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

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

Index: Protein Expr. Purif. 77 , 140-145, (2011)

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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.Copyright © 2011 Elsevier Inc. All rights reserved.