Hirabayashi J, Kasai K
Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
J Mol Recognit. 1990 Oct-Dec;3(5-6):204-7. doi: 10.1002/jmr.300030506.
A characteristic property of anhydrotrypsin, i.e., its ability to strongly bind C-terminal arginine, proved to be useful as a tool for specific enrichment of a recombinant protein. An arginine tail was introduced at the C-terminus, for example, of a human beta-galactoside-binding lectin by site-directed mutagenesis. The resulting mutant recombinant lectin, which retained sugar-binding activity as high as the wild-type lectin, became recognizable by anhydrotrypsin. It was adsorbed on an anhydrotrypsin-agarose column and eluted with benzoylglycylarginine. The added arginine tail could be specifically removed by carboxypeptidase B. When E. coli lyzate containing the mutant lectin was applied to the column more than 10-fold enrichment of the mutant lectin was attained. This procedure should be generally applicable and may be advantageous because the addition of a single arginine residue may have minimal effect on the structure and function of the target protein.
脱水胰蛋白酶的一个特性，即其与C端精氨酸强烈结合的能力，被证明是一种用于特异性富集重组蛋白的有用工具。例如，通过定点诱变在人β-半乳糖苷结合凝集素的C端引入精氨酸尾。所得的突变重组凝集素保留了与野生型凝集素一样高的糖结合活性，能被脱水胰蛋白酶识别。它被吸附在脱水胰蛋白酶-琼脂糖柱上，并用苯甲酰甘氨酰精氨酸洗脱。添加的精氨酸尾可以被羧肽酶B特异性去除。当将含有突变凝集素的大肠杆菌裂解物应用于该柱时，突变凝集素的富集倍数超过了10倍。该方法应具有普遍适用性，且可能具有优势，因为添加单个精氨酸残基对靶蛋白的结构和功能可能影响最小。
