title: A strategy for targeting recombinant proteins to protein storage vacuoles by fusion to Brassica napus napin in napin-depleted seeds.
abstract: Seeds are capable of accumulating high levels of seed storage proteins (SSP), as well as heterologous proteins under certain conditions. Arabidopsis thaliana was used to develop a strategy to deplete seeds of an endogenous SSP and then replenish them with the same protein fused to a heterologous protein. In several other studies, competition with endogenous SSP for space and metabolic resources was shown to affect the accumulation of recombinant proteins in seeds. We used RNAi to reduce the expression of the five napin genes and deplete the seeds of this SSP. Targeting a recombinant protein to a vacuole or structure within the seed where it can be protected from cytosolic proteases can also promote its accumulation. To achieve this, a synthetic Brassica napus napin gene (Bn napin) was designed that was both impervious to the A. thaliana napin (At napin) RNAi construct and permitted fusion to a heterologous protein, in this case green fluorescent protein (GFP). GFP was placed in several strategic locations within Bn napin with consideration to maintaining structure, processing sites and possible vacuolar targeting signals. In transgenic A. thaliana plants, GFP was strongly localized to the seed protein storage vacuole in all Bn napin fusion configurations tested, but not when expressed alone. This SSP depletion-replenishment strategy outlined here would be applicable to expression of recombinant proteins in industrial crops that generally have large repertoires of endogenous SSP genes.
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada. Electronic address: Dwayne.Hegedus@agr.agr.ca.
authors.forename: Dwayne D
authors.initials: DD
authors.lastname: Hegedus
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada.
authors.forename: Marcus
authors.initials: M
authors.lastname: Baron
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada.
authors.forename: Natalie
authors.initials: N
authors.lastname: Labbe
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada.
authors.forename: Cathy
authors.initials: C
authors.lastname: Coutu
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada.
authors.forename: Derek
authors.initials: D
authors.lastname: Lydiate
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada.
authors.forename: Helen
authors.initials: H
authors.lastname: Lui
authors.affiliation: Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK S7N OX2, Canada.
authors.forename: Kevin
authors.initials: K
authors.lastname: Rozwadowski
mesh_terms: D055516:2S Albumins, Plant / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D029688:Brassica napus / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D015202:Protein Engineering; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
chemical_list: D055516:2S Albumins, Plant; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C050363:napin protein, Brassica napus
keywords: GFP fusion; Napin; Plant host; RNAi; Recombinant protein expression; Seeds