Jiang F, Mannervik B
Department of Biochemistry, Uppsala University, Biomedical Center, Uppsala, S-751 23, Sweden.
Protein Expr Purif. 1999 Feb;15(1):92-8. doi: 10.1006/prep.1998.0986.
Glutathione reductase (GR) from the cyanobacterium Anabaena PCC 7120 was heterologously expressed in Escherichia coli SG5. Silent random mutations were introduced in the 5' region of DNA encoding the enzyme in order to generate a high-level expression clone. To maximize protein expression, the culture conditions were also optimized. In the high-level expression clones selected, E. coli-preferred codons were selectively used at certain positions. Under the optimal expression conditions, a yield of 17 mg recombinant protein per liter was obtained, which is about 10-fold higher than that of the wild-type enzyme. A hexahistidine tag was added at the C-terminal of the protein in order to allow IMAC affinity purification. This strategy simplified the purification process and provided a homogeneous enzyme for functional characterization. Anabaena GR uses NADPH as a coenzyme, like most of the GRs from other sources, but the KM values for NADPH and GSSG are higher than those of enzymes previously studied. The Anabaena enzyme also shows significant activity when NADH is used as a reductant.
来自蓝藻鱼腥藻PCC 7120的谷胱甘肽还原酶（GR）在大肠杆菌SG5中进行了异源表达。为了获得一个高水平表达克隆，在编码该酶的DNA的5'区域引入了沉默随机突变。为了使蛋白质表达最大化，还对培养条件进行了优化。在所选的高水平表达克隆中，在某些位置选择性地使用了大肠杆菌偏好的密码子。在最佳表达条件下，每升可获得17毫克重组蛋白，这比野生型酶的产量高约10倍。在蛋白质的C末端添加了一个六组氨酸标签，以便进行IMAC亲和纯化。该策略简化了纯化过程，并提供了一种用于功能表征的均一酶。与大多数其他来源的GR一样，鱼腥藻GR使用NADPH作为辅酶，但NADPH和GSSG的KM值高于先前研究的酶。当使用NADH作为还原剂时，鱼腥藻酶也显示出显著活性。
