2010年11月
A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant
PROTEIN EXPRESSION AND PURIFICATION
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- 巻
- 74
- 号
- 1
- 開始ページ
- 80
- 終了ページ
- 87
- 記述言語
- 英語
- 掲載種別
- 研究論文(学術雑誌)
- DOI
- 10.1016/j.pep.2010.04.018
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
Recent high resolution structures of modified G-protein coupled receptors (GPCRs) have provided major insight into the mechanisms of receptor-ligand binding. However understanding of the complete mechanism of GPCR function remains limited. This study characterised C-terminally truncated versions of the human adenosine A(2A) receptor (A(2A)R) with a view to producing protein suitable for structural studies. The constructs terminated at residue A316, removing the intracellular C-terminal tail, or V334, producing a C-terminal tail equivalent in length to that of rhodopsin. Higher levels of functional receptor before and after solubilisation were obtained for both C-terminally truncated constructs compared to the wild-type receptor (WT) as assessed by radioligand binding analysis using [H-3]ZM241385. The construct which yielded the highest level of functional receptor, V334 A(2A)R, was purified in DDM to high homogeneity with a final yield of 2 mg/L. Binding analysis revealed that the purified receptor had a specific activity of 20.2 +/- 1.2 nmol/mg, close to the theoretical maximum. Pure V334 A(2A)R was resistant to degradation over 15 days when stored at 4 degrees C or 20 degrees C and showed remarkable functional stability when stored at 4 degrees C, retaining 84% of initial functionality after 30 days. This construct is an excellent candidate for structural studies. (C) 2010 Elsevier Inc. All rights reserved.
- リンク情報
- ID情報
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- DOI : 10.1016/j.pep.2010.04.018
- ISSN : 1046-5928
- eISSN : 1096-0279
- J-Global ID : 201002246277505624
- Web of Science ID : WOS:000281495100011