Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using “2A” Self-Cleaving Peptides

Simultaneous stoichiometric expression of multiple genes plays a major part in modern research and biotechnology. Traditional methods for incorporating multiple transgenes (or “gene stacking”) have drawbacks such as long time frames, uneven gene expression, gene silencing, and segregation derived from the use of multiple promoters. 2A self-cleaving peptides have emerged over the last two decades as a functional gene stacking method and have been used in plants for the co-expression of multiple genes under a single promoter. Here we describe design features of multicistronic polyproteins using 2A peptides for co-expression in plant cells and targeting to the endoplasmic reticulum (ER). We designed up to quad-cistronic vectors that could target proteins in tandem to the ER. We also exemplify the incorporation of self-excising intein domains within 2A polypeptides, to remove residue additions. These features could aid in the design of stoichiometric protein co-expression strategies in plants in combination with targeting to different subcellular compartments.