Cell-free expression systems exploit the cellular protein synthesis machinery for direct protein synthesis outside intact cells using exogenous messenger RNA (mRNA) or DNA as templates. This is achieved by combining the lysate from growing cells with the exogenous supply of essential amino acids, nucleotides, salts, energy-generating factors, cofactors and so on. The lysates contain the necessary elements required for transcription, translation, and protein folding, such as ribosomes, tRNAs, aminoacyl-tRNA synthetases, and initiation, elongation and termination factors. The most commonly used cell-free systems are based on extracts from Escherichia coli (E. coli), wheat germ embryos, and rabbit reticulocytes respectively. Cell-free systems can function either in a coupled manner, where DNA is used as a template, or in an uncoupled manner, which requires mRNA template produced from native sources or by in vitro transcription. The DNA template must contain a promoter and a translation initiation signal and may be in the form of a plasmid or polymerase chain reaction (PCR) fragment. In order to increase the expression level, a transcription and translation termination region is also required. Cell-free systems can be used to express single proteins and multiple proteins in high-throughput applications.
Cell-free protein synthesis has distinct advantages over conventional cell-based protein expression methods due to the open reaction environment, which allows for easy manipulation, direct monitoring, and rapid sampling. Cell-free systems can produce proteins directly from a PCR fragment or an mRNA template without the need for E. coli cloning, allowing them applicable for high throughput protein synthesis. In addition, cell-free systems can simultaneously express multiple templates, allowing a protein population to be generated in a single reaction. These systems generally synthesize soluble and functional proteins, whereas cell-based methods produce insoluble aggregates for many proteins. Cell-free systems can also express toxic, proteolytically sensitive or unstable proteins, which are not physiologically tolerated by the living cell. Therefore, cell-free systems offer a rapid route to functional protein analysis. The protein synthesis conditions in a cell-free system can be regulated and controlled by the addition of helper molecules, providing a defined environment for correct protein folding. Furthermore, cell-free systems allow the efficient incorporation of non-natural or chemically-modified amino acids into the expressed protein at desired positions during translation.
Numerous highly active cell-free expression systems are available, and the choice of the system is generally determined by the biochemical properties and downstream application of the target protein. Amerigo Scientific offers high-quality cell-free protein expression systems for the production of protein libraries, therapeutic proteins, evolved proteins, membrane proteins, and virus-like particles.