Optimization of the Human Adenosine A2a Receptor Yields in Saccharomyces cerevisiae

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• 作者：Alison Wedekind ; Michelle A. O'Malley ; Ronald T. Niebauer ; Anne S. Robinson
• 刊名：Biotechnology Progress
• 出版年：2006
• 出版时间：October 2006
• 年：2006
• 卷：22
• 期：5
• 页码：1249 - 1255
• 全文大小：244K
• 年卷期：v.22,no.5(October 2006)
• ISSN：1520-6033

文摘

G-protein coupled receptors (GPCRs) have been implicated in many human diseases and haveemerged as important drug targets. Despite their medical relevance, knowledge about GPCRstructure is limited, mainly due to difficulties associated with producing large amounts offunctional protein and isolating this protein in functional form. However, our previous resultsindicate that when the human adenosine A₂a receptor (A₂aR) is expressed in Saccharomycescerevisiae, high yields can be achieved. In light of these initial results and in anticipation offuture purification efforts, experiments were conducted to optimize the system for maximumtotal protein yield. Emphasis was placed on not only producing large quantities of A₂aR in eachcell 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 onboth cell growth (as measured by optical density, OD₆₀₀) and per cell A₂aR expression levels.For these studies, the A₂aR expression levels were determined using a previously describedA₂aR-green fluorescent protein (GFP) fusion, so that expression could be monitored byfluorescence. Overall the data indicate that at late times (~60 h of expression) approximately75% higher total batch protein yields can be achieved using lower expression temperatures or60% higher using elevated induction cell density. The highest yields correspond to approximately28 mg per liter of culture of total A₂aR. Amounts of functional receptor were shown to increaseon a per cell basis by decreasing expression temperature up to 25 h of expression, but at latetime points (~60 h) functional yields did not appreciably improve. When compared to otherreports of GPCR expression in yeast it is clear that this system is among those producing thehighest GPCR protein yields per culture both before and after optimization.