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.