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Use of Ubiquitin Fusions to Augment Protein Expression in Transgenic Plants1

David Hondred2, 3, Joseph M. Walker2, Dennis E. Mathews4, and Richard D. Vierstra*

Cellular and Molecular Biology Program and the Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706

A major goal of plant biotechnology is the production of genetically engineered crops that express natural or foreign proteins at high levels. To enhance protein accumulation in transgenic plants, we developed a set of vectors that express proteins and peptides as C-terminal translational fusions with ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabacum) showed that: (a) proteins can be readily expressed in plants as UBQ fusions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused protein moieties in free forms; (c) the synthesis of a protein as a UBQ fusion can significantly augment its accumulation; (d) proper processing and localization of a protein targeted to either the apoplast or the chloroplast is not affected by the N-terminal UBQ sequence; and (e) single amino acid substitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps. Noncleavable UBQ fusions of beta -glucuronidase became extensively modified, with additional UBQs in planta. Because multiubiquitinated proteins are the preferred substrates of the 26S proteasome, noncleavable fusions may be useful for decreasing protein half-life. Based on their ability to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.

1   This work was supported the U.S. Department of Agriculture funded through the Consortium for Plant Biotechnology Research (grant no. 92-34190-6941) and the North Central Biotechnology Initiative (grant no. 94-34190-1204), Pioneer Hi-Bred International, Proctor & Gamble, ICI Seeds, Rhône Poulenc S.A., Agrigenetics, Dow Elanco, Northrup King, the Graduate School of the University of Wisconsin, and the Research Division of the University of Wisconsin, College of Agriculture and Life Sciences, Madison.
2   These authors contributed equally to this work.
3   Present address: Pioneer Hi-Bred International, 7300 NW 62nd Street, B.P.O. Box 1004, Johnston, IA 50131-1004.
4   Present address: Department of Plant Biology, 46 College Road, University of New Hampshire, Durham, NH 03824.
*   Corresponding author; e-mail vierstra{at}facstaff.wisc.edu; fax: 1-608-262-4743.

Plant Physiol. (1999) 119: 713-724
Copyright Clearance Center:   0032-0889/99/119//12
© 1999 American Society of Plant Physiologists




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