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

Result code in 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>
Result on the web
<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>

Result language
angličtina
Original language name
Increasing recombinant protein production in E. coli via FACS-based selection of N-terminal coding DNA libraries
Original language description
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.
Czech name
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Czech description
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Type
J<sub>imp</sub> - Article in a specialist periodical, which is included in the Web of Science database
CEP classification
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OECD FORD branch
10608 - Biochemistry and molecular biology

Project
<a href="/en/project/LX22NPO5102" target="_blank" >LX22NPO5102: National institute for cancer research</a><br>
Continuities
I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year
2024
Confidentiality
S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

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