Expression and functional purification of a glycosylation deficient version of the human adenosine 2a receptor for structural studies

Protein Expression and Purification
Niall J Fraser

Abstract

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...Continue Reading

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Citations

Jun 15, 2012·Microbial Cell Factories·Mitsunori ShiroishiTakuya Kobayashi
Dec 11, 2013·Microbial Cell Factories·Mouna GuerfalNico Callewaert
Jan 31, 2009·Microbial Cell Factories·Nicklas BonanderRoslyn M Bill
Dec 31, 2009·Microbial Cell Factories·Maria FreigassnerAnton Glieder
Jul 30, 2014·Applied Microbiology and Biotechnology·Anita EmmerstorferHarald Pichler
Oct 4, 2015·Methods : a Companion to Methods in Enzymology·Sarah J RoutledgeRoslyn M Bill
Oct 7, 2008·Protein Expression and Purification·Thierry MagninRenaud Wagner
Apr 3, 2007·Biotechnology Progress·Emily C McCuskerAnne Skaja Robinson
Nov 5, 2019·Protein Expression and Purification·Daniel N WisemanAlan D Goddard
Jun 20, 2017·ACS Medicinal Chemistry Letters·Hideyuki MuranakaTomonaga Ozawa

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