Fu J Y, Lyga A, Shi H, Blue M L, Dixon B, Chen D
Department of Cancer and Osteoporosis Research, Bayer Corporation, West Haven, Connecticut 06516, USA.
Protein Expr Purif. 2001 Mar;21(2):268-74. doi: 10.1006/prep.2000.1376.
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.
巨噬细胞金属弹性蛋白酶（MMP - 12）与许多疾病的病理过程有关，如肺气肿、主动脉病变和癌症。最近，已从小鼠和人巨噬细胞中克隆并纯化出MMP - 12。我们在此报告大鼠MMP - 12全长和催化结构域在大肠杆菌中的表达以及纯化酶的特性。用一种新方法对表达的大鼠MMP - 12催化结构域的包涵体进行变性和复性，然后用锌螯合琼脂糖亲和纯化至近乎同质。纯化的大鼠MMP - 12催化结构域在消化底物方面具有高活性，K(m)为12微摩尔，最适pH为7.5 - 8.5。在研究天然底物特异性时，我们发现大鼠MMP - 12催化结构域能够完全降解Ⅴ型胶原，部分降解Ⅰ型胶原，但不能消化Ⅳ型胶原。该酶还能降解骨连接蛋白、玻连蛋白和纤连蛋白，但不能降解层粘连蛋白和白蛋白。大鼠MMP - 12催化结构域的催化特性和天然底物特异性与人MMP - 12催化结构域不同。
