In this study, a human thymosin-alpha 1 (hT alpha 1) fusion protein was overexpressed in Escherichia coli (E. coli). The hexahistidine-tagged hT alpha 1 fusion protein was obtained in soluble form in cells of the engineered E. coli strain BL21 (DE3)/pET-28a-hT alpha 1 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 hT alpha 1 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 hT alpha 1 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 pl of approximately 4.0, and full wavelength screening showed an optimal absorbance wavelength at around 214 nm. (c) 2011 Elsevier Inc. All rights reserved.