Functional human insulin-degrading enzyme can be expressed in bacteria
Functional human insulin-degrading enzyme can be expressed in bacteria
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DOI：
10.1006/prep.2000.1217
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发表时间：
2000-06-01
影响因子：
1.6
通讯作者：
Rosner, MR
中科院分区：
文献类型：
--
作者：
Chesneau, V;Rosner, MR
胰岛素降解酶(IDE)已被证明可以降解许多重要的生物多肽，包括胰岛素和与阿尔茨海默病有关的淀粉样β蛋白。然而，由于缺乏一种简便的方法来产生纯化的酶和相关的突变体，因此很难研究IDE在清除这些多肽中的确切作用，因此，我们确定了重组野生型和突变型人IDE是否可以作为功能性酶在细菌中过表达。构建了3个携带N端多组氨酸标记的重组IDES编码基因的载体，并用金属亲和层析纯化了在大肠杆菌中表达的蛋白(最终产量约为8 mg/L培养物)。重组IDES与内源性哺乳动物酶一样，迁移时的表观分子质量为110 kDa。十二烷基硫酸钠-聚丙烯酰胺凝胶，在凝胶过滤中约为200 kDa。Native PAGE进一步分析表明，IDE可以形成不同复杂程度的多聚体。野生型重组内肽酶降解胰岛素，其效率与从哺乳动物组织中纯化的酶相似。纯化的IDE在4摄氏度下至少稳定1个月。纯化的重组蛋白被用来产生免疫沉淀天然哺乳动物IDE的特异性多克隆抗体，因此，所描述的方法允许快速生产大量纯化的IDE，并证明在没有其他潜在相互作用的哺乳动物蛋白的情况下，IDE可以以活性形式产生。(C)2000年学术出版社。
Insulin-degrading enzyme (IDE) has been shown to degrade a number of biologically important peptides, including insulin and the amyloid-beta protein implicated in Alzheimer's disease. However, lack of a facile method to generate purified enzyme and related mutants has made it difficult to study the precise role of IDE in the clearance of these peptides, Therefore, we determined whether recombinant wild-type and mutant human IDEs can be overexpressed as functional enzymes in bacteria. Three vectors carrying cDNAs encoding N-terminally polyhistidine-tagged recombinant IDEs were constructed, and the proteins expressed in Escherichia coli were purified by metal affinity chromatography (final yield approximate to 8 mg per liter of culture). The recombinant IDEs, Like the endogenous mammalian enzyme, migrate with 110-kDa apparent molecular masses in. SDS-polyacrylamide gels and as a approximate to 200-kDa species in gel filtration. Further analysis by native PAGE indicates that IDE can form multimers of different complexities. The wild-type recombinant endopeptidase degrades insulin, with an efficiency similar to that of the enzyme purified from mammalian tissues. Purified IDEs are stable at 4 degrees C for at least 1 month. Purified recombinant protein, was used to raise specific polyclonal antibodies that can immunoprecipitate native mammalian IDE, Thus, the procedure described allows the rapid production of large amounts of purified IDE and demonstrates that IDE can be produced in an active form in the absence of other potential interacting mammalian proteins. (C) 2000 Academic Press.