Optimization of mRNA-peptide conjugate formation
The formation rate of mRNA-peptide conjugate using PUREfrex®1.0 was optimized through iterations of three different reaction conditions: translation time, salt mix concentration, and incubation time after salt mix addition. All reactions were performed at 10 μl scale reaction. For the translation time, we tested 0, 5, 15, 30 and 60 minutes at 37 ℃, prior to the addition of salt mix (final conc, 32.5 mM MgCl2 and 375 mM KCl) followed by 60 minutes of incubation. For the optimization of salt mix concentration, we first conducted 30 minutes translation at 37 ℃, then either added no salt or 1x, 1/2x, 1/4x, 1/8x concentration of 65 mM MgCl2 and 750 mM KCl concentration originally reported to promote covalent bond between puromycin and polypeptide chain (Naimuddin et al., 2011; Naimuddin & Kubo, 2016). For the incubation time after salt addition, we initially performed 30 minutes translation, added salt to final 32.5 mM MgCl2, 375 mM KCl concentration, then tested 0, 10, 30, 60 and 90 minutes of incubation time.