<italic>In vitro</italic> synthetic biology: Cell-free protein synthesis

Abstract

Synthetic biology is an emerging interdisciplinary field covering the subjects of biology, chemistry and engineering, which promotes the progress from understanding life to creating living systems. As one of important forefront directions of synthetic biology, in vitro synthetic biology (IVSB) focuses on studying the phenomenon and rules of life activities in testing tubes, not relying on living organisms. Cell-free protein synthesis (CFPS) is an effective research platform for IVSB, which utilizes exogenous DNA or mRNA as genetic information for synthesizing protein in vitro , instead of traditional protein expression in cells. CFPS possesses superior advantages over cell-based systems including convenience, simplicity, rapidness, excellent controllability and green economy. Moreover, CFPS is a robust research model for exploration of life evolution and creation of artificial living system. Since 1958, it has been demonstrated for the first time that proteins could be synthesized from cell extract; and afterwards, CFPS has emerged to not only a promising platform for proteomics and pharmaceutics, but also a significant tool for revelation of chemical basis of cells and exploration of origins of life. Cell extracts contained basic transcription and translation machineries, energy regeneration substrates, amino acids, nucleotides and cofactors. A variety of cell extracts have been created from different cell sources such as Escherichia coli ( E.coli ), wheat germ, yeast and rabbit reticulocyte. E.coli was the earliest prokaryotic system and most widely used due to its high production yield, short production cycle and low cost. The protein yields of E. coli system could reach up to several milligrams per milliliter of reaction. Eukaryotic systems such as wheat germ and rabbit reticulocyte although less productive, provided more robust platform for studying protein functions, in particular for post-translationally modified proteins. Different production formats of CFPS have been developed and classified according to how the reactions were fed, including batch, continuous-flow, continuous-exchange, bilayer and hydrogel. Batch was the simplest format, but the key limitation was the short lifetimes and the consequent low yield. This was primarily because of the rapid depletion of the high-energy phosphate pool, leading to accumulation of free phosphates and further inhibition of protein synthesis. Continuous-flow overcame the problem in batch format because of the continuous supply of energy and substrates and the continuous removal of the by-products. As a result, the reaction time could be extended to 20 hours, and the product yield was increased by two orders of magnitude in comparison with batch format. In continuous-exchange format, passive exchange of substrates and by-products further extended the reaction lifetime. However, continuous formats were not easily applicable to high-throughput processes, in which miniaturization and automation were required. Bilayer format was simple, efficient and high-throughput-friendly. Very recently, hydrogel formats have been developed because of the gel niche resembling the physicochemical nature of cells, subsequently leading to high yield. One of the promising applications of CFPS was to synthesize biologically active proteins. CFPS could produce proteins that were difficult or impossible to express with cell-based systems, such as membrane proteins, cell-toxic proteins, isotope-labeling proteins and proteins with unnatural amino acids incorporated. The open nature and high versatility of the CFPS platform enabled high-throughput tools for genomics and proteomics, allowing for biopharmaceutical applications. Through the high- throughput screening of proteins in CFPS the efficacy of protein drugs could be significantly improved by addition of unnatural amino acids, which provided excellent pharmacological properties and prolonged the half-life of drugs in plasma. In this review, we therefore summarize the development history, compositions, categories and production formats of CFPS, as well as applications in the synthesis of functional proteins, high throughput synthesis and screening of protein, and production of protein drugs.