Utilization of a tetracycline-inducible system for high-level expression of recombinant proteins in Francisella tularensis LVS

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60162694%3AG44__%2F21%3A00557003" target="_blank" >RIV/60162694:G44__/21:00557003 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0147619X21000111?via%3Dihub" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0147619X21000111?via%3Dihub</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.plasmid.2021.102564" target="_blank" >10.1016/j.plasmid.2021.102564</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Utilization of a tetracycline-inducible system for high-level expression of recombinant proteins in Francisella tularensis LVS

Popis výsledku v původním jazyce

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.

Název v anglickém jazyce

Utilization of a tetracycline-inducible system for high-level expression of recombinant proteins in Francisella tularensis LVS

Popis výsledku anglicky

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.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

—

Návaznosti

S - Specificky vyzkum na vysokych skolach<br>I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Plasmid

ISSN

0147-619X

e-ISSN

1095-9890

Svazek periodika

115

Číslo periodika v rámci svazku

May

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

8

Strana od-do

102564

Kód UT WoS článku

000649111200005

EID výsledku v databázi Scopus

2-s2.0-85101554649