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A purified C-terminally truncated human adenosine A(2A) receptor construct is functionally stable and degradation resistant
DOI:
10.1016/j.pep.2010.04.018
Authors:
Shweta
Singh
(Imperial College London)
,
Diana
Hedley
(University of Reading)
,
Elodie
Kara
(University of Reading)
,
Adrien
Gras
(Imperial College London)
,
So
Iwata
(Imperial College London; Diamond Light Source; Japan Science and Technology Agency; Kyoto University)
,
Jonathan
Ruprecht
(Imperial College London)
,
Phillip G.
Strange
(University of Reading)
,
Bernadette
Byrne
(Imperial College London)
Co-authored by industrial partner:
No
Type:
Journal Paper
Journal:
Protein Expression And Purification
, VOL 74 (1)
, PAGES 80-87
State:
Published (Approved)
Published:
January 2010
Abstract: 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.
Journal Keywords: G-protein coupled receptor; Adenosine A2A receptor; C-terminally truncated constructs; Functionality; Stability; Degradation resistant; Structural studies
Subject Areas:
Biology and Bio-materials
Diamond Offline Facilities:
Membrane Protein Laboratory (MPL)
Technical Areas:
Added On:
23/09/2010 10:52
Discipline Tags:
Life Sciences & Biotech
Technical Tags: