Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization.
Purification and refolding of recombinant human proMMP-7 (pro-matrilysin) expressed in Escherichia coli and its characterization.
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在大肠杆菌中表达的重组人 proMMP-7（pro-matrilysin）的纯化和重折叠及其表征。
DOI：
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发表时间：
1996
期刊：
影响因子：
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通讯作者：
M. Seiki
中科院分区：
文献类型：
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作者：
M. Itoh;K. Masuda;Y. Ito;T. Akizawa;M. Yoshioka;K. Imai;Y. Okada;H. Sato;M. Seiki
人基质金属蛋白酶-7 （MMP-7 =基质溶酶）作为重组酶原（31 kDa）在大肠杆菌中过量产生，其c端含有人工六组氨酸。大部分酶是从细胞裂解物的不溶性部分分离出来的，用8 M尿素溶液溶解沉淀后，用Ni-NTA树脂一步纯化。将树脂结合的重组蛋白重新折叠成可被对氨基苯基醋酸汞以自催化方式激活的形式。活化酶在报道的MMP-7位点切割合成肽底物。重组蛋白酶消化羧甲基化转铁蛋白（MMP-7的天然底物）产生的片段与天然纯化的MMP-7具有相同的肽图。自催化活化和酶反应完全依赖于钙和锌离子的存在。切割羧甲基转铁蛋白的酶活性被金属蛋白酶组织抑制剂-1和-2 （mmp特异性抑制剂）抑制。重组MMP-7的活性也被半胱氨酸开关部分合成的肽抑制，该开关维持酶原处于无活性状态。因此，我们在这里报道了一种制备大量重组proMMP-7的简单方法，可用于研究激活机制和筛选合成抑制剂。
Human matrix metalloproteinase-7 (MMP-7 = matrilysin) was overproduced in Escherichia coli as a recombinant zymogen (31 kDa), the C-terminus of which bears artificial hexa-histidines. Most of the enzyme was isolated from the insoluble fraction of the cell lysate and purified by a single step using Ni-NTA resin after solubilization of the precipitates with 8 M urea solution. The resin-bound recombinant protein was refolded into a form that is activatable by p-amino-phenylmercuric acetate in an autocatalytic manner. The activated enzyme cleaved a synthetic peptide substrate at the reported site for MMP-7. Digestion of carboxymethylated transferrin (a natural substrate of MMP-7) by the recombinant proteinase generated fragments with the same peptide map as in the case of native purified MMP-7. The autocatalytic activation and enzyme reaction were entirely dependent on the presence of calcium and zinc ions. The enzyme activity to cleave carboxymethylated transferrin was inhibited by tissue inhibitors of metalloproteinases-1 and -2, MMP-specific inhibitors. The activity of the recombinant MMP-7 was also inhibited by a synthetic peptide derived from a part of the cysteine switch that maintains the zymogen in an inactive state. Thus, we report here a simple means of preparing a large quantity of recombinant proMMP-7 that can be used to study the activation mechanism and to screen synthetic inhibitors.