Exploring the Potential of Cell-Free Protein Synthesis for Extending the Abilities of Biological Systems

Abstract

Cell-free protein synthesis (CFPS) system is a simple, rapid, and sensitive tool that is devoid of membrane-bound barriers, yet contains all the mandatory substrates, biomolecules, and machineries required for the synthesis of the desired proteins. It has the potential to overcome loopholes in the current in vivo production systems and is a promising tool in both basic and applied scientific research. It facilitates a simplified organization of desired experiments with a variety of reaction conditions, making CFPS a powerful tool in biological research. It has been used for the expansion of genetic code, assembly of viruses, and in metabolic engineering for production of toxic and complex proteins. Subsequently, CFPS systems have emerged as potent technology for high-throughput production of membrane proteins, enzymes, and therapeutics. The present review highlights the recent advances and uses of CFPS systems in biomedical, therapeutic, and biotechnological applications. Additionally, we highlight possible solutions to the potential biosafety issues that may be encountered while using CFPS technology.

Keywords: CFPS; biocontainment; high-throughput proteins; synthetic biology; therapeutics; virus-like particles.

Figure 1

Schematic representation of a CFPS system performed in a single tube which requires cellular lysate, energy sources, nucleotides, amino acids, salts, cofactors, linear or plasmid DNA, and water/buffer to maintain the reaction. Such a system could be used to synthesize viruses, antibodies, therapeutic and high-throughput proteins.

Figure 2

A comparison of a conventional in vivo system and a CFPS system. The in vivo system is capable of producing recombinant proteins or therapeutic compounds like the CFPS system, although it takes more experimental steps and time to achieve the equivalent result.

Figure 3

The numerous potential applications of CFPS systems for assisting high-throughput protein production, including the production of antibodies, proteins with unnatural amino acids, therapeutics, viruses and virus-like particles, and gene circuits.

Figure 4

For incorporation of non-natural amino acids, amber-suppressing tRNAs are used. A strain having RF1 would create a competition between amber suppression tRNA and RF1 to bind with site-specific amber codon, which is represented in the figure. To avoid this competitive binding, cell extract is prepared using a strain lacking RF1. Figure reproduced with permission from Schoborg and Jewett (2018) © John Wiley and Sons, Inc.

Figure 5

To produce membrane proteins through CFPS platforms, several membrane-mimicking structures are used. (A) Lipid bilayer, (B) liposome, (C) micelle, (D) bicelle, (E) nanodisc, and (F) tethered bilayer lipid membrane. Figure reproduced with permission from (Schoborg and Jewett, 2018) © John Wiley and Sons, Inc.