Presentation Information
[4DSP-08]Rational design versus random selection of optimal signal peptides for secretion and downstream processing of recombinant proteins
○Jan Maarten van Dijl1 (1. University of Groningen, University Medical Center Groningen (Netherlands))
Keywords:
downstream processing,protein secretion,signal peptide,random selection,rational design
Purpose: Recombinant proteins are of major importance in healthcare, food and feed, and a sustainable bio-based economy. To facilitate their downstream processing, such proteins are preferably secreted into the fermentation broth from which they can be readily harvested. Effective recombinant protein secretion relies on appropriate cell factories with high-performance secretion machinery, secretion-competence of the protein-of-interest (POI), and an effective N-terminal signal peptide that directs the POI into the path for protein secretion. Due to their relatively simple cell envelope structure and potent protein secretion machinery, monoderm Gram-positive bacteria, as exemplified by the bacterium Bacillus subtilis, are attractive cell factories for secretory production of recombinant POIs.
Method: In B. subtilis the secretory (Sec) pathway is the major route for protein export from the cytoplasm to the fermentation broth. Equally important as the Sec pathway is an effective signal peptide that directs the POI from the ribosome across the cytoplasmic membrane and cell wall into the fermentation broth. Although signal peptides show structural conservation across species, different POIs present different signal peptide requirements. To obtain the best possible signal peptides for a particular POI, two approaches can be followed, namely random selection from a large signal peptide library or, unprecedently, rational signal peptide design.
Results: Our research shows that both approaches can be successful in delivering highly effective signal peptides for protein secretion by B. subtilis. For random signal peptide selection, we developed a pipeline involving a library of ~12,000 signal peptides and high-throughput screening with so-called nanoliter reactors [1]. Rational signal peptide design involved in silico interaction studies between signal peptides and the signal recognition particle. Both rational design and random selection approaches yielded signal peptides that were more effective in enzyme secretion than their natural counterparts.
Conclusion: Rational design and random selection approaches can yield highly effective signal peptides for super secretion and downstream processing of recombinant proteins.
[1] Grasso et al. (2023). Signal peptide efficiency: from high-throughput data to prediction and explanation. ACS Synth Biol. 12: 390-404. doi: 10.1021/acssynbio.2c00328.
Method: In B. subtilis the secretory (Sec) pathway is the major route for protein export from the cytoplasm to the fermentation broth. Equally important as the Sec pathway is an effective signal peptide that directs the POI from the ribosome across the cytoplasmic membrane and cell wall into the fermentation broth. Although signal peptides show structural conservation across species, different POIs present different signal peptide requirements. To obtain the best possible signal peptides for a particular POI, two approaches can be followed, namely random selection from a large signal peptide library or, unprecedently, rational signal peptide design.
Results: Our research shows that both approaches can be successful in delivering highly effective signal peptides for protein secretion by B. subtilis. For random signal peptide selection, we developed a pipeline involving a library of ~12,000 signal peptides and high-throughput screening with so-called nanoliter reactors [1]. Rational signal peptide design involved in silico interaction studies between signal peptides and the signal recognition particle. Both rational design and random selection approaches yielded signal peptides that were more effective in enzyme secretion than their natural counterparts.
Conclusion: Rational design and random selection approaches can yield highly effective signal peptides for super secretion and downstream processing of recombinant proteins.
[1] Grasso et al. (2023). Signal peptide efficiency: from high-throughput data to prediction and explanation. ACS Synth Biol. 12: 390-404. doi: 10.1021/acssynbio.2c00328.
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