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 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.