Presentation Information
[P03-358]Development of Novel Translation-Enhancing Peptides to Improve recombinant protein productivity in Bacillus subtilis
○Khairil Anwar1, Hideo Nakano1, Teruyo Ojima-Kato1 (1. Graduate School of Bioagricultural Sciences, Nagoya University (Japan))
Keywords:
Bacillus subtilis,recombinant protein production,translational arrest,arrest peptide,translation-enhancing peptides
[Purpose]
Bacillus subtilis has been widely used as a host for producing various heterologous proteins and has become an essential tool in industry and biotechnological research owing to its generally recognized as safe (GRAS) and robust capacity to secrete proteins. However, several essential proteins are challenging to express in B. subtilis, resulting in low yields of protein. Our previous studies have demonstrated that inserting an SKIK (Ser–Lys–Ile–Lys) peptide-encoding peptide tag immediately downstream of the start codon enhanced protein expression and reduced ribosomal stalling mediated by arrest peptides (APs), such as consecutive prolines in E. coli.
[Method]
In this study, we report a new approach to enhance protein productivity in the B. subtilis expression system by using novel translation-enhancing peptides (TEPs). First, screening of TEPs in B. subtilis was performed based on the capability of the peptides to attenuate AP-mediated translational arrest.
[Results]
A total of 380 samples, including positive and negative clones, were analyzed, revealing that TEP sequences exhibited varying translation-enhancing activities, as reflected by differences in reporter sfGFP fluorescent intensities. The top five TEPs showing the highest fluorescence were subsequently validated using an alternative reporter, mCherry. The results confirmed that these selected TEPs consistently increase mCherry production in B. subtilis.
[Consideration]
This study reports for the first time that newly identified TEPs can alleviate AP-mediated translational arrest and improve protein production in B. subtilis.
[Conclusion]
This strategy presents a promising approach for preventing ribosomal stalling and enhancing protein production in B. subtilis for broad applications in synthetic biology and industrial biotechnology.
Bacillus subtilis has been widely used as a host for producing various heterologous proteins and has become an essential tool in industry and biotechnological research owing to its generally recognized as safe (GRAS) and robust capacity to secrete proteins. However, several essential proteins are challenging to express in B. subtilis, resulting in low yields of protein. Our previous studies have demonstrated that inserting an SKIK (Ser–Lys–Ile–Lys) peptide-encoding peptide tag immediately downstream of the start codon enhanced protein expression and reduced ribosomal stalling mediated by arrest peptides (APs), such as consecutive prolines in E. coli.
[Method]
In this study, we report a new approach to enhance protein productivity in the B. subtilis expression system by using novel translation-enhancing peptides (TEPs). First, screening of TEPs in B. subtilis was performed based on the capability of the peptides to attenuate AP-mediated translational arrest.
[Results]
A total of 380 samples, including positive and negative clones, were analyzed, revealing that TEP sequences exhibited varying translation-enhancing activities, as reflected by differences in reporter sfGFP fluorescent intensities. The top five TEPs showing the highest fluorescence were subsequently validated using an alternative reporter, mCherry. The results confirmed that these selected TEPs consistently increase mCherry production in B. subtilis.
[Consideration]
This study reports for the first time that newly identified TEPs can alleviate AP-mediated translational arrest and improve protein production in B. subtilis.
[Conclusion]
This strategy presents a promising approach for preventing ribosomal stalling and enhancing protein production in B. subtilis for broad applications in synthetic biology and industrial biotechnology.
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