Enhanced expression of B-subunit of Escherichia coli heat-labile enterotoxin in tobacco by optimization of coding sequence.
Escherichia coli heat-labile toxin (LT) is a potent mucosal immunogen and immunoadjuvant for coadministered antigens. We synthesized a gene encoding the B-subunit of LT (LTB) adapted to the coding sequence of tobacco plants and fused to the endoplasmic reticulum retention signal SEKDEL to enhance its level of expression in plants. The synthetic LTB gene was cloned into a plant expression vector adjacent to the CaMV 35S promoter and was introduced into tobacco by Agrobacterium-mediated transformation. The amount of LTB protein detected in transgenic tobacco leaves was 2.2% of the total soluble plant protein, which is approx 200-fold higher than in previous reports of native LTB gene expression in transgenic plants. Enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, suggesting that the LTB subunits formed active pentamers.
Kang
Tae-Jin
TJ
Institute of Basic Sciences, Chonbuk National University, Jeonju 561-756, South Korea.
Han
So-Chon
SC
Jang
Mi-Ok
MO
Kang
Kui-Hyeon
KH
Jang
Yong-Suk
YS
Yang
Moon-Sik
MS
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001427:Bacterial Toxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D004768:Enterotoxins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D005813:Genes, Synthetic; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D005677:G(M1) Ganglioside; C027538:heat-labile enterotoxin, E coli