Overview of PURE system-based display method
We have refined and optimized the mRNA and cDNA display method utilizing the commercially available PUREfrex1.0 system (in vitro TX-TL system with recombinant elements (Y Shimizu et al., 2001; Yoshihiro Shimizu, Kanamori, & Ueda, 2005) ) and screened approximately 1012 peptide sequences in a single experiment (Figure 1). One of the important features of our refined method is the stability of the mRNA-tag product during translation via RNase-free PUREfrex and two separate gel purification steps that assure the high purity of mRNA-tag for translation as well as peptide-conjugated products. Elimination of PURE components (RNA, enzyme, polyamine, cofactors, and ions) from the mRNA and cDNA-peptide conjugates avoids non-specific interaction among components, peptide conjugates and target during the downstream binding assay. Anti-FLAG M2 antibody was chosen as a suitable target to test the performance of our refined display method given the nature of its short octapeptide FLAG epitope (DYKDDDDK), known crystal structure (Roosild et al., 2006), and the evidence that previously three different display methods (phage display, DNA display and ribosome display) were able to enrich the core FLAG epitope motif (Miceli, DeGraaf, & Fischer, 1994; Osada, Shimizu, Akbar, Kanamori, & Ueda, 2009; Srila & Yamabhai, 2013; Yonezawa et al., 2003). The overall procedure takes total approximately 21 hours (mRNA display) or 22 hours (cDNA display), requiring standard four working days for one round of selection (Figure 1). High-throughput sequencing was performed after collecting multiple rounds of DNA library samples.