Cell-free protein systems and in vitro display methods as compelling tools for high-throughput screening

Synthetic biology has become a promising field that aims at developing and using tools to work with recombinant DNA. In this field, cell-free protein expression systems have become a valuable asset to enable the in vitro transcription-translation of recombinant proteins, as functional elements of synthetic biology. These systems are not dependent on a living organism and consequently offer full control of the reactions’ composition and environment, thus enabling protein expression in situations where in vivo systems would not perform efficiently. In this work, we aimed to explore their applications for in vitro display techniques, for protein and peptide evolution in drug discovery. Cell-free systems have the potential to allow for a higher number of library candidates to be selected and to enable the use of recombinant or unnatural candidates. These unnatural candidates are elements not used by organisms naturally, whether because they are toxic, they don’t have the metabolisms to process them or just because they are fully synthetic. The most researched targets for drugs are membrane proteins, but they are also some of the most challenging, as they require a proper lipid membrane to fold and settle correctly. The work presented in this thesis is focused on linking cell-free systems, in vitro display methods and membrane proteins, by characterising the effects of specific components on performance in a systematic step-by-step manner. The thesis first describes the uncovering of the underlying dynamics of protein expression in two different types of cell-free systems, namely cell-extracts and purified components. A set of T7 promoter variants was constructed and tested in both systems, and the protein expression levels recorded and analysed. Both systems are driven by different expression dynamics for protein and mRNA. These expression dynamics represent the behaviour of certain parameters involved in synthesis, regulation, degradation, bottlenecks, etc … The limiting factors of both systems were identified for optimization of protein expression. Following conclusions from this analysis, purified components for protein translation were adopted and applied to both mRNA and cDNA display techniques. The results demonstrated the ability of the cell-free systems to provide a screening/selection method producing highly stable peptide conjugates and high sample purification. This proof of concept was tested and verified with the FLAG epitope, as a thoroughly characterised system. Several motifs with high affinity were obtained after 4 rounds of selection and further sequenced. Building further on these developments, cell-free systems were used to produce CX3CR1, a membrane protein from the G-protein-coupled receptors (GPCRs) family, within two types of synthetic lipid membranes, liposomes and nanodiscs. The thesis finishes by providing potential directions for the possible use of the cell-free expression systems, mRNA display and GPCR proteins for the creation of a peptide screening and selection method that could be used in the future for drug screening of membrane proteins