Increasing recombinant protein production in E. coli via FACS-based selection of N-terminal coding DNA libraries

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F86652036%3A_____%2F24%3A00617020" target="_blank" >RIV/86652036:_____/24:00617020 - isvavai.cz</a>

Výsledek na webu

<a href="https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/febs.17376" target="_blank" >https://febs.onlinelibrary.wiley.com/doi/epdf/10.1111/febs.17376</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1111/febs.17376" target="_blank" >10.1111/febs.17376</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Increasing recombinant protein production in E. coli via FACS-based selection of N-terminal coding DNA libraries

Popis výsledku v původním jazyce

Here, we present a previously undescribed approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated that the nucleotides immediately following the start codon can significantly influence protein expression. However, the impact of these sequences is construct-specific and is not universally applicable to all proteins. Most of the previous research has been limited to selecting from a few rationally designed sequences. In contrast, we used a directed evolution-based methodology, screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increased expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.

Název v anglickém jazyce

Increasing recombinant protein production in E. coli via FACS-based selection of N-terminal coding DNA libraries

Popis výsledku anglicky

Here, we present a previously undescribed approach to modify N-terminal sequences of recombinant proteins to increase their production yield in Escherichia coli. Prior research has demonstrated that the nucleotides immediately following the start codon can significantly influence protein expression. However, the impact of these sequences is construct-specific and is not universally applicable to all proteins. Most of the previous research has been limited to selecting from a few rationally designed sequences. In contrast, we used a directed evolution-based methodology, screening large numbers of diversified sequences derived from DNA libraries coding for the N-termini of investigated proteins. To facilitate the identification of cells with increased expression of the target construct, we cloned a GFP gene at the C-terminus of the expressed genes and used fluorescent activated cell sorting (FACS) to separate cells based on their fluorescence. By following this systematic workflow, we successfully elevated the yield of soluble recombinant proteins of multiple constructs up to over 30-fold.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10608 - Biochemistry and molecular biology

Projekt

<a href="/cs/project/LX22NPO5102" target="_blank" >LX22NPO5102: Národní ústav pro výzkum rakoviny</a><br>

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2024

Kód důvěrnosti údajů

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

Název periodika

FEBS Journal

ISSN

1742-464X

e-ISSN

1742-4658

Svazek periodika

neuveden

Číslo periodika v rámci svazku

DEC 2024

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

16

Strana od-do

—

Kód UT WoS článku

001383431600001

EID výsledku v databázi Scopus

2-s2.0-85213012521