Optimized Loopable Translation as a Platform for the Synthesis of Repetitive Proteins

Abstract

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.

Microbial Synthesis of High-Molecular-Weight, Highly Repetitive Protein Polymers

“…cmRNAs are cyclized, single-stranded RNA derived from back-splicing of precursor mRNAs. A cmRNA can be generated by fusion of a self-splicing intron (e.g., the td gene from T4 phage) to a mRNA that encodes the target protein [ 21 , 22 ]. Once transcribed, the self-splicing intron catalyzes a spontaneous splicing–ligation reaction to form cmRNA that contains a stop-codon-free coding RNA ( Figure 2 ).…”

“…Second, because cmRNAs do not have free 5′ and 3′ ends, which are recognition sites of common ribonucleases, cmRNAs are more stable (less prone to RNase degradation) than their linear mRNA counterparts. The higher RNA stability allows more proteins to be translated per mRNA ( Figure 2 ) [ 21 , 22 ].…”

“…The advantages of cmRNAs have encouraged material scientists to explore them for synthesis of repetitive PBMs. Lee et al first engineered cmRNAs to express spider dragline silk protein MaSp1 from Trichonephila clavipes in E. coli [ 21 ]. Although cmRNA encoding short 16× silk repeats (48.5 kDa) was successfully transcribed, the translated silk proteins have less than 32 repeats, meaning translation terminated before completing one around of translation and protein yield was very low [ 21 ].…”

“…Lee et al first engineered cmRNAs to express spider dragline silk protein MaSp1 from Trichonephila clavipes in E. coli [ 21 ]. Although cmRNA encoding short 16× silk repeats (48.5 kDa) was successfully transcribed, the translated silk proteins have less than 32 repeats, meaning translation terminated before completing one around of translation and protein yield was very low [ 21 ]. Success was later obtained by Liu et al who used cmRNA that contained a relatively shorter MaSp1sequence (encoding a 5.1 kDa protein monomer).…”

“…Lastly, translation efficiency can be strongly affected by unwanted interactions between target proteins. If the produced proteins strongly self-associate or aggregate, their interaction may make the ribosome disengage from the cmRNA template, resulting in early translation termination and low-MW products, which do have desirable material properties [ 21 ].…”