Anchorage to the cytosolic face of the endoplasmic reticulum membrane: a new strategy to stabilize a cytosolic recombinant antigen in plants.
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.
Barbante
Alessandra
A
Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle Ricerche, Via Bassini 15, 20133 Milan, Italy.
Irons
Sarah
S
Hawes
Chris
C
Frigerio
Lorenzo
L
Vitale
Alessandro
A
Pedrazzini
Emanuela
E
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
D000956:Antigens, Viral; D015693:Gene Products, nef; D011993:Recombinant Fusion Proteins; D015786:Cytochromes b5