His-tagged protein purification by metal-chelate affinity extraction with nickel-chelate reverse micelles.
Di(2-ethylhexyl) phosphoric acid (HDEHP) was used as a transition metal ion chelator and introduced to the nonionic reverse micellar system composed of equimolar Triton X-45 and Span 80 at a total concentration of 30 mmol/L. Ni(II) ions were chelated to the HDEHP dimers in the reverse micelles, forming a complex denoted as Ni(II)R(2). The Ni(II)-chelate reverse micelles were characterized for the purification of recombinant hexahistidine-tagged enhanced green fluorescent protein (EGFP) expressed in Escherichia coli. The affinity binding of EGFP to Ni(II)R(2) was proved by investigation of the forward and back extraction behaviors of purified EGFP. Then, EGFP was purified with the affinity reverse micelles. It was found that the impurities in the feedstock impeded EGFP transfer to the reverse micelles, though they were little solubilized in the organic phase. The high specificity of the chelated Ni(2+) ions toward the histidine tag led to the production of electrophoretically pure EGFP, which was similar to that purified by immobilized metal affinity chromatography. A two-stage purification by the metal-chelate affinity extraction gave rise to 87% recovery of EGFP. Fluorescence spectrum analysis suggests the preservation of native protein structure after the separation process, indicating the system was promising for protein purification.
Dong
Xiao-Yan
XY
Dept. of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.
Feng
Xu-Dong
XD
Sun
Yan
Y
D002614:Chelating Agents / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D008823:Micelles; D009532:Nickel / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D002614:Chelating Agents; D008823:Micelles; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006639:Histidine; D009532:Nickel