Charge engineering of a protein domain to allow efficient ion-exchange recovery
Charge engineering of a protein domain to allow efficient ion-exchange recovery
复制标题
DOI：
10.1093/protein/13.10.703
复制
发表时间：
2000-10-01
期刊：
影响因子：
--
通讯作者：
Hober, S
中科院分区：
文献类型：
--
作者：
Gräslund, T;Lundin, G;Hober, S
我们已经创造了具有极端表面电荷的蛋白质结构域。这些突变的结构域允许在有利于选择性和有效捕获的条件下进行离子交换色谱，使用大肠杆菌作为宿主生物体。葡萄球菌蛋白A衍生结构域Z（Z（wt））被用作构建两个突变体Z（basic 1）和Z（basic 2）的支架，具有高的正表面电荷。远紫外圆二色性测量表明，它们具有与母体分子Z（wt）相当的二级结构含量。虽然工程化结构域的解链温度（Tm）低于野生型Z结构域的解链温度，但两种突变体都可以在大肠杆菌中成功地以胞内全长产物的形式产生。大肠杆菌中表达，并通过离子交换层析纯化至均一。对Z（碱性1）和Z（碱性2）进行的进一步研究表明，即使在pH值为9至11的范围内，它们也能够与阳离子交换剂结合。Z（basic 2）和酸性人血清白蛋白结合结构域（ABD），来自链球菌蛋白G之间的基因融合，也被构建。基因产物Z（basic 2）-ABD可以使用阳离子交换层析从全细胞裂解物纯化至大于90%的纯度。
We have created protein domains with extreme surface charge. These mutated domains allow for ion-exchange chromatography under conditions favourable for selective and efficient capture, using Escherichia coli as a host organism. The staphylococcal protein A-derived domain Z (Z(wt)) was used asa scaffold when constructing two mutants, Z(basic1) and Z(basic2), with high positive surface charge. Far-ultraviolet circular dichroism measurements showed that they have a secondary structure content comparable to the parental molecule Z(wt). Although melting temperatures (T-m) of the engineered domains were lower than that of the wild-type Z domain, both mutants could be produced successfully as intracellular full-length products in E. coli and purified to homogeneity by ion-exchange chromatography. Further studies performed on Z(basic1) and Z(basic2) showed that they were able to bind to a cation exchanger even at pH values in the 9 to 11 range. A gene fusion between Z(basic2) and the acidic human serum albumin binding domain (ABD), derived from streptococcal protein G, was also constructed. The gene product Z(basic2)-ABD could be purified using cation-exchange chromatography from a whole cell lysate to more than 90% purity.