EXPRESSION AND MUTAGENESIS OF RECOMBINANT HUMAN AND MURINE ERYTHROPOIETINS IN ESCHERICHIA-COLI
EXPRESSION AND MUTAGENESIS OF RECOMBINANT HUMAN AND MURINE ERYTHROPOIETINS IN ESCHERICHIA-COLI
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DOI：
10.1016/0167-4781(94)00213-m
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发表时间：
1995-03-14
期刊：
影响因子：
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通讯作者：
LAPPIN, TRJ
中科院分区：
文献类型：
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作者：
BILL, RM;WINTER, PC;LAPPIN, TRJ
研究了四种细菌表达载体在大肠杆菌中对促红细胞生成素（EPO）的表达。利用聚合酶链反应（PCR）扩增编码人和鼠EPO的互补dna，并将其克隆到谷胱甘肽- s -转移酶（GST）融合载体pGEX-2T中。将人EPO DNA克隆到载体pET14b、pIN III-Omp A2和pT7/7中。利用pGEX-2T构建体获得人和小鼠EPO的表达。对于基于其他载体的构建，尽管尝试优化条件，EPO的表达还是缺失或低水平。人和鼠EPO以GST融合蛋白的形式表达，部分可溶，具有EPO生物活性。可溶性GST-EPO融合蛋白在固定化谷胱甘肽上亲和纯化。不溶性蛋白也可以通过SDS-PAGE凝胶片的洗脱纯化，得到融合蛋白或凝血酶处理后的未修饰的EPO，既可溶又具有生物活性。pGEX表达系统是通过体外诱变分析EPO结构-功能关系的一种手段。构建了3个人和3个小鼠EPO突变体，并将其表达为GST融合蛋白。纯化后，用生物活性、免疫活性和GST活性测定法评价生物活性。pGEX表达系统补充了之前描述的EPO表达的真核系统，并应提供有关激素结构-功能关系的许多有用信息。
Expression of the polypeptide hormone erythropoietin (EPO) in Escherichia coli by four bacterial expression vectors was examined. Complementary DNAs encoding human and murine EPO were amplified by polymerase chain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T. Human EPO DNA was also cloned into the vectors, pET14b, pIN III-Omp A2 and pT7/7. Expression of human and murine EPO was obtained using constructs based on pGEX-2T. For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions. Human and murine EPO, expressed as fusion proteins with GST, were partially soluble and displayed EPO bioactivity. Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione. Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO which was both soluble and bioactive. The pGEX expression system was evaluated as a means of analysing the structure-function relationship of EPO by in vitro mutagenesis. Three human and three murine EPO mutants were constructed and expressed as GST fusion proteins. Following purification, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity. The pGEX expression system complements eukaryotic systems described previously for expression of EPO and should provide much useful information about the structure-function relationships of the hormone.