Widersten M, Huang M, Mannervik B
Department of Biochemistry, Uppsala University, Sweden.
Protein Expr Purif. 1996 Jun;7(4):367-72. doi: 10.1006/prep.1996.0054.
An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.
已开发出一种用于大规模生产多态性人谷胱甘肽转移酶（GST）M1-1的表达克隆。在大肠杆菌中的异源表达每3升培养基可产生100毫克GST M1-1，与亲本表达构建体相比，产量提高了约10倍。该酶的过量生产取决于编码谷胱甘肽转移酶M1-1的DNA序列5'区域的密码子使用情况。通过在编码序列中选定的摆动位置进行随机沉默突变以及从随机突变体文库中进行克隆的免疫选择相结合，产生了高水平表达克隆。所使用的策略通常适用于重组蛋白的生产，前提是有合适的选择程序可用于鉴定所需的突变体。
