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Utilization of a tetracycline-inducible system for high-level expression of recombinant proteins in Francisella tularensis LVS
Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic.
Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic.
Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Klinisk bakteriologi.
Department of Molecular Pathology and Biology, Faculty of Military Health Sciences, University of Defence, Hradec Kralove, Czech Republic.
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2021 (Engelska)Ingår i: Plasmid, ISSN 0147-619X, E-ISSN 1095-9890, Vol. 115, artikel-id 102564Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Francisella tularensis is a Gram-negative intracellular pathogen causing tularemia. A number of its potential virulence factors have been identified, but their biology and functions are not precisely known. Understanding the biological and immunological functions of these proteins requires adequate genetic tools for homologous and heterologous expression of cloned genes, maintaining both original structure and post-translational modifications. Here, we report the construction of a new multipurpose shuttle plasmid – pEVbr – which can be used for high-level expression in F. tularensis. The pEVbr plasmid has been constructed by modifying the TetR-regulated expression vector pEDL17 (LoVullo, 2012) that includes (i) a strong F. tularensis bfr promoter, and (ii) two tet operator sequences cloned into the promoter. The cloned green fluorescent protein (GFP), used as a reporter, demonstrated almost undetectable basal expression level under uninduced conditions and a highly dynamic dose-dependent response to the inducer. The utility of the system was further confirmed by cloning the gapA and FTT_1676 genes into the pEVbr vector and quantifying proteins expression in F. tularensis LVS, as well as by studying post-translational modification of the cloned genes. This study demonstrates that high levels of recombinant native-like Francisella proteins can be produced in Francisella cells. Hence, this system may be beneficial for the analysis of protein function and the development of new treatments and vaccines.

Ort, förlag, år, upplaga, sidor
Elsevier, 2021. Vol. 115, artikel-id 102564
Nyckelord [en]
ATc inducible, Expression plasmid, Francisella tularensis, Regulated bfr promoter
Nationell ämneskategori
Biokemi och molekylärbiologi Mikrobiologi inom det medicinska området
Identifikatorer
URN: urn:nbn:se:umu:diva-181586DOI: 10.1016/j.plasmid.2021.102564ISI: 000649111200005Scopus ID: 2-s2.0-85101554649OAI: oai:DiVA.org:umu-181586DiVA, id: diva2:1538575
Tillgänglig från: 2021-03-19 Skapad: 2021-03-19 Senast uppdaterad: 2023-09-05Bibliografiskt granskad

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Golovliov, Igor

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