title: Optimization of the human adenosine A2a receptor yields in Saccharomyces cerevisiae.
abstract: G-protein coupled receptors (GPCRs) have been implicated in many human diseases and have emerged as important drug targets. Despite their medical relevance, knowledge about GPCR structure is limited, mainly due to difficulties associated with producing large amounts of functional protein and isolating this protein in functional form. However, our previous results indicate that when the human adenosine A(2)a receptor (A(2)aR) is expressed in Saccharomyces cerevisiae, high yields can be achieved. In light of these initial results and in anticipation of future purification efforts, experiments were conducted to optimize the system for maximum total protein yield. Emphasis was placed on not only producing large quantities of A(2)aR in each cell but also achieving high cell density in batch culture. Therefore, temperature, media pH, inducer concentration in the media, and induction cell density were tested for their effects on both cell growth (as measured by optical density, OD(600)) and per cell A(2)aR expression levels. For these studies, the A(2)aR expression levels were determined using a previously described A(2)aR-green fluorescent protein (GFP) fusion, so that expression could be monitored by fluorescence. Overall the data indicate that at late times ( approximately 60 h of expression) approximately 75% higher total batch protein yields can be achieved using lower expression temperatures or 60% higher using elevated induction cell density. The highest yields correspond to approximately 28 mg per liter of culture of total A(2)aR. Amounts of functional receptor were shown to increase on a per cell basis by decreasing expression temperature up to 25 h of expression, but at late time points ( approximately 60 h) functional yields did not appreciably improve. When compared to other reports of GPCR expression in yeast it is clear that this system is among those producing the highest GPCR protein yields per culture both before and after optimization.
authors.affiliation: Chemical Engineering Department, University of Delaware, Newark, Delaware 19716, USA.
authors.forename: Alison
authors.initials: A
authors.lastname: Wedekind
authors.forename: Michelle A
authors.initials: MA
authors.lastname: O'Malley
authors.forename: Ronald T
authors.initials: RT
authors.lastname: Niebauer
authors.forename: Anne S
authors.initials: AS
authors.lastname: Robinson
mesh_terms: D001709:Biotechnology / Q000379:methods*; D005690:Galactose / Q000737:chemistry; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013997:Time Factors
chemical_list: D008024:Ligands; D043705:Receptor, Adenosine A2A; D049452:Green Fluorescent Proteins; D005690:Galactose