Expression of cholera toxin B subunit oligomers in transgenic potato plants.
A gene encoding the cholera toxin B subunit protein (CTB), fused to an endoplasmic reticulum (ER) retention signal (SEKDEL) was inserted adjacent to the bi-directional mannopine synthase P2 promoter in a plant expression vector containing a bacterial luciferase AB fusion gene (luxF) linked to the P1 promoter. Potato leaf explants were transformed by Agrobacterium tumefaciens carrying the vector and kanamycin-resistant plants were regenerated. The CTB-SEKDEL fusion gene was identified in the genomic DNA of bioluminescent plants by polymerase chain reaction amplification. Immunoblot analysis indicated that plant-derived CTB protein was antigenically indistinguishable from bacterial CTB protein, and that oligomeric CTB molecules (M(r) approximately 50 kDa) were the dominant molecular species isolated from transgenic potato leaf and tuber tissues. Similar to bacterial CTB, plant-synthesized CTB dissociated into monomers (M(r) approximately 15 kDa) during heat or acid treatment. The maximum amount of CTB protein detected in auxin-induced transgenic potato leaf and tuber tissues was approximately 0.3% of total soluble plant protein. Enzyme-linked immunosorbent assay methods indicated that plant-synthesized CTB protein bound specifically to GM1-ganglioside, the natural membrane receptor of cholera toxin. In the presence of the SEKDEL signal, CTB protein accumulates in potato tissues and is assembled into an oligomeric form that retains native biochemical and immunological properties. The expression of oligomeric CTB protein with immunological and biochemical properties identical to native CTB protein in edible plants opens the way for preparation of inexpensive food plant-based oral vaccines for protection against cholera and other pathogens in endemic areas throughout the world.
Arakawa
T
T
Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, CA 92350, USA.
Chong
D K
DK
Merritt
J L
JL
Langridge
W H
WH
D016960:Agrobacterium tumefaciens; D002772:Cholera Toxin / Q000096:biosynthesis*; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D011198:Solanum tuberosum
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin; D008156:Luciferases; D006836:Hydro-Lyases; C070395:mannopine synthase