High-throughput E. coli cell-free expression: From PCR product design to functional validation of GPCR

Abstract

This chapter outlines a protocol to express GPCRs libraries for screening of targets. High-throughput screening of GPCR expression raised a big interest in the development of proteomic drug candidates, protein engineering, and microarrays. However, GPCRs represent a large family of difficult-to-express proteins which can be successfully produced by cell-free systems in the presence of liposomes. The open and flexible nature of this in vitro expression system allows the manipulation of transcription and translation as well as the modulation of the cell-free reaction environment by the addition of any adjuvant or the incorporation of unnatural amino acid for example. The compatibility of PCR fragments with cell-free protein synthesis and using SPRi as multiplex analytical platform offer an effective method to rapidly select different targets. Large-scale expression and purification of GPCRs into proteoliposome format are discussed at the end of this chapter.