[Two-step chromatographic method for the separation and purification of recombinant porcine beta2-adrenoceptors].
Beta2-Adrenoceptors are the members of cell surface receptors which perform their signal transduction to the interior of the cells by coupling to heterotrimeric G proteins. On the foundation of successful clone and expression of beta2-adrenoceptors, a two-step chromatographic method using Ni-chelated Sepharose High Performance affinity media and Quaternary Sepharose Fast Flow anion exchangers was established to prepare recombinant beta2-adrenoceptors expressed in E. coli BL21 (DE3) as histidine-tagged protein. In the affinity chromatographic column, the buffer A was consisted of 20 mmol/L phosphate buffered saline (PBS) containing 500 mmol/L NaCl (pH 7.4), and the buffer B was consisted of buffer A with the addition of 0.5 mol/L imidazole (pH 7.4); in anion chromatographic column, the buffer A was 20 mmol/L PBS (pH 7.4), and the buffer B was consisted of buffer A with 800 mmol/L NaCl (pH 7.4). The analysis results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and high performance size-exclusion chromatography (Shim-pack Diol-300) showed that the purity of obtained beta2-adrenoceptors was about 95%. Furthermore, the bioactivity of beta2-adrenoceptors was studied by receptor ligand combination test, and the results assured the object protein possessed good bioactivity. Finally the conclusion can be reached that the method can effectively separate active recombined beta2-adrenoceptors.
Xu
Lihua
L
College of Chemical Engineering, Northwest University, Xi'an 710069, China.
Wang
Shixiang
S
Zheng
Xiaohui
X
Bian
Liujiao
L
D000818:Animals; D002845:Chromatography / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D013552:Swine
D018343:Receptors, Adrenergic, beta-2; D011994:Recombinant Proteins