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).