Use of ubiquitin fusions to augment protein expression in transgenic plants

A major goal of plant biotechnology is the production of genetically engine ered crops that express natural or foreign proteins at high levels. To enha nce protein accumulation in transgenic plants, we developed a set of vector s that express proteins and peptides as C-terminal translational fusions wi th ubiquitin (UBQ). Studies of several proteins in tobacco (Nicotiana tabac um) showed that: (a) proteins can be readily expressed in plants as UBQ fus ions; (b) by the action of endogenous UBQ-specific proteases (Ubps), these fusions are rapidly and precisely processed in vivo to release the fused pr otein moieties in free forms; (c) the synthesis of a protein as a UBQ fusio n can significantly augment its accumulation; (d) proper processing and loc alization of a protein targeted to either the apoplast or the chloroplast i s not affected by the N-terminal UBQ Sequence; and (e) single amino acid su bstitutions surrounding the cleavage site can inhibit in vivo processing of the fusion by Ubps. Noncleavable UBQ fusions of p-glucuronidase became ext ensively modified, with additional UBQs in planta. Because multiubiquitinat ed proteins are the preferred substrates of the 26S proteasome, noncleavabl e fusions may be useful for decreasing protein half-life. Based on their ab ility to augment protein accumulation and the sequence specificity of Ubps, UBQ fusions offer a versatile way to express plant proteins.