A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant

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...

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Bibliographic Details
Published in:Protein expression and purification 2010-11, Vol.74 (1), p.80-87
Main Authors: Singh, Shweta, Hedley, Diana, Kara, Elodie, Gras, Adrien, Iwata, So, Ruprecht, Jonathan, Strange, Philip G, Byrne, Bernadette
Format: Article
Language:English
Subjects:
Cell Line
Cholesterol Esters - metabolism
Detergents
Gene Expression
Humans
Protein Stability
Receptor, Adenosine A2A - genetics
Receptor, Adenosine A2A - isolation & purification
Receptor, Adenosine A2A - metabolism
Solubility
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Summary: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 [(3)H]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.
ISSN:1096-0279
DOI:10.1016/j.pep.2010.04.018