Book Chapter

Gene stacking and stoichiometric expression of ER-targeted constructs using “2A” self-cleaving peptides

Abstract

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.

Authors

Spatola Rossi, Tatiana
Fricker, Mark
Kriechbaumer , Verena

Oxford Brookes departments

Department of Biological and Medical Sciences

Dates

Year of publication: 2024
Date of RADAR deposit: 2024-03-06

---

All rights reserved.

---

Related resources

This RADAR resource is the Accepted Manuscript of Gene Stacking and Stoichiometric Expression of ER-Targeted Constructs Using “2A” Self-Cleaving Peptides
This RADAR resource is Part of The plant endoplasmic reticulum: methods and protocols [ISBN: 9781071637098] / edited by Verena Kriechbaumer (Springer, 2024).