Frimpong K, Darnay B G, Rodwell V W
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153.
Protein Expr Purif. 1993 Aug;4(4):337-44. doi: 10.1006/prep.1993.1044.
When overexpressed in Escherichia coli, the catalytic domain of Syrian hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) is catalytically active, but exhibits major heterogeneity. This heterogeneity reflects deletion of about 60 aminoacyl residues from the C-terminus, presumably a result of proteolytic cleavage or premature termination of translation. With the intent of separating the intact and truncated proteins via immunoaffinity chromatography, we constructed the expression phagemid pKFT7-21. This construct encodes the catalytic domain of Syrian hamster HMG-CoA reductase with the C-terminal extension Glu-Glu-Phe, an epitope recognized by a specific antibody. Following overexpression, the modified catalytic domain RcatEEF had high catalytic activity and exhibited no heterogeneity. It therefore was possible to purify RcatEEF to over 95% homogeneity without resorting to immunoaffinity chromatography. The yield of homogeneous protein averaged 20-25 mg per liter of cells with a final specific activity of up to 40 mumol NADPH oxidized per minute per milligram. The EEF modification thus should prove useful for the purification of the catalytic domains of other eukaryotic HMG-CoA reductases which exhibit heterogeneity.
当叙利亚仓鼠3-羟基-3-甲基戊二酰辅酶A还原酶（HMG-CoA还原酶，EC 1.1.1.34）的催化结构域在大肠杆菌中过表达时，具有催化活性，但表现出主要的异质性。这种异质性反映了C末端约60个氨酰基残基的缺失，推测是蛋白水解切割或翻译提前终止的结果。为了通过免疫亲和色谱法分离完整和截短的蛋白质，我们构建了表达噬菌粒pKFT7-21。该构建体编码带有C末端延伸Glu-Glu-Phe的叙利亚仓鼠HMG-CoA还原酶的催化结构域，Glu-Glu-Phe是一种被特异性抗体识别的表位。过表达后，修饰后的催化结构域RcatEEF具有高催化活性且无异质性。因此，无需借助免疫亲和色谱法就有可能将RcatEEF纯化至超过95%的同质性。每升细胞中均一蛋白质的产量平均为20-25毫克，最终比活性高达每分钟每毫克氧化40微摩尔NADPH。因此，EEF修饰对于纯化表现出异质性的其他真核HMG-CoA还原酶的催化结构域应该是有用的。
