Factors Influencing GPCR Overexpression

Although the choice of expression system is the key factor in obtaining sufficient quantities of high-quality GPCR, a number of other factors have to be considered and modulated to optimize GPCR expression levels. For structural studies, the development of constructs that are compatible with the method of structural determination is crucial. The presence of large loops and extended N- and C-termini can hamper crystallization, increase proteolysis, and reduce the overall protein yield [96] . Expression levels can also be improved using mutagenesis. For example, the expression of turkey p1AR was significantly improved following a Cys116Leu mutation [127]. Directed evolution is another alternative given that a 12-fold increase in expression of the NTR receptor was achieved over wild-type receptor [128], and as previously mentioned, the use of fusion partners has also been used to increase expression levels [64] . While modifying the construct can lead to increases in expression levels and reduced heterogeneity, care has to be taken to not dramatically affect the pharmacology of the receptor in a detrimental way.

Finally, manipulation of culture conditions also offers a route to improve expression levels. Optimization of the culture conditions through the addition of additives, such as ligands [65, 129], amino acids [130], and DMSO [130, 131], and by altering temperature [130] can increase expression by nearly 10-fold [130] .

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