EUKARYOTIC PROTEINS EXPRESSED IN ESCHERICHIA-COLI - AN IMPROVED THROMBIN CLEAVAGE AND PURIFICATION PROCEDURE OF FUSION PROTEINS WITH GLUTATHIONE-S-TRANSFERASE
EUKARYOTIC PROTEINS EXPRESSED IN ESCHERICHIA-COLI - AN IMPROVED THROMBIN CLEAVAGE AND PURIFICATION PROCEDURE OF FUSION PROTEINS WITH GLUTATHIONE-S-TRANSFERASE
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DOI：
10.1016/0003-2697(91)90534-z
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发表时间：
1991-02-01
影响因子：
2.9
通讯作者：
DIXON, JE
中科院分区：
文献类型：
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作者：
GUAN, KL;DIXON, JE
已经开发了几个系统来允许快速和有效地纯化在细菌中表达的重组蛋白。在含有谷胱甘肽S转移酶(GST)的框架内表达多肽，可以通过谷胱甘肽琼脂糖亲和层析从非变性条件下的细菌粗提物中纯化融合蛋白(D.B.Smith和K.S.Johnson，1988，Gene67，31-40)。该载体表达系统还加入了特定的蛋白酶裂解位点，以促进细菌融合蛋白的蛋白分解。在我们手中，这些融合蛋白在凝血酶裂解部位的裂解进行得很慢。为了促进融合蛋白的切割，我们引入了一个富含甘氨酸的连接体(甘氨酸连接蛋白)，它包含紧接在凝血酶切割位点之后的序列P·G·I·S·G·G。这种甘氨酸激活剂极大地提高了几种融合蛋白的凝血酶切割效率。将甘氨酸激活剂引入融合蛋白，可以在融合蛋白连接到亲和树脂上时切割融合蛋白，从而实现重组蛋白的一步纯化。当使用蛋白质酪氨酸磷酸酶作为测试蛋白质时，在几小时内从相当于100毫升的细菌培养物中获得了超过2毫克的高纯度蛋白质。载体pGEX-KG也进行了修饰，以便于在所有阅读框架中克隆各种cDNA，并已成功地用于表达几种真核蛋白。
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, Gene67, 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.