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It is true that cell-free protein synthesis works with genetic materials such as plasmid DNA but also with PCR-amplified linear DNA sequences. However, when cell-free expression assays started with PCR fragments, the expression yields were much lower than from plasmid DNA. This was due to the DNase activity present in the cell extract that decreased the lifetime of linear DNA.
However, as protein production from PCR-products was already much faster due to the elimination of cloning, screening and selection steps, several approaches have been investigated to improve productivity by decreasing linear DNA degradation or by stabilizing mRNA (Lee et al., 2018; Schinn et al. 2016).
Today, cell-free systems achieve similar yields from both plasmid DNA and PCR fragments.
One such approach is to remove gene encoding endonuclease I and exonuclease V, two major nucleases of double-stranded DNA, from the genome of E. coli strain used for cell extract preparation (Michel-Reydellet et al., 2005). In this approach, endA gene encoding for endonuclease I was suppressed and the recCBD operon, which is part of the E. coli recombination system and encodes for the exonuclease V, was replaced by the lambda phage Red recombination system. Bacterial growth rate was not affected by these mutations and PCR product stability was increased. As a result, CAT (chloramphenicol acetyltransferase) was produced with similar yields (500-600 μg/mL) from PCR products or from plasmids.
Other approaches improved productivity using linear DNA templates: for example the addition of bacteriophage lambda Gam protein, an inhibitor of RecBCD protein (Sitaraman et al., 2004) or the introduction of chi-sites in linear DNA. Since RecBCD stops on DNA chi-sites as part of homologous recombination (Marshall et al., 2017), both strategies increased the stability of the linear DNA in the bacterial cell extract.
Prolonging the lifetime of the mRNA is another approach that aims to improve productivity when using PCR products. By preparing a cell extract lacking RNase E activity and introducing a stem-loop structure at the 3’-end of the mRNA (Ahn et al., 2005), mRNA were protected from endonucleases and exonucleases, mRNA half-lives were significantly prolonged and translation occurred even with the rapid degradation of DNA template. CAT was produced at 685 μg/mL, a yield comparable to that observed when using plasmid DNA.
Thus, the enhanced productivity in cell-free systems, using PCR fragments, enables its use as a high-throughput tool to screen libraries or engineered proteins. Indeed, the proteins can be rapidly produced from a linear PCR product through microscale reactions allowing high throughput screening of a protein library in 384 well-plates format, as well as by using plasmids. These protocols can be automated with the use of a liquid handling robot but can also be performed manually.