Purification of matrix metalloproteinases by column chromatography
Purification of matrix metalloproteinases by column chromatography
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DOI：
10.1038/nprot.2008.74
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发表时间：
2008-01-01
期刊：
影响因子：
14.8
通讯作者：
Okada, Yasunori
中科院分区：
文献类型：
--
作者：
Imai, Kazushi;Okada, Yasunori
基质金属蛋白酶(MMPs)是由23个成员组成的锌内肽酶，属于梅津菌素超家族的一个亚家族。它们通过降解细胞外基质(ECM)和非ECM蛋白并与多种分子相互作用，在许多病理生理事件中发挥重要作用，包括发育、器官发生、血管生成、组织重塑和破坏，以及癌细胞的增殖和进展。在这里，我们提出了使用常规柱层析纯化天然proMMPs(proMMP1、-2、-3、-7、-9和-10)和重组MT1-MMPs(MMP14)的标准方法。纯化步骤包括第一步[二乙氨基乙基(DEAE)-纤维素柱，Green A Dyematrex凝胶和明胶Sepharose柱]，第二步用免疫亲和柱(抗-MMP1和/或抗MMP3Ig-Sepharose柱)去除非靶向proMMPs，最后一步进一步纯化(Ig-Sepharose柱，DEAE-纤维素柱，Zn2+-螯合-Sepharose柱和/或凝胶过滤柱)。纯化的ProMMPs和MMPs具有功能活性，适合生化分析。从培养基中提纯的基本程序类似于7-10天。
Matrix metalloproteinases (MMPs) are zinc endopeptidases composed of 23 members in humans, which belong to a subfamily of the metzincin superfamily. They play important roles in many pathophysiological events including development, organogenesis, angiogenesis, tissue remodeling and destruction, and cancer cell proliferation and progression by degradation of extracellular matrix (ECM) and non-ECM proteins and interaction with various molecules. Here, we present standard protocols for purification of native proMMPs (proMMP-1, -2, -3, -7, -9 and -10) and recombinant MT1-MMP (MMP-14) using conventional column chromatography. Purification steps comprise the initial common step [diethylaminoethyl (DEAE)-cellulose, Green A Dyematrex gel and gelatin Sepharose columns], the second step for removal of nontarget proMMPs by immunoaffinity columns (anti-MMP-1 and/or anti-MMP-3 IgG-Sepharose columns) and the final step for further purification (IgG-Sepharose, DEAE-cellulose, Zn2+-chelate-Sepharose and/or gel filtration columns). Purified proMMPs and MMP are functionally active and suitable for biochemical analyses. The basic protocol for the purification from culture media takes similar to 7-10 d.