用于蛋白质纯化的高效液相色谱系统。

Efficient high-performance liquid chromatographic system for protein purification.

作者信息

Yagi S, Izawa K, Nakagawa T, Tanaka H, Yoshitake A, Mohri Z

机构信息

Faculty of Pharmaceutical Sciences, Kyoto University, Japan.

出版信息

J Chromatogr. 1989 Aug 25;493(1):27-33. doi: 10.1016/s0378-4347(00)82705-x.

DOI:10.1016/s0378-4347(00)82705-x

PMID:2778019

Abstract

An efficient high-performance liquid chromatographic system, consisting of an affinity column and a high-performance size-exclusion column, was developed and applied to the purification of growth hormone receptors from rabbit livers. When a 6-ml sample of Triton X-100 extracts containing 16 mg of protein was applied to the system, 1200-fold purified receptor with a 10% recovery of binding activity from homogenates was obtained within 3-4 h. The purified receptor exhibited one main band on sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the affinity constant (Ka = 6.0.10(9) M-1) was found to be comparable with that of 1% Triton X-100 extract (4.4.10(9) M-1). The injection of 1 ml of 3 M urea solution prior to receptor elution with 10 ml of 6 M urea solution was effective in removing non-specific binding proteins.

摘要

开发了一种高效液相色谱系统，该系统由亲和柱和高效尺寸排阻柱组成，并将其应用于从兔肝脏中纯化生长激素受体。当将含有16 mg蛋白质的6 ml Triton X-100提取物样品应用于该系统时，在3-4小时内从匀浆中获得了1200倍纯化的受体，其结合活性回收率为10%。纯化后的受体在十二烷基硫酸钠聚丙烯酰胺凝胶电泳上呈现一条主带，并且发现其亲和常数（Ka = 6.0×10⁹ M⁻¹）与1% Triton X-100提取物的亲和常数（4.4×10⁹ M⁻¹）相当。在用10 ml 6 M尿素溶液洗脱受体之前，注入1 ml 3 M尿素溶液可有效去除非特异性结合蛋白。