title: The expression, refolding, and purification of the catalytic domain of human collagenase-3 (MMP-13).
abstract: We have cloned, expressed, and purified a recombinant C-terminal truncated form (residues Glu103-Asn274) of human collagenase-3 (MMP-13) in Escherichia coli. The molecule contains the catalytic domain of the enzyme and is expressed almost exclusively as inclusion bodies. Using a combination of rapid dilution and diafiltration, the enzyme has been successfully refolded from these inclusion bodies. The protein was purified to homogeneity using cation-exchange and size-exclusion chromatography. The purified enzyme is a monomer with a Mr of approximately 19,600 and was characterized using a variety of techniques including, SDS-PAGE, RP-HPLC, LC-MS, amino acid analysis, and dynamic light scattering. Microheterogeneity at the NH2-terminus of the refolded, purified protein disappeared after incubating for 30-60 min at 37 degreesC. The enzyme was highly active using a fluorescent peptide substrate and was found to release S-GAG from bovine nasal cartilage chips.
authors.affiliation: Core Technologies, Arthritis Biology, Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, New Jersey, 07901, USA.
authors.forename: N
authors.initials: N
authors.lastname: Pathak
authors.forename: S I
authors.initials: SI
authors.lastname: Hu
authors.forename: J A
authors.initials: JA
authors.lastname: Koehn
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017364:Collagenases / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies / Q000382:microbiology; D053509:Matrix Metalloproteinase 13; D008970:Molecular Weight; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13