Cloning, expression, purification, and characterization of rat MMP-12.
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.
Department of Cancer and Osteoporosis Research, Bayer Corporation, West Haven, Connecticut 06516, USA.
J Y
JY
Fu
A
A
Lyga
H
H
Shi
M L
ML
Blue
B
B
Dixon
D
D
Chen
D000818:Animals; D020134:Catalytic Domain; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D020869:Gene Expression Profiling; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D053508:Matrix Metalloproteinase 12; D008666:Metalloendopeptidases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D020673:Protein Renaturation; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
D016326:Extracellular Matrix Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D008666:Metalloendopeptidases; C507122:MMP12 protein, human; D053508:Matrix Metalloproteinase 12; D015032:Zinc