Expression and functional purification of a glycosylation deficient version of the human adenosine 2a receptor for structural studies.
A glycosylation deficient (dG) version of the human adenosine 2a receptor (hA2aR) was made in Pichia pastoris strain SMD1163. Under optimal conditions, expression levels of between 8 and 12pmol receptor/mg membrane protein were obtained routinely. In a shake flask, this is equivalent to ca. 0.2mg of receptor per litre of culture. The level of functional receptor produced was essentially independent of the pH of the yeast media. In contrast to this, addition of the hA2aR antagonist theophylline to the culture media caused a twofold increase in receptor expression. A similar effect on dG hA2aR production was also observed when the induction temperature was reduced from 29 to 22 degrees C. In P. pastoris membranes, dG hA2aR had native-like pharmacological properties, binding antagonists with rank potency ZM241385>XAC>theophylline, as well as the agonist NECA. Furthermore, the receptor was made with its large (ca. 120 amino acid) C-terminal domain intact. dG hA2aR was purified to homogeneity in three steps, and its identity confirmed by electrospray tandem mass spectrometry following digestion with trypsin. The secondary structure of the entire receptor is largely (ca. 81%) alpha-helical. Purified dG hA2aR bound [(3)H]ZM241385 in a saturable manner with a B(max) of 18.1+/-0.5 nmol/mg protein, close to the theoretical B(max) value for pure protein (21.3 nmol/mg protein), showing that the receptor had retained its functionality during the purification process. Regular production of pure dG hA2aR in milligram quantities has enabled crystallisation trials to be started.
Fraser
Niall J
NJ
Division of Biochemistry and Molecular Biology, IBLS, Glasgow Biomedical Research Centre, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK. niall.fraser@bio.gla.ac.uk <niall.fraser@bio.gla.ac.uk>
D002462:Cell Membrane / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D010957:Plasmids / Q000235:genetics; D017433:Protein Structure, Secondary; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D012995:Solubility; D014170:Transformation, Genetic / Q000235:genetics
D043705:Receptor, Adenosine A2A; D011993:Recombinant Fusion Proteins