Guan K L, Dixon J E
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907.
Anal Biochem. 1991 Feb 1;192(2):262-7. doi: 10.1016/0003-2697(91)90534-z.
Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.
已经开发了几种系统，以实现对细菌中表达的重组蛋白的快速高效纯化。与谷胱甘肽S-转移酶（GST）读框内表达多肽，可通过在谷胱甘肽琼脂糖上进行亲和层析，在非变性条件下从细菌粗提物中纯化融合蛋白（D.B.史密斯和K.S.约翰逊，1988年，《基因》67卷，31 - 40页）。该载体表达系统还引入了特定的蛋白酶切割位点，以促进细菌融合蛋白的蛋白水解。在我们的实验中，这些融合蛋白在凝血酶切割位点的切割进行得很慢。为了促进融合蛋白的切割，我们引入了一个富含甘氨酸的接头（甘氨酸扭结），其序列为P.G.I.S.G.G.G.G.G，紧接在凝血酶切割位点之后。这个甘氨酸扭结大大提高了几种融合蛋白的凝血酶切割效率。将甘氨酸扭结引入融合蛋白中，可使融合蛋白在附着于亲和树脂时被切割，从而实现重组蛋白的一步纯化。当使用蛋白酪氨酸磷酸酶作为测试蛋白时，在几小时内从相当于100毫升细菌培养物中获得了超过2毫克的高度纯化蛋白。载体pGEX-KG也经过了修饰，以利于在所有读框中克隆多种cDNA，并已成功用于表达几种真核蛋白。
