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Concept
Cell-free protein synthesis
Résumé
Cell-free protein synthesis, also known as in vitro protein synthesis or CFPS, is the production of protein using biological machinery in a cell-free system, that is, without the use of living cells. The in vitro protein synthesis environment is not constrained by a cell wall or homeostasis conditions necessary to maintain cell viability. Thus, CFPS enables direct access and control of the translation environment which is advantageous for a number of applications including co-translational solubilisation of membrane proteins, optimisation of protein production, incorporation of non-natural amino acids, selective and site-specific labelling. Due to the open nature of the system, different expression conditions such as pH, redox potentials, temperatures, and chaperones can be screened. Since there is no need to maintain cell viability, toxic proteins can be produced.
Common components of a cell-free reaction include a cell extract, an energy source, a supply of amino acids, cofactors such as magnesium, and the DNA with the desired genes. A cell extract is obtained by lysing the cell of interest and centrifuging out the cell walls, DNA genome, and other debris. The remains are the necessary cell machinery including ribosomes, aminoacyl-tRNA synthetases, translation initiation and elongation factors, nucleases, etc.
Two types of DNA can be used in CFPS: plasmids and linear expression templates (LETs). Plasmids are circular, and only made inside cells. LETs can be made much more effectively via PCR , which replicates DNA much faster than raising cells in an incubator. While LETs are easier and faster to make, plasmid yields are usually much higher in CFPS. Because of this, much research today is focused on optimizing CFPS LET yields to approach the yields of CFPS with plasmids.
An energy source is an important part of a cell-free reaction. Usually, a separate mixture containing the needed energy source, along with a supply of amino acids, is added to the extract for the reaction.
Source officielle
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