SINGLE-STEP PURIFICATION OF POLYPEPTIDES EXPRESSED IN ESCHERICHIA-COLI AS FUSIONS WITH GLUTATHIONE S-TRANSFERASE
SINGLE-STEP PURIFICATION OF POLYPEPTIDES EXPRESSED IN ESCHERICHIA-COLI AS FUSIONS WITH GLUTATHIONE S-TRANSFERASE
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DOI：
10.1016/0378-1119(88)90005-4
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发表时间：
1988-07-15
期刊：
影响因子：
3.5
通讯作者：
JOHNSON, KS
中科院分区：
文献类型：
--
作者：
SMITH, DB;JOHNSON, KS
已经构建了质粒表达载体，其指导外源多肽在大肠杆菌中作为与Sj 26的C末端的融合物的合成，Sj 26是由寄生蠕虫日本血吸虫编码的26-kDa谷胱甘肽S-转移酶（GST; EC 2.5.1.18）。在大多数情况下，融合蛋白可溶于水溶液中，并且可以在非变性条件下通过在固定化谷胱甘肽上的亲和色谱法从粗细菌裂解物中纯化。使用分批洗涤程序，几种融合蛋白可以在2小时内平行纯化，产率高达15 μ g蛋白/ml培养物。载体已经被工程化，使得GST载体可以通过用位点特异性蛋白酶如凝血酶或凝血因子Xa消化而从融合蛋白切割，随后，载体和任何未切割的融合蛋白可以通过在谷胱甘肽-琼脂糖上吸收而去除。该系统已成功用于30多种不同真核多肽的表达和纯化。
Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum. In the majority of cases, fusion proteins are soluble in aqueous solutions and can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilised glutathione. Using batch wash procedures several fusion proteins can be purified in parallel in under 2 h with yields of up to 15 .mu.g protein/ml of culture. The vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin or blood coagulation factor Xa, following which, the carrier and any uncleaved fusion protein can be removed by absporption on glutathione-agarose. This system has been used successfully for the expression and purification of more than 30 different eukaryotic polypeptides.