Pasquinelli R S, Shepherd R E, Koepsel R R, Zhao A, Ataai M M
Department of Chemical Engineering, Center for Biotechnology and Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15219, USA.
Biotechnol Prog. 2000 Jan-Feb;16(1):86-91. doi: 10.1021/bp990139h.
Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to be superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. For example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper we have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.
亲和标签常用于通过固定化金属亲和色谱法来完成重组蛋白的纯化。标签的成功使用取决于所使用的螯合金属以及宿主细胞蛋白的洗脱曲线。由于锌（II）-亚氨基二乙酸（Zn（II）-IDA）对细胞蛋白的结合亲和力相对较低，它可能比固定化铜或镍更具优势。例如，几乎所有大肠杆菌蛋白在pH 7.5至7.0之间从Zn（II）-IDA柱上洗脱下来，在pH值低于7.0时只有很少的细胞蛋白出现。因此，Zn（II）-IDA洗脱曲线的很大一部分可能没有污染蛋白，这可用于从粗细胞提取物中一步纯化目标蛋白。在本文中，我们鉴定了几种融合标签，它们可以将目标蛋白的洗脱引导至Zn（II）-IDA洗脱曲线的低背景区域。这些标签允许将蛋白质靶向洗脱曲线的不同区域，便于在温和条件下进行纯化。
