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

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Abstract
Summary: 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.

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