UNC Libraries · 2020 · 18 citations · 0 references
Life SciencesBiotechnologyMolecular BiologyGene ExpressionProtein EngineeringSynthetic BiologyExpression Vector DesignProtein Expression EngineeringRecombinant Protein ExpressionCardiac ReprogrammingCell Fate ConversionCleavage EfficiencyDirect Cardiac ReprogrammingDirect ReprogrammingFluorescent Cell LabelingFluorescent LabelingGene Position Effects
Cloning multiple genes into a single vector, especially with 2A self‑cleaving peptides, has become a key strategy for co‑expressing complex proteins due to their small size and high cleavage efficiency. This study aims to systematically compare the performance of different 2A peptides, alone or in combination, in polycistronic constructs. The authors evaluated how gene order and 2A choice affect protein expression in bi‑, tri‑, and quad‑cistronic vectors, and tested the impact on fluorescent labeling and cardiac reprogramming efficiency. Fluorophore expression declined toward the 3′ end, with optimal reprogramming when the fluorophore occupied the second position, and quad‑cistronic TPE2A constructs outperformed 3P2A and PTE2A, providing guidance for future 2A peptide use.
Cloning of multiple genes in a single vector has greatly facilitated both basic and translational studies that require co-expression of multiple factors or multi-units of complex protein. Many strategies have been adopted, among which 2A “self-cleaving” peptides have garnered increased interest for their polycistronic nature, small size and high “cleavage” efficiency. However, broad application of 2 A peptides is limited by the lack of systematic comparison of different 2As alone or in combination. Here we characterized the effect of varying gene position and 2As on the expression of proteins encoded in bi-, tri-, or quad-cistronic constructs. Using direct cardiac reprogramming as an example, we further determined the effect of varied 2As on the efficiency of fluorescent cell labeling and cell fate conversion. We found that the expression of fluorophores decreased as it was moved towards the end of the construct while reprogramming was most efficient with the fluorophore at the second position. Moreover, quad-cistronic TPE2A constructs resulted in more efficient reprogramming than 3P2A or PTE2A constructs. We expect that the bi-, tri-, and quad-cistronic vectors constructed here and our results on protein expression ratios from different 2A constructs could serve to guide future utilization of 2A peptides in basic research and clinical applications.