With excessive demands on sample amounts progressively decreasing, cell-free protein synthesis (CFPS) systems are gaining momentum as tools for structural biology applications to complex proteins. Out of the available CFPS systems, wheat germ cell-free protein synthesis (WG-CFPS) merges the highest yields, compatible with structural approaches, with the use of a eukaryotic ribosome. This makes it an excellent approach for the study of complex viral proteins including, for example, large protein complexes and membrane proteins. 

Studying biological systems represents a challenging task due to the inherent complexity of living cells and, still, the lack of quantitative and reproducible data sets. Attempts to create membrane proteins outside living cells, namely from cell lysates, have brought us to an alternative route of protein synthesis, eventually bypassing the inherent regulation processes of protein synthesis pathways occurring in living cells. Such novel combinations of cell-free protein synthesis with artificial membranes with an optimized translation initiation model are subsumed as a bottom-up synthesis of natural versus non-natural systems aiming to understand membrane proteins.

A decisional tool was developed to evaluate a cell-free synthesis system's economic and operational performance compared to an E. coli process used to manufacture highly potent biotherapeutic proteins. The real-world simulated scenarios highlighted areas where the cell-free platform offered up to 35% savings. Ultimately, the advantage of identifying when the cell-free platform outperforms was equivalent to reducing process development times by up to 18 person-months—potentially accelerating life-saving medicines to patients. 

The cotranslational insertion of nascent GPCRs and other membrane proteins into MSP or SapA-based nanoparticles by cell-free protein synthesis generates high-quality samples for functional and structural studies. We demonstrate strategies for cell-free GPCR/nanoparticle formation, their lipid-dependent functional analysis, and the cryo-electron microscopy characterization of GPCR/G-protein complexes. GPCRs were further transferred from nanoparticles into membranes of living cells to study their activity and interactions with endogenous proteins.

Traditionally, transient gene expression (TGE) has been the technology used for production of therapeutic proteins at early drug development stages as it allows for rapid production of high-quality material. In this study, two anti-viral mAbs were produced using AstraZeneca’s proprietary CHO-based transient expression system. To assess the suitability of transiently-generated material for clinical studies, batch-to-batch product quality consistency as well as process scalability were investigated.

Targets for drug discovery projects are becoming more diverse and challenging. They are chosen based on evidence linking them to human disease and not on the challenges, which need to be overcome to express these proteins in suitable quantity and quality to support drug discovery projects. A number of examples of recent AstraZeneca projects will be presented, in which difficult expression/purification challenges have been overcome.

Advances in cell-free protein synthesis (CFPS) offer the prospect of industrially-relevant production processes for stratified and personalised protein therapeutics, with the potential for greater development and production speed, control over process environment, and improved consistency. We present studies of how a membrane reactor can be used to improve the economics, titre, and product quality, by recreating the localised enzyme concentrations seen in the cellular environment.

Pseudomonas aeruginosa is the second leading cause of nosocomial infections and pneumonia in hospitals. The outer membrane protein OprF is a well conserved and immunogenic porin playing an important role in quorum sensing and in biofilm formation. We used a bacterial cell-free expression system to reconstitute OprF under its native forms and in active open oligomerized state in liposomes and we demonstrated that the resulting OprF proteoliposomes can be used as a fully functional recombinant vaccine against P. aeruginosa. Our approach thus represents an easy and efficient way for producing bacterial membrane antigens exposing native epitopes for vaccine purposes.