Synthesizing Long and Repetitive Proteins Via a “Loopable Translator” System

Abstract

The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we present a system that generates circular mRNA in vivo by circularly permuting the self-splicing thymidylate synthase (TD) intron from T4 bacteriophage, resulting in “loopable” translation. In this system, the order of exons and intron are rearranged such that the identical natural RNA splicing events result in a circular mRNA. The self-splicing group I intron was selected because it does not require an enzyme for its activity. We demonstrated that circularization successfully occurs in vivo, and under the optimal conditions (identified to date) the looping construct achieves 15% of the GFP production relative to the positive control. Successful mRNA circularization was confirmed by SDS-PAGE gel electrophoresis. The data suggest that protein yield may be limited by inefficient initiation, though ribosomes can perform many transits on a circular mRNA template per initiation event. This circularizing translation system may contribute to the production of protein-based polymers with diverse applications.