cellfree

Cell-free systems, or cell-free expression (CFPS), are biochemical platforms that produce proteins outside living cells by using intracellular transcription-translation machinery extracted from cells. In CFPS, a crude or purified cell extract containing ribosomes, tRNAs, amino acids, nucleotides, cofactors, and energy sources is supplied with a DNA or mRNA template, enabling protein synthesis in a test tube or microplate without cellular growth. CFPS allows rapid protein production and direct manipulation of reaction conditions, offering an open environment for synthesis and screening.

Common sources include Escherichia coli-based systems (the most widely used), as well as eukaryotic extracts from yeast, insect, plant, and mammalian cells. Each system varies in capacity for post-translational modifications and folding quality. Reactions are typically performed in small volumes and can run from tens of minutes to several hours; energy regeneration modules and coupled transcription-translation components are often optimized for yield.

History: early cell-free protein synthesis experiments in the 1960s, notably by Nirenberg and Matthaei, demonstrated translation of RNA in crude extracts. Since the 2000s, CFPS has matured with commercial kits and enhanced systems enabling higher yields, longer reaction times, and incorporation of noncanonical amino acids.

Applications include rapid protein prototyping, toxic or membrane proteins, enzyme screening, pathway engineering, and education. The open format allows direct control of reaction composition, easier incorporation of nonstandard amino acids, and integration with high-throughput screening or microfluidics. Limitations include costs, scalability challenges for large-scale production, incomplete post-translational modification for some systems, and variability between batches of extracts.

Cell-free systems are a growing area of synthetic biology and biomanufacturing, with ongoing work to improve yields, cost efficiency, and PTMs, and to broaden accessibility for research, industry, and education.