Abstract
Studies on the structure and functions of membrane proteins are impeded by their lability, hydrophobicity, difficulty in purification and low yields. Human chemokine receptor 3 (CCR3) is a G protein-coupled receptor related to allergic diseases. A Triton X-100/PEG20000 two-phase system was employed for enrichment separation of CCR3 over-expressed in E. coli. Optimal CCR3 partitioning with partition coefficient around 8 was obtained at pH 7.0, ionic strength of 0.3 mol/kg and 3 h equilibration time. Total recovery of CCR3 reached 102 ± 15%, which was much higher than 32 ± 5% of the normally used ultracentrifugation method. The recovered CCR3 was finally purified by two chromatography steps giving a final protein of 87 kDa.
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Abbreviations
- DM:
-
Dodecyl-β-d-maltoside
- OG:
-
Octyl-glucoside
- FC-14:
-
Fos-Choline 14
- Trt:
-
Triton
- TW:
-
Tween
- Dx:
-
Detran
- PEG 6K:
-
PEG6000
- PEG 20K:
-
PEG20000
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Acknowledgment
We gratefully thanked Dr Ren Hui and Zhang Shuguang (Center for Biological Engineering, MIT) who kindly provided biosynthesized human CCR3 gene. We also thanked Prof Huang Fang for his helpful discussion and improvement on written language.
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Ge, B., Li, W. & Yin, Y. Enrichment separation of recombinant human CCR3 using detergent/polymer two-phase system. Biotechnol Lett 32, 393–397 (2010). https://doi.org/10.1007/s10529-009-0169-0
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DOI: https://doi.org/10.1007/s10529-009-0169-0