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Two-step column chromatography separation and purification of recombinant porcine β_2- adrenergic receptor

Author: XuLiHua
Tutor: BianLiuJiao
School: Northwestern University
Course: Biochemical Engineering
Keywords: β2-Adrenoceptors column chromatography separation and purification
CLC: Q503
Type: Master's thesis
Year: 2010
Downloads: 65
Quote: 1
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Abstract


β2-Adrenoceptors is a member of a large super-family of cell surface receptors that carry out their signal transduction to the interior of the cell by coupling to heterotrimeric G proteins, it is related to a lot of physiologica and pathological in bodies, and drug play a pharmacodynamics by combining with acceptors, so, using this feature we can screen a large number of drug fast.On the foundation of successfully colony and expression ofβ2-Adrenoceptors, a two-step chromatographic method consisting of a Ni-chelated Sepharose High Performance (Sepharose H.P.) affinity medium and a Quaternary Sepharose Fast-Flow (Quaternary Sepharose F.F.) anion exchanger was established to prepare recombinantβ2-Adrenoceptors expressed in E. coli BL21(DE3) as histidine-tagged protein. In affinity chromatographic column, the buffer A was consisted of 20.0 mmol/L phosphate (PB)+500.0mmol/L NaCl (pH 7.4) and the buffer B was buffer A with the addition of 500.0 mmol/L imidazole, we can get three fractions from Sepharose H.P. medium, but there are a lot of hybrid protein in target fraction through analyzing by SDS-PAGE. In anion chromatographic column, the buffer A was 20.0 mmol/L phosphate (pH 7.4) and the buffer B was consisted of buffer A with 800.0 mmol/L NaCl, using buffer A+18% buffer B balance the Quaternary Sepharose F.F., and separately we choose a liner gradient of buffer A+18% Buffer B～100% buffer B for 30.0mL, five fractions were obtained totally, the analysis results of SDS-PAGE and high performance size-exclusion (Shim-pack Diol-300) showed that the purity of fraction V of obtained from Quaternary Sepharose F.F. was about 95%. Furthermore, we studied the bioactivity ofβ2-Adrenoceptors by receptor ligand combination test and the results assured the object protein possessed good bioactivity. Finally the conclusion could be reached that our methods could effectively separate active recombinedβ2-Adrenoceptors easily.

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