Barrett M P, Phillips C, Adams M J, Le Page R W
University of Cambridge, Department of Pathology, United Kingdom.
Protein Expr Purif. 1994 Feb;5(1):44-9. doi: 10.1006/prep.1994.1006.
The gene encoding 6-phosphogluconate dehydrogenase (EC 1.1.1.44) from Trypanosoma brucei was cloned into the overexpression vector pET3a which utilizes the T7 polymerase gene expression system. Up to 40% of total cell protein consisted of the trypanosome enzyme when expression was induced in Escherichia coli host strains at 28 degrees C. The enzyme was rapidly degraded at temperatures higher than 30 degrees C. The T. brucei enzyme was purified to near homogeneity (as judged by SDS-polyacrylamide gel electrophoresis) using a two-step purification method, involving a DE-52 cellulose batch preparation followed by 2' AMP-agarose affinity chromatography. The purified protein crystallized readily. A molecular model of the trypanosome enzyme based on its mammalian counterpart revealed differences between the two enzymes in residues involved in cofactor binding.
将布氏锥虫编码6-磷酸葡萄糖酸脱氢酶（EC 1.1.1.44）的基因克隆到利用T7聚合酶基因表达系统的过表达载体pET3a中。当在28℃的大肠杆菌宿主菌株中诱导表达时，高达40%的总细胞蛋白由锥虫酶组成。该酶在高于30℃的温度下迅速降解。使用两步纯化方法将布氏锥虫酶纯化至接近均一（通过SDS-聚丙烯酰胺凝胶电泳判断），该方法包括DE-52纤维素批量制备，然后进行2'-AMP-琼脂糖亲和色谱。纯化后的蛋白质很容易结晶。基于其哺乳动物对应物的锥虫酶分子模型揭示了两种酶在参与辅因子结合的残基上存在差异。
