Lilius G, Persson M, Bülow L, Mosbach K
Pure and Applied Biochemistry, Chemical Centre, University of Lund, Sweden.
Eur J Biochem. 1991 Jun 1;198(2):499-504. doi: 10.1111/j.1432-1033.1991.tb16042.x.
In this study, galactose dehydrogenase (EC 1.1.1.48) was chosen as a prototype target protein to investigate the capability of metal affinity precipitation to facilitate the purification of genetically engineered proteins. A DNA fragment encoding five histidine residues was fused to the 3'-terminal end of the galactose dehydrogenase gene from Pseudomonas fluorescens and thereafter expressed in Escherichia coli. The additional five histidines functioned as an affinity tail and the modified enzyme could be purified using metal affinity precipitation when the metal-chelate complex with ethylene glycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra-acetic acid, EGTA(Zn)2, was added to the protein solution. The affinity tail could also be applied for the purification of the fusion protein utilising immobilised metal affinity chromatography. After purification, the pentahistidine affinity tail could be removed enzymatically by carboxypeptidase A. Furthermore, growth rate experiments demonstrated that the expression of the metal-binding affinity tail in E. coli cells enhanced the tolerance to zinc ions when added to the growth medium.
在本研究中，选择半乳糖脱氢酶（EC 1.1.1.48）作为原型靶蛋白，以研究金属亲和沉淀促进基因工程蛋白纯化的能力。将编码五个组氨酸残基的DNA片段融合到荧光假单胞菌半乳糖脱氢酶基因的3'末端，然后在大肠杆菌中表达。额外的五个组氨酸作为亲和尾，当向蛋白质溶液中加入与乙二醇双（β-氨基乙醚）N，N，N'，N'-四乙酸形成的金属螯合物EGTA（Zn）2时，修饰后的酶可通过金属亲和沉淀进行纯化。亲和尾也可用于利用固定化金属亲和色谱法纯化融合蛋白。纯化后，可通过羧肽酶A酶促去除五组氨酸亲和尾。此外，生长速率实验表明，当向生长培养基中添加锌离子时，大肠杆菌细胞中金属结合亲和尾的表达增强了对锌离子的耐受性。
