The t2a peptide, initially discovered in foot-and-mouth disease virus (FMDV), mediates polycistronic expression of multiple genes through an efficient self-cleaving mechanism. Compared to other strategies for co-expression of multiple genes, the 2A peptide provides higher levels of downstream protein expression and less risk of interfering with function of other genes. It also enables the synthesis of a large number of proteins in a single vector assembly step, a significant advantage in the field of genetic engineering and synthetic biology.
In addition to their high expression levels, the 2A peptides produce a range of different products that may be important for cellular function. For example, in some viruses the 2A peptides produce proteins that act as enzymes, thereby contributing to host cell metabolism, whereas other 2A peptides have a role in cellular defense or signaling mechanisms.
Despite their versatility, the construction of polycistronic expression vectors utilizing 2A peptides remains challenging as the ligation of the 2A encoding DNA fragment and CDS is laborious, time-consuming and inefficient. This is because traditional cloning methods that use Type IIP endonucleases have difficulty selecting restriction sites to ligate the 2A and CDS without disrupting the ORF.
To alleviate these limitations, we have developed a robust and efficient modular system that allows the rapid and flexible construction of polycistronic vectors for plants. The modular vector is Golden Gate cloning compatible and provides the ability to express a large number of proteins simultaneously.
To evaluate the effectiveness of our modular vector system, we cloned a GFP-FP2A-mCherry-CAAX construct into the NotI/SalI sites of a pShuttle vector and subsequently transfected into HEK293T cells. Western blotting and flow cytometry were used to assess the level of protein expression mediated by each of the three FPs at various positions along the tri-cistronic constructs (Fig. 3C). FP expression was greatest at the first gene position and smallest at the second and third gene positions. This is largely consistent with the theory that naturally occurring ribosomes drop-off at a gradual rate, resulting in a reduction of protein expression along a vector.