Cloning, expression, purification, and characterization of rat MMP-12.

Abstract

Macrophage metalloelastase (MMP-12) is implicated in the pathology of many diseases such as emphysema, aortic lesions and cancer. Recently, MMP-12 was cloned and purified from mouse and human macrophages. We report here the expression of the full-length and catalytic domain of rat MMP-12 in Escherichia coli and characterization of the purified enzyme. Inclusion bodies of expressed rat MMP-12 catalytic domain were denatured and refolded using a new method, and then affinity purified to near homogeneity with zinc-chelating Sepharose. The purified rat MMP-12 catalytic domain was highly active in digesting substrates, having a K(m) of 12 microM and optimal pH of 7.5--8.5. During investigation of natural substrate specificity, we found that rat MMP-12 catalytic domain was able to completely degrade collagen-V, partially degrade collagen-I, but it was unable to digest collagen-IV. The enzyme could also degrade osteonectin, vitronectin, and fibronectin, but not laminin and albumin. The catalytic properties and natural substrate specificity of rat MMP-12 catalytic domain differed from those of human MMP-12 catalytic domain.

• Authors: J Y Fu, A Lyga, H Shi, M L Blue, B Dixon, D Chen
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 21 Issue 2 Pg. 268-74 (Mar 2001) ISSN: 1046-5928 [Print] United States
• PMID: 11237688 (Publication Type: Journal Article)
• Copyright: Copyright 2001 Academic Press.

Chemical References

• Extracellular Matrix Proteins
• RNA, Messenger
• Recombinant Proteins
• Metalloendopeptidases
• MMP12 protein, human
• Matrix Metalloproteinase 12
• Zinc

Topics

• Animals
• Catalytic Domain
• Cell Line
• Chromatography, Affinity
• Cloning, Molecular
• Extracellular Matrix Proteins (metabolism)
• Gene Expression Profiling
• Humans
• Hydrogen-Ion Concentration
• Kinetics
• Macrophages (enzymology)
• Matrix Metalloproteinase 12
• Metalloendopeptidases (genetics, isolation & purification, metabolism)
• Protein Renaturation
• RNA, Messenger (analysis, genetics)
• Rats
• Recombinant Proteins (isolation & purification, metabolism)
• Substrate Specificity
• Zinc (metabolism)