oc1c00574_si_001.pdf (3.08 MB)
Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins
journal contributionposted on 2021-09-24, 18:39 authored by Sea On Lee, Qi Xie, Stephen D. Fried
The expression of long proteins with repetitive amino acid sequences often presents a challenge in recombinant systems. To overcome this obstacle, we report a genetic construct that circularizes mRNA in vivo by rearranging the topology of a group I self-splicing intron from T4 bacteriophage, thereby enabling “loopable” translation. Using a fluorescence-based assay to probe the translational efficiency of circularized mRNAs, we identify several conditions that optimize protein expression from this system. Our data suggested that translation of circularized mRNAs could be limited primarily by the rate of ribosomal initiation; therefore, using a modified error-prone PCR method, we generated a library that concentrated mutations into the initiation region of circularized mRNA and discovered mutants that generated markedly higher expression levels. Combining our rational improvements with those discovered through directed evolution, we report a loopable translator that achieves protein expression levels within 1.5-fold of the levels of standard vectorial translation. In summary, our work demonstrates loopable translation as a promising platform for the creation of large peptide chains, with potential utility in the development of novel protein materials.
prone pcr methodnovel protein materialslarge peptide chainsidentify several conditionsstandard vectorial translationoptimize protein expressionoptimized loopable translationcircularized mrnas couldcircularized mrnasloopable translatorcircularized mrnavivo translational efficiencyt4 bacteriophagesplicing intronribosomal initiationrepetitive proteinsrecombinant systemsrational improvementspotential utilitymodified errorlong proteinslimited primarilyinitiation regiongenetic constructdirected evolutiondata suggestedconcentrated mutationscircularizes mrnabased assay