Cell-Free Protein Synthesis: A New Direction for Future Drug Development

Protein, the almighty molecule that reigns supreme in the intricate web of living organisms, holds within its versatile structure a myriad of awe-inspiring functions. In the age-old pursuit of unlocking its secrets, researchers have relied upon the steadfast cell expression systems, wherein they meticulously cultivate cells to produce the proteins for thorough analysis. However, these systems encounter an arduous gauntlet of obstacles, shackled by the chains of restrained cell growth, the relentless need for upkeep, and contamination. Such bottlenecks prove especially vexing when striving for high-throughput production and custom protein synthesis.

To provide more options for efficient protein preparation and research, cell-free protein expression technology has emerged. Powered by the latest technological advancements, it was bestowed upon the power of high-yield protein expression. The cell-free expression system differs from the conventional cell expression system by enabling protein synthesis in a controlled in vitro environment. This innovative technology offers remarkable advantages, including high efficiency, convenience, flexibility, and exceptional purity. It has quickly emerged as a cutting-edge solution that commands significant attention in the realms of biomedicine and basic research.

Fig.1 Cell-free and cell-based protein synthesis systems (Brookwell A., et al. 2021).

Cell-Free Protein Synthesis System

In order to synthesize proteins with new properties, structures and functions, the DNA or mRNA template encoding the protein, as well as amino acids and nucleotides, should be added to the cell-free protein synthesis (CFPS) system to start the transcription and translation process. The CFPS system uses crude extracts of microorganisms, plants or animal cells to produce ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, ribosome release factors and other elements necessary for transcription, translation, protein folding and energy metabolism. Once initiated, cell-free protein synthesis typically continues until one of the substrates (e.g., ATP, cysteine, etc.) is depleted or the accumulation of by-products (e.g., inorganic phosphate) reaches inhibitory concentrations. To date, a variety of CFPS systems for extracts have been developed. Each system has its own advantages and disadvantages. It is necessary to select a more suitable system based on the target protein to be expressed, weighing its requirements such as yield, cost and post-translational modification.

Advantages of Cell-Free Protein Expression

Compared with traditional living cell protein expression technology, cell-free protein expression technology has the following significant advantages.

Higher protein expression: Traditional live cell protein expression technology faces limitations imposed by various factors inherent to the cells, which ultimately restricts the quantity of protein that can be expressed. The cell-free protein expression technology not only avoids the limitations faced by traditional living cell expression by performing synthesis reactions in an environment with high substrate concentration in vitro, it can also control the reaction system well, so as to obtain proteins with higher expression.

Faster expression speed: Traditional live cell protein expression requires cells to grow and reach an optimal density for protein expression, and this process often takes several days. The cell-free protein expression technology usually only takes a few hours to complete the protein expression, which is significantly faster than the traditional living cell expression technology.

More accurate protein synthesis: Cell-free protein expression technology performs protein synthesis in vitro, which can precisely control parameters such as substrate concentration, reaction temperature, and reactant ratio, so that customized proteins can be synthesized more accurately, which is of great significance for research and application.

More flexible control: In cell-free protein expression technology, protein synthesis can be performed using separated component systems, the ratio of substrates and reactants can be controlled, and custom modifications can be performed under appropriate reaction conditions, such as protein Labeling, pharmacodynamic analysis, etc. These advantages make cell-free protein expression technology more flexible, controllable, and applicable to a wider range of applications.

References

1. Brookwell A.; et al. Biotechnology applications of cell-free expression systems. Life. 2021, 11(12): 1367.
2. Dondapati S.; et al. Cell-free protein synthesis: A promising option for future drug development. BioDrugs. 2020, 34(3): 327-348.