Matrix Metalloproteinase 13 (MMP-13), His-tagged
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Human, recombinant matrix metalloproteinase is expressed in Sf9-insect cells using the baculovirus expression vector system and purified from cell culture supernatant. The protein contains in addition to the 452 amino acids of full length procollagenase-3 a C-terminal His-tag. The resulting Mr is 52.520 Da. Due to N-linked glycosylation the proenzyme appears as a band of about 60.000 Da in SDS-PAGE. Procollagenase-3 is solublized in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM CaCl2, 0.05% Brij-35. Upon incubation with APMA active collagenase-3 with an apparent Mr of 48.000 Da is formed.
Overview
Matrix metalloproteinases (MMP) are Zn2+- and Ca2+- dependent endopeptidases [1]. Main subfamilies of MMP are collagenases, gelatinases, stromelysins and membrane-type matrix metalloproteinases [2]. Three homologous collagenases have been identified in human tissues: interstitial collagenase, neutrophil collagenase and collagenase-3. The three enzymes cleave fibrillar collagens at a single site, generating fragments of approximately ¾ and ¼ the size of the original molecules.
Procollagenase-3 consists of 452 amino acids with a calculated Mr of 51.680 Da [3]. Due to N-linked glycosylation the apparent Mr is about 60.000 Da [4]. Within the protein the following domains and sequence regions can be distinguished [3, 4]: an N-terminal propeptide, which confers latency to the proenzyme, a Ca2+- and Zn2+- ions binding catalytic domain, a hinge region, and a C-terminal hemopexin-like domain.
Procollagenase-3 can be activated by proteases as stromelysin [4], gelatinase A, membranetype 1 matrix metalloproteinase and plasmin [5] or by incubation with organomercurials (e.g. APMA) [4].
Active collagenase-3 begins with the N-terminal sequence YNVFPRTL [4]. Collagenase-3 hydrolyzes type II collagen 5- to 6-times faster than type I and type III collagens. The enzyme exhibits also high activity towards gelatin and it degrades SERPINS as 1-antichymotrypsin and plaminogen activator inhibitor-2 [4]. Collagenase-3 is inhibited in a 1:1 stoichiometric fashion by TIMP-1, TIMP-2 and TIMP-3. Collagenase-3 is expressed during fetal bone development [6]. In adult human tissues collagenase-3 has been detected only in pathological conditions: in malignant tumors [3], in chronic ulcers [7], in arthritic cartilage [8] and synovium [9].
Data/Specifications
Purity: Recombinant procollagenase-3 appears as a single protein band of about 60.000 Da in SDS-PAGE (> 95% of total protein). Due to autoproteolytic activity minor bands of activated collagenase-3 may be visible in the enzyme preparation.
Specific Activity: The specific activity of collagenase-3 is ≥ 200 mU/mg. 1 U is the activity that hydrolyzes 1 mol peptide (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-(3-[2, 4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg) within 1 min under the assay conditions described below.
Inhibitors: Collagenase-3 is inhibited by tissue inhibitors of matrix metalloproteinases (TIMP) and by chelators of divalent cations as EDTA or o- phenanthroline.
Stability & Storage: Recombinant procollagenase-3 table until the expiry date given on the label if stored at -70 °C. The proenzyme can be kept at -20 °C for several weeks. Repeated freezing and thawing should be avoided.
Applications: Recombinant procollagenase-3 can serve as antigen standard in immunochemical analyses. The active enzyme may be used to study the degradation of extracellular matrix proteins, to screen inhibitors of matrix metalloproteinases and to characterize inhibitor actions.