PURE mRNA display and cDNA display provide rapid detection of consensus binding motif via high-throughput sequencing

Abstract

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 10 12 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.