利用亲和色谱和高效液相色谱法分离纯化大肠杆菌表达的人胸腺素α1 。

Separation and purification of Escherichia coli-expressed human thymosin-α1 using affinity chromatography and high-performance liquid chromatography.

作者信息

Zhang Hong-Ying, Chen Pei-Fu, Xu Jia-Ming, Dai Quan-Min, Xu Feng, Han Qing-Wang, Wang Jian-Jun, Jin Hei-Ying

机构信息

Nanjing Center for Disease Control and Prevention, Nanjing 210003, China.

出版信息

Protein Expr Purif. 2011 Jun;77(2):140-5. doi: 10.1016/j.pep.2011.01.004. Epub 2011 Jan 21.

DOI:10.1016/j.pep.2011.01.004

PMID:21256964

Abstract

In this study, a human thymosin-α1 (hTα1) fusion protein was overexpressed in Escherichia coli (E. coli). The hexahistidine-tagged hTα1 fusion protein was obtained in soluble form in cells of the engineered E. coli strain BL21 (DE3)/pET-28a-hTα1 that had been induced with isopropyl -D-1-thiogalactopyranoside (IPTG). The recombinant protein accounted for approximately 50-60% of the total protein. We then developed and validated a separation method for hTα1 from E. coli cells based on thermal denaturation, nickel-resin affinity chromatography and high-performance liquid chromatography. The purification method showed good reproducibility and was easy to operate. Purified recombinant hTα1 of high homogeneity was characterized and found to be of high purity (over 99%), as determined by high-voltage electrophoresis and high-performance liquid chromatography analysis. Isoelectric focusing analysis indicated a pI of approximately 4.0, and full wavelength screening showed an optimal absorbance wavelength at around 214nm.

摘要

在本研究中，人胸腺素α1（hTα1）融合蛋白在大肠杆菌（E. coli）中过表达。带有六聚组氨酸标签的hTα1融合蛋白以可溶形式在经异丙基 -D-1-硫代半乳糖苷（IPTG）诱导的工程化大肠杆菌菌株BL21（DE3）/pET-28a-hTα1的细胞中获得。重组蛋白约占总蛋白的50 - 60%。然后，我们基于热变性、镍树脂亲和色谱和高效液相色谱开发并验证了一种从大肠杆菌细胞中分离hTα1的方法。该纯化方法具有良好的重现性且易于操作。经高压电泳和高效液相色谱分析确定，纯化后的高均一性重组hTα1具有高纯度（超过99%）。等电聚焦分析表明其pI约为4.0，全波长扫描显示最佳吸光波长约为214nm。