In vitro translation using cell-free system for mRNA
display
Two μg of the mRNA-tag sample was used for in vitro translation
using PUREfrex 1.0 (Gene Frontier) in the 100 µl reaction mix. The
optimized condition for efficient mRNA-peptide conjugate formation are
as follows: initial translation at 37 ℃ for 30 min, followed by addition
of a salt mix to a final concentration of 32.5 mM MgCl2and 375 mM KCl, and then a second incubation at 37 ℃ for 60 min. After
this step, an equal volume of 2x Laemmli sample buffer (Bio-Rad) was
added, mixed and centrifuged at 10,000 x g, 1 min at room temperature to
remove the salt precipitant. The supernatant containing the translated
product was recovered and further resolved on a 3.5% stacking - 10%
resolving polyacrylamide SDS-PAGE gel at constant 50 mA. 8 M urea was
added to the resolving gel only. Afterwards, the gel band corresponding
to mRNA-peptide conjugate was excised, purified using the
Electro-Eluter. and precipitated with ethanol, as described in the
previous section.