Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

Abstract

Proteins are the main source of drug targets and some of them possess therapeutic potential themselves. Among them, membrane proteins constitute approximately 50% of the major drug targets. In the drug discovery pipeline, rapid methods for producing different classes of proteins in a simple manner with high quality are important for structural and functional analysis. Cell-free systems are emerging as an attractive alternative for the production of proteins due to their flexible nature without any cell membrane constraints. In a bioproduction context, open systems based on cell lysates derived from different sources, and with batch-to-batch consistency, have acted as a catalyst for cell-free synthesis of target proteins. Most importantly, proteins can be processed for downstream applications like purification and functional analysis without the necessity of transfection, selection, and expansion of clones. In the last 5 years, there has been an increased availability of new cell-free lysates derived from multiple organisms, and their use for the synthesis of a diverse range of proteins. Despite this progress, major challenges still exist in terms of scalability, cost effectiveness, protein folding, and functionality. In this review, we present an overview of different cell-free systems derived from diverse sources and their application in the production of a wide spectrum of proteins. Further, this article discusses some recent progress in cell-free systems derived from Chinese hamster ovary and Sf21 lysates containing endogenous translocationally active microsomes for the synthesis of membrane proteins. We particularly highlight the usage of internal ribosomal entry site sequences for more efficient protein production, and also the significance of site-specific incorporation of non-canonical amino acids for labeling applications and creation of antibody drug conjugates using cell-free systems. We also discuss strategies to overcome the major challenges involved in commercializing cell-free platforms from a laboratory level for future drug development. Key PointsCell-free protein synthesis has the potential to become an alternative method for the rapid and highly parallel expression of a diverse range of proteins.Cell-free systems utilize the translation machinery of the cells, bypassing the constraints of the cell membrane and thus offer openness and configurational flexibility even for the synthesis of difficult-to-express proteins.In comparison to traditional approaches, cell-free systems can be time-saving, from initial synthesis to downstream applications.

Druggable Transient Pockets in Protein Kinases

“…A wide range of cell-free systems are commercially available; these systems are based on cell lysates derived from various sources or the PURE system. The cell-free protein synthesis system has continuously been improved [ 86 ].…”

Optimising protein synthesis in cell‐free systems, a review

“…Many constraints must be considered for translation efficiency starting from codon context. The presence of rare codons can impact the translation of a given protein [53], alternatively, their optimised version despite improving protein yield could lead to a decrease in protein function due to misfolding [54]. Another important concern is regarding the choice of RBS sequences.…”

Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology

“…It was suggested that such systems could be used more in the future for the production of dedicated pharma proteins, for example, incorporating noncanonical amino acids ( Hong et al, 2014 ; Quast et al, 2015 ; Wu et al, 2020 ), preparation of dedicated proteins that were produced under more defined conditions than possible in cell-based systems ( Oza et al, 2015 ), or allowing for the “on-demand” production of protein therapeutics in the clinic ( Mohr et al, 2016 ; Sullivan et al, 2016 ; Timm et al, 2016 ). The diverse features of CFPS systems promoted also their recent use in teaching ( Stark et al, 2019 ), protein engineering ( Kido et al, 2020 ), and synthetic biology ( Tinafar et al, 2019 ), which holds great promises for studies on genetic networks or rapid prototyping ( Karim et al, 2020 ) in metabolic engineering ( Perez et al, 2016 ) as well as future drug development ( Dondapati et al, 2020 ). Moreover, the in vitro reaction format of CFPS systems allows for full automation, miniaturization ( Ayoubi-Joshaghani et al, 2020 ), and working with large sample numbers ( Zhu et al, 2015 ).…”

“…Eukaryotic ribosomes from plants are better adapted for protein folding during synthesis than prokaryotic ribosomes from E. coli extracts, notably when eukaryotic proteins are targeted. Besides those established CFPS systems ( Rosenblum and Cooperman, 2014 ; Zemella et al, 2015 ; Dondapati et al, 2020 ), new systems were developed for rapid protein expression that better match the features of cell-based systems, for instance, using extracts from HeLa ( Mikami et al, 2008 ) or Chinese Hamster Ovary (CHO) cells ( Brodel et al, 2015 ; Thoring et al, 2016 ). Other advancing systems are based on extracts from Saccharomyces cerevisiae ( Gan and Jewett, 2014 ), Pichia pastoris ( Spice et al, 2020 ), tobacco BY-2 cells ( Buntru et al, 2015 ), rice ( Suzuki et al, 2020 ), or modified E. coli strains ( Seki et al, 2009 ; Cole et al, 2020 ) to name a few.…”