Expression and purification of the extracellular domains of human chemokine receptor CCR5

Objective To express and purify the first extracellular domain and extracellular loop 2(ECL-2) of human chemokine receptor CCR5.Methods Gene fragments of the first extracellular domain and ECL-2 were amplified respectively and then spliced by Splicing of overlapping extension(SOE) PCR.This fragment was inserted into the prokaryotic expression vector pET-21b(+) and transformed into E.coli strain BL21(DE3),and then inducted with IPTG.The recombinant protein fusing with 6His-tag,named CCR5-N-E2,was detected by Western blot analysis.Metal-chelating affinity chromatography was performed to purify CCR5-N-E2.Results SDS-PAGE analysis showed that the recombinant protein(Mr 8 500) was expressed in E.coli in the form of inclusion body at the expression level over 50% of the total cell protein.After metal-chelating affinity chromatography,the recombinant protein reached the purity over 95%.Result of Western blotting showed that the recombinant protein could react with anti-human CCR5 antibody.Conclusion Recombinant protein including the first extracellular domain and ECL-2 of human chemokine receptor CCR5 is expressed and highly purified,which will facilitate the study of inhibitor agents against HIV-1 targeting CCR5.