Optimization of conjugate formation during in vitrotranslation
Recovered mRNA-tag was subjected to in vitro translation using a
commercial cell-free PUREfrex 1.0 kit to form mRNA-peptide conjugates.
PUREfrex is known for its high purity for eliminating nucleases and
proteases and thus ideal for the application for mRNA display. Indeed,
throughout our experiment we never encountered mRNA-tag degradation
during the translation reaction. The formation rate of the mRNA-peptide
conjugate also depends on the incubation conditions during and after the
translation to promote efficient incorporation of puromycin to the
stalled ribosome at the mRNA-DNA boundary. Previous studies have applied
rare codons at the 3’ end of mRNA coding region (Nagumo et al., 2016) or
used long linker in the DNA tag (Naimuddin & Kubo, 2016) to improve the
conjugate formation. In this study, we have adopted both of these
features by placing rare GGA codon at the 3’ end and included polyA (18
nt) in the puromycin-FITC DNA tag. By using the gel purified 10aa-random
mRNA-tag as a substrate, we carried out the optimization of conjugate
formation via iteration of three different parameters: translation time,
salt mix (KCl and MgCl2) concentration, and incubation
time after salt addition. Translation time reflects the ribosome
loading, translation of the coding region which ends up stalling at the
3’ end due to lack of stop codon. We tested translation at 37 ℃ from 0
to 60 minutes and found that 5 minutes of translation is sufficient to
reach over 40% conjugate formation and the extent of conjugation does
not significantly increase with longer incubation times (Figure 2A).
Next, various salt mix concentrations were tested during post
translation incubation. Previously, a high concentration of KCl has been
shown to improve the accessibility of ribosome-bound peptidyl-tRNA to
puromycin (Van Der Mast & Bloemers, 1973) and has been adopted in the
recent mRNA and cDNA display methods (Naimuddin & Kubo, 2016; Seelig,
2011; Yamaguchi et al., 2009). We found that without salt addition,
conjugate formation is less than 10%, however the addition of 32.5 mM
MgCl2 and 375 mM KCl greatly increased the formation
rate to above 40% (Figure 2B). Furthermore, we found the incubation
time after salt mix addition to be important for puromycin to react with
peptidyl-tRNA to form covalent link between mRNA and peptide. Here, 60
minutes of incubation at 37℃ gave the highest yield with a formation
rate of 48.5% (Figure 2C). With the above optimized condition,
mRNA-peptide conjugates were synthesized, and gel purified from the
SDS-urea polyacrylamide gel. Throughout all conditions we tested, we did
not observe any mRNA degradation nor mRNA-peptide conjugate degradation
during the translation and incubation steps (Figure 2A and 2C).