Bill R M, Winter P C, McHale C M, Hodges V M, Elder G E, Caley J, Flitsch S L, Bicknell R, Lappin T R
Dyson Perrins Laboratory, University of Oxford, UK.
Biochim Biophys Acta. 1995 Mar 14;1261(1):35-43. doi: 10.1016/0167-4781(94)00213-m.
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 relationships 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.
利用四种细菌表达载体检测了多肽激素促红细胞生成素（EPO）在大肠杆菌中的表达。通过聚合酶链反应（PCR）扩增编码人和鼠EPO的互补DNA，并克隆到谷胱甘肽-S-转移酶（GST）融合载体pGEX-2T中。人EPO DNA也被克隆到载体pET14b、pIN III-Omp A2和pT7/7中。使用基于pGEX-2T的构建体获得了人和鼠EPO的表达。对于基于其他载体的构建体，尽管尝试优化条件，但EPO的表达不存在或水平很低。作为与GST的融合蛋白表达的人和鼠EPO部分可溶，并显示出EPO生物活性。可溶性GST-EPO融合蛋白在固定化谷胱甘肽上进行亲和纯化。不溶性蛋白也可以通过SDS-PAGE后从凝胶切片上洗脱来纯化，以产生融合蛋白，或者在用凝血酶处理后产生既可溶又具有生物活性的未修饰EPO。评估了pGEX表达系统作为通过体外诱变分析EPO结构-功能关系的一种手段。构建了三个人和三个鼠EPO突变体，并作为GST融合蛋白表达。纯化后，使用生物活性、免疫活性和GST活性测定法评估生物活性。pGEX表达系统补充了先前描述的用于EPO表达的真核系统，应该能提供许多关于该激素结构-功能关系的有用信息。
