCTURE.	Protein Folding, Globular,Folding, Globular Protein,Folding, Protein,Foldings, Globular Protein,Foldings, Protein,Globular Protein Folding,Globular Protein Foldings,Protein Foldings,Protein Foldings, Globular
D057809	HEK293 Cells	A cell line generated from human embryonic kidney cells that were transformed with human adenovirus type 5.	293T Cells,HEK 293 Cell Line,HEK 293 Cells,Human Embryonic Kidney Cell Line 293,Human Kidney Cell Line 293,293 Cell, HEK,293 Cells, HEK,293T Cell,Cell, 293T,Cell, HEK 293,Cell, HEK293,Cells, 293T,Cells, HEK 293,Cells, HEK293,HEK 293 Cell,HEK293 Cell
D020778	Matrix Metalloproteinase 2	A secreted endopeptidase homologous with INTERSTITIAL COLLAGENASE, but which possesses an additional fibronectin-like domain.	Gelatinase A,72-kDa Gelatinase,72-kDa Type IV Collagenase,MMP-2 Metalloproteinase,MMP2 Metalloproteinase,Matrix Metalloproteinase-2,72 kDa Gelatinase,72 kDa Type IV Collagenase,Gelatinase, 72-kDa,MMP 2 Metalloproteinase,Metalloproteinase 2, Matrix,Metalloproteinase, MMP-2,Metalloproteinase, MMP2