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PURE mRNA display and cDNA display provide rapid detection of consensus binding motif via high-throughput sequencing
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  • Sabrina Galinanes Reyes,
  • Yutetsu Kuruma,
  • Mai Fujimi,
  • Masako Yamazaki,
  • Sumie Eto,
  • Satoshi Tamaki,
  • Asaki Kobayashi,
  • Ryo Mizuuchi,
  • Lynn Rothschild,
  • Mark Ditzler,
  • Kosuke Fujishima
Sabrina Galinanes Reyes
University of Glasgow

Corresponding Author:[email protected]

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Yutetsu Kuruma
Japan Agency for Marine-Earth Science and Technology
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Mai Fujimi
Tokyo Institute of Technology
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Masako Yamazaki
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Sumie Eto
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Satoshi Tamaki
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Asaki Kobayashi
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Ryo Mizuuchi
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Lynn Rothschild
NASA Ames Research Center
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Mark Ditzler
NASA Ames Research Center
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Kosuke Fujishima
Tokyo Institute of Technology
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The recombinant in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable mRNA and cDNA-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. As a proof-of-concept, we chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 x 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG binding motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. The consistency of two different display methods achieved by commercially available PURE system will be useful for future studies to explore sequence and functional space of diverse polypeptides.
04 Jun 2020Submitted to Biotechnology and Bioengineering
04 Jun 2020Submission Checks Completed
04 Jun 2020Assigned to Editor
09 Jun 2020Reviewer(s) Assigned
17 Jul 2020Editorial Decision: Revise Major
17 Jul 2020Review(s) Completed, Editorial Evaluation Pending
31 Dec 20201st Revision Received
02 Jan 2021Submission Checks Completed
02 Jan 2021Assigned to Editor
04 Jan 2021Reviewer(s) Assigned
17 Jan 2021Review(s) Completed, Editorial Evaluation Pending
17 Jan 2021Editorial Decision: Accept