Over-expression of a functionally active human GM2-activator protein in Escherichia coli.
Over-expression of a functionally active human GM2-activator protein in Escherichia coli.
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功能活性人类 GM2 激活蛋白在大肠杆菌中的过度表达。
DOI：
10.1042/bj2920571
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Sandhoff,K
中科院分区：
文献类型：
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作者：
Klima,H;Klein,A;vanEchten,G;Schwarzmann,G;Suzuki,K;Sandhoff,K
将人gm2激活蛋白cDNA克隆到表达载体pHX17中。该质粒编码一种融合蛋白，其n端有一条六组氨酸尾部和一个因子Xa切割位点。利用金属离子亲和层析法从变性条件下的细胞匀浆中一步纯化重组蛋白，然后进行折叠。六组氨酸尾部可在需要时用Xa因子消化去除。在功能试验中，从大肠杆菌中生成的gm2激活蛋白经过再生，无论是否含有六组氨酸尾部，其活性都与从人体组织中纯化的gm2激活蛋白一样。当添加到培养基中时，携带六组氨酸尾部的重组无糖GM2激活剂可以有效地吸收，并使GM2-神经节脂病AB型突变成纤维细胞的神经节脂苷GM2降解率恢复到正常水平，GM2-神经节脂病的特征是GM2激活剂蛋白的遗传缺陷。原核表达系统可用于生产毫克量的纯功能活性gm2激活剂。
The cDNA of the human GM2-activator protein was cloned into the expression vector pHX17. The plasmid encodes a fusion protein with a hexahistidine tail and a Factor Xa cleavage site at its N-terminus. The recombinant protein was purified from cell homogenates under denaturing conditions by metal-ion affinity chromatography in a single step and then was refolded. The hexahistidine tail could be removed when desired by digestion with Factor Xa. In a functional assay, the GM2-activator thus generated from Escherichia coli and renatured, with or without the hexahistidine tail, was as active as the native GM2-activator protein that was purified from human tissue. When added to the culture medium, the recombinant carbohydrate-free GM2-activator, carrying the hexahistidine tail, could be taken up efficiently and restored the degradation of ganglioside GM2 to normal rates in mutant fibroblasts with the AB variant of GM2-gangliosidosis, which is characterized by a genetic defect in the GM2-activator protein. The prokaryotic expression system is useful for producing milligram quantities of a pure and functionally active GM2-activator.