Separation and purification of Escherichia coli-expressed human thymosin-α1 using affinity chromatography and high-performance liquid chromatography.
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.
Nanjing Center for Disease Control and Prevention, Nanjing 210003, China.
Hong-Ying
HY
Zhang
Pei-Fu
PF
Chen
Jia-Ming
JM
Xu
Quan-Min
QM
Dai
Feng
F
Xu
Qing-Wang
QW
Han
Jian-Jun
JJ
Wang
Hei-Ying
HY
Jin
D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007525:Isoelectric Focusing; D007544:Isopropyl Thiogalactoside / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results; D012995:Solubility; D000077596:Thymalfasin; D013947:Thymosin / Q000031:analogs & derivatives* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D013947:Thymosin; D000077596:Thymalfasin