Integrative refolding and purification of histidine-tagged protein by like-charge facilitated refolding and metal-chelate affinity adsorption.
This work proposed an integrative method of protein refolding and purification by like-charged resin facilitated refolding and metal-chelate affinity adsorption. Hexahistidine-tagged enhanced green fluorescence protein (EGFP) was overexpressed in Escherichia coli as inclusion bodies (IBs), and then the protein was refolded and purified from urea-solubilized IBs by this method. A metal-chelating resin was fabricated by coupling iminodiacetic acid (IDA) to agarose gel (Sepharose FF). The anionic resin was used to facilitate the refolding of like-charged EGFP from IBs. After refolding, nickel ions were introduced for the affinity purification of the target protein by metal-chelating adsorption. It was found that the resin was effective in facilitating EGFP refolding. For 0.1mg/mL EGFP IBs refolding, the fluorescence recovery (FR) by direct dilution was only 64%; addition of only 0.05 g/mL resin increased the FR to over 90%. Moreover, the FR increased with increasing resin concentration. Owning to the shielding effect of the oppositely charged impurities embedded in IBs on the surface charges of the IDA resin, more resin particles were required to exert an aggregation inhibition effect in the IBs protein refolding. Additionally, compared with direct-dilution refolding, inclusion of like-charged resins not only offered an enhanced FR of EGFP, but also bound some opposite-charged contaminant proteins, leading to a preliminary purification effect. Afterwards, the refolded EGFP was recovered by metal-chelating adsorption at an FR of 85% and purity of 93%. This work has thus extended the like-charge facilitated protein refolding strategy to the integrative protein refolding and purification.
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Hu
H
Liu
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
Wen-Jie
WJ
Du
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China.
Xiao-Yan
XY
Dong
Department of Biochemical Engineering and Key Laboratory of Systems Bioengineering of Ministry of Education, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, China. Electronic address: ysun@tju.edu.cn.
Yan
Y
Sun
D000327:Adsorption; D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods; D056831:Coordination Complexes / Q000737:chemistry*; D003300:Copper; D005782:Gels; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D007098:Imino Acids / Q000737:chemistry*; D008670:Metals; D009532:Nickel; D009842:Oligopeptides / Q000737:chemistry*; D058849:Protein Refolding; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012117:Resins, Synthetic; D012685:Sepharose / Q000737:chemistry
D002614:Chelating Agents; D056831:Coordination Complexes; D005782:Gels; C471213:His-His-His-His-His-His; D007098:Imino Acids; D008670:Metals; D009842:Oligopeptides; D011506:Proteins; D011994:Recombinant Proteins; D012117:Resins, Synthetic; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006639:Histidine; D003300:Copper; D009532:Nickel; D012685:Sepharose; C008109:iminodiacetic acid
Affinity adsorption; Inclusion bodies; Like-charge; Metal chelate; Protein refolding; Purification