A ubiquitin-based vector for the co-ordinated synthesis of multiple proteins in plants.
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 beta-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.
Walker
Joseph M
JM
Department of Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA.
Vierstra
Richard D
RD
D017930:Genes, Reporter; D005822:Genetic Vectors; D005966:Glucuronidase / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014026:Nicotiana / Q000235:genetics*; D025801:Ubiquitin / Q000235:genetics* / Q000378:metabolism
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D008156:Luciferases; D005966:Glucuronidase