title: Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants.
abstract: The levels of accumulation of recombinant vaccines in transgenic plants are protein specific and strongly influenced by the subcellular compartment of destination. The human immunodeficiency virus protein Nef (negative factor), a promising target for the development of an antiviral vaccine, is a cytosolic protein that accumulates to low levels in transgenic tobacco and is even more unstable when introduced into the secretory pathway, probably because of folding defects in the non-cytosolic environment. To improve Nef accumulation, a new strategy was developed to anchor the molecule to the cytosolic face of the endoplasmic reticulum (ER) membrane. For this purpose, the Nef sequence was fused to the C-terminal domain of mammalian ER cytochrome b5, a long-lived, tail-anchored (TA) protein. This consistently increased Nef accumulation by more than threefold in many independent transgenic tobacco plants. Real-time polymerase chain reaction of mRNA levels and protein pulse-chase analysis indicated that the increase was not caused by higher transcript levels but by enhanced protein stability. Subcellular fractionation and immunocytochemistry indicated that Nef-TA accumulated on the ER membrane. Over-expression of mammalian or plant ER cytochrome b5 caused the formation of stacked membrane structures, as observed previously in similar experiments performed in mammalian cells; however, Nef-TA did not alter membrane organization in tobacco cells. Finally, Nef could be removed in vitro by its tail-anchor, taking advantage of an engineered thrombin cleavage site. These results open up the way to use tail-anchors to improve foreign protein stability in the plant cytosol without perturbing cellular functions.
authors.affiliation: Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133 Milan, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Barbante
authors.forename: Sarah
authors.initials: S
authors.lastname: Irons
authors.forename: Chris
authors.initials: C
authors.lastname: Hawes
authors.forename: Lorenzo
authors.initials: L
authors.lastname: Frigerio
authors.forename: Alessandro
authors.initials: A
authors.lastname: Vitale
authors.forename: Emanuela
authors.initials: E
authors.lastname: Pedrazzini
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism / Q000648:ultrastructure; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015693:Gene Products, nef / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000956:Antigens, Viral; D015693:Gene Products, nef; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5