ubiquitin-based vector for the co-ordinated synthesis of multiple proteins in plants

• Published in: Plant biotechnology journal 2007-05, Vol.5 (3), p.413-421
• Main Authors: Walker, Joseph M, Vierstra, Richard D
• Format: Article
• Language: English
• Subjects: expression vectors; Genes, Reporter; Genetic Vectors; Glucuronidase - metabolism; Luciferases - metabolism; Nicotiana - genetics; Plants, Genetically Modified - genetics; Plants, Genetically Modified - metabolism; Protein Engineering - methods; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; RNA, Messenger - chemistry; RNA, Messenger - metabolism; Saccharomyces cerevisiae - genetics; transgenic crops; ubiquitin; Ubiquitin - genetics; Ubiquitin - metabolism
• ISSN: 1467-7644; 1467-7652
• DOI: 10.1111/j.1467-7652.2007.00250.x

The genetic engineering of complex traits into crop plants will ultimately require strategies to co-express more than one protein at the same time. Here, we report the development of a ubiquitin (Ub)-based expression method that can generate two proteins from a single transcript. It contains coding regions for the proteins of interest, separated in-frame by the coding region for the C-terminal end of Ub followed by a full-length Ub. On expression in tobacco, this polycistronic messenger RNA (mRNA) is translated to produce a chimeric protein that is rapidly processed by endogenous deubiquitinating proteases to release the two proteins plus a Ub moiety in intact forms. The C-terminal protein domain is released without additional amino acids, whereas the N-terminal protein domain retains the short C-terminal end of Ub. The analysis of vectors with progressively shorter C-terminal ends indicates that only the last six C-terminal amino acids of the proximal Ub domain are needed for efficient processing in plants. By comparing the levels of luciferase and β-glucuronidase simultaneously expressed by this method in multiple independent tobacco transformants, we synthesized consistently similar ratios of the two proteins over a wide range of protein amounts. Ub-based polyprotein vectors should facilitate the genetic engineering of crops by providing a simple method for the co-ordinated and stoichiometric synthesis of two or more proteins.