Cell-Free protein synthesis from exonuclease-deficient cellular extracts utilizing linear DNA templates
2022
Sabeti Azad, Mahnaz | Cardoso Batista, Angelo | Faulon, Jean-Loup | Beisel, Chase, L | Bonnet, Jérôme | Kushwaha, Manish | MICrobiologie de l'ALImentation au Service de la Santé (MICALIS) ; AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE) | Helmholtz Centre for Infection Research (HZI) | University Hospital of Würzburg | Centre de Biologie Structurale [Montpellier] (CBS) ; Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM) | ANR-17-CE07-0046,SINAPUV,(Bio)synthèse et étude des propriétés physico-chimiques et biologiques d'analogues du malate de sinapoyl: de nouvelles molécules anti-UV non-toxiques et biosourcées pour l'industrie cosmétique(2017) | ANR-18-CE33-0015,VR-MARS,Réalité Virtuelle, Assistance et Secours Médical pour Spationautes(2018) | ANR-21-CE48-0003,DREAMY,Algorithmes distribués pour les systèmes microbiologiques(2021)
International audience
اظهر المزيد [+] اقل [-]إنجليزي. Cell-free protein synthesis (CFPS) has recently become very popular in the field of synthetic biology due to its numerous advantages. Using linear DNA templates for CFPS will further enable the technology to reach its full potential, decreasing the experimental time by eliminating the steps of cloning, transformation, and plasmid extraction. Linear DNA can be rapidly and easily amplified by PCR to obtain high concentrations of the template, avoiding potential in vivo expression toxicity. However, linear DNA templates are rapidly degraded by exonucleases that are naturally present in the cell extracts. There are several strategies that have been proposed to tackle this problem, such as adding nuclease inhibitors or chemical modification of linear DNA ends for protection. All these strategies cost extra time and resources and are yet to obtain near-plasmid levels of protein expression. A detailed protocol for an alternative strategy is presented here for using linear DNA templates for CFPS. By using cell extracts from exonuclease-deficient knockout cells, linear DNA templates remain intact without requiring any end-modifications. We present the preparation steps of cell lysate from Escherichia coli BL21 Rosetta2 ΔrecBCD strain by sonication lysis and buffer calibration for Mg-glutamate (Mg-glu) and K-glutamate (K-glu) specifically for linear DNA. This method is able to achieve protein expression levels comparable to that from plasmid DNA in E. coli CFPS.
اظهر المزيد [+] اقل [-]المعلومات البيبليوغرافية
تم تزويد هذا السجل من قبل Institut national de la recherche agronomique