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Updated: Oct 25, 2026

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells
Published on: November 13, 2014
A Two-step Gene Expression Method for In Vitro Evolution of Translational Proteins
Akari Sakurai1, Kentaro Shoji1, Norikazu Ichihashi1,2,3
1Department of Life Science, Graduate School of Arts and Sciences, The University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo153-8902, Japan.
Abstract:
Improving the reconstituted translation system is a key requirement for bottom-up synthetic biology. Here, we developed a two-step in vitro evolutionary method that can be used for improving translational proteins. In this method, two distinct conditions were sequentially applied while maintaining genotype-phenotype linkage in water-in-oil droplets. Using this method, we performed in vitro evolution of four translation factors, IleRS, PheRS, EF-G, and EF-Tu, and identified mutations that modestly enhanced translation activity in in vitro expression assays. One of the EF-G mutations (P610S) increased activity per protein approximately 2-fold for the recombinant protein purified from E. coli. This selection method is useful for improving translational proteins for bottom-up synthetic biology.
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