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.
Highlights
Proteins serve as the building blocks for functional, tunable materials across the tree of life
Ares and co-workers demonstrated that the self-splicing group I intron within the thymidylate synthase gene of T4 phage retains activity following circular permutation at the P6a stem (Figure 2A).[22−26] In this reorganized topology, the same two phosphoryl transfer reactions result in the circularization of an internal region, in contrast with the wild-type intron which catalyzes the splicing of two flanking exons
Guanosine is required because the intron uses a two-step mechanism that begins with the free nucleoside cleaving the phosphodiester linkage between the 5′exon and the intron by prepending itself to the 5′-most nucleotide of the intron, and divalent cations are required to stabilize the tertiary fold of the catalytic core.[25,31−34] We found that green fluorescent protein (GFP) expression from pBAD-tdTEVDB was not strongly dependent on guanosine (Figure 3A); fluorescence could be markedly improved by 147% by increasing the concentration of MgCl2 in the growth media up to 20 mM (Figure 3B)
Summary
Proteins serve as the building blocks for functional, tunable materials across the tree of life. There have been several successes in the design of novel globular cagelike protein materials, generally based on motifs that self-assemble through symmetry.[17−20] On the other hand, natural fibrous proteins typically consist of highly repetitive low-complexity regions within polypeptides with long chain lengths and display self-assembly on many length scales, spanning from the nanometer (protein−protein interactions) to the micrometer (phase separation) and the millimeter (filamentization). Proteins of this type are generally less amenable to rational design and are typically challenging to express. A short repetitive unit is autoligated into tandem repeats by availing of self-complementary sticky ends from two restriction enzymes.[1,6] The judicious choice of two enzymes that create identical sticky ends but recognize distinct recognition sites enables directionality and prevents the formation of inverse repeats (Figure 1A)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
