E. coli chromosomal-driven expression of NADK2 from A. thaliana: A preferable alternative to plasmid-driven expression for challenging proteins

Autor: Matthieu Goussé, Elisa Dell’Aglio, Gilles Curien, Stéphanie Borland, Sébastien Renoud, Caroline Ranquet, Alexia Chandor-Proust
Přispěvatelé: BGene Genetics, Physiologie cellulaire et végétale (LPCV), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA), Light Photosynthesis & Metabolism (Photosynthesis), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), start-up Bgene
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: Protein Expression and Purification
Protein Expression and Purification, 2022, 195-196, pp.106090. ⟨10.1016/j.pep.2022.106090⟩
ISSN: 1046-5928
1096-0279
DOI: 10.1016/j.pep.2022.106090⟩
Popis: International audience; The expression and purification of large recombinant proteins or protein complexes is problematic for somebiotechnology laboratories. Indeed, it is often difficult to obtain enough active proteins to perform biologicalcharacterization or reach commercialization, when large proteins or protein complexes are expressed in E. colivia the popular T7-based plasmid-driven expression system. There is also an industrial demand to decrease our dependence on plasmid-driven expression, because of itsdrawbacks, such as: i) the common use of antibiotics to maintain the plasmid, ii) the issue of plasmid copynumber, and iii) the risk of overloading the expression system. Despite all these issues, alternative solutions, suchas gene integration in the bacterial chromosome, are rarely employed and their advantages are still a matter ofdebate.Plant plastidial NAD kinases (NADK; ATP:NAD 2′-phosphotransferase, EC 2.7.1.23) are a classic example ofproteins with high molecular weight, that are difficult to express and purify with traditional T7-based tech-nology. We therefore compared plasmid-driven and chromosomal-driven expression of the Arabidopsis thalianaNADK2 protein, using a proprietary counter-selection tool, COLIBELT®, that allows scar-free and marker-freechromosomal modifications.Here we show that chromosomal-driven expression allowed recovery of more active NADK2 protein thanclassic T7 expression systems, as well as better production, thus confirming that expression from one singlechromosomal copy is preferable to plasmid-driven expression and might be appealing for both basic and appliedresearch.
Databáze: OpenAIRE
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