Tuning Vector Stability and Integration Frequency Elevates Functional GPCR Production and Homogeneity in...

Membrane proteins play a valuable role in biotechnology, yet the difficulty of producing high yields of functional membrane protein limits their use in synthetic biology. The practical application of G protein-coupled receptors in whole cell biosensors, for example, is restricted to those that are functionally produced at the cell surface in the chosen host, limiting the range of detectable molecules. Here, we present a facile approach to significantly improve the yield and homogeneity of functional membrane proteins in Saccharomyces cerevisiae by altering only the choice of expression vector. Expression of a model GPCR, the human adenosine A₂a receptor, from commonly used centromeric and episomal vectors leads to low yields and cellular heterogeneity due to plasmid loss in 20-90% of the cell population. In contrast, homogeneous production of GPCR is attained using a multisite integrating vector or a novel, modified high copy vector that does not require genomic integration or addition of any selection agents. Finally, we introduce a FACS-based screen, which enables rapid isolation of cells with 4- to 15-fold increases in gene dosage and up to a 9-fold increase in functional protein yield without loss of homogeneity compared to a strain isolated through conventional, low-throughput methods. These results can be extended to improve the cellular homogeneity and yield of other membrane proteins, expanding the repertoire of useful receptors for synthetic biology applications.