Expression of the native cholera toxin B subunit gene and assembly as functional oligomers in transgenic tobacco chloroplasts

Abstract

The B subunits of enterotoxigenic Escherichia coli (LTB) and cholera toxin of Vibrio cholerae (CTB) are candidate vaccine antigens. Integration of an unmodified CTB-coding sequence into chloroplast genomes (up to 10,000 copies per cell), resulted in the accumulation of up to 4.1 % of total soluble tobacco leaf protein as functional oligomers (410-fold higher expression levels than that of the unmodified LTB gene expressed via the nuclear genome). However, expression levels reported are an underestimation of actual accumulation of CTB in transgenic chloroplasts, due to aggregation of the oligomeric forms in unboiled samples similar to the aggregation observed for purified bacterial antigen. PCR and Southern blot analyses confirmed stable integration of the CTB gene into the chloroplast genome. Western blot analysis showed that the chloroplast- synthesized CTB assembled into oligomers and were antigenically identical with purified native CTB. Also, binding assays confirmed that chloroplast-synthesized CTB binds to the intestinal membrane GM1-ganglioside receptor, indicating correct folding and disulfide bond formation of CTB pentamers within transgenic chloroplasts. In contrast to stunted nuclear transgenic plants, chloroplast transgenic plants were morphologically indistinguishable from untransformed plants, when CTB was constitutively expressed in chloroplasts. Introduced genes were inherited stably in subsequent generations, as confirmed by PCR and Southern blot analyses. Increased production of an efficient transmucosal carrier molecule and delivery system, like CTB, in transgenic chloroplasts makes plant-based oral vaccines and fusion proteins with CTB needing oral administration commercially feasible. Successful expression of foreign genes in transgenic chromoplasts and availability of marker-free chloroplast transformation techniques augurs well for development of vaccines in edible parts of transgenic plants. Furthermore, since the quaternary structure of many proteins is essential for their function, this investigation demonstrates the potential for other foreign multimeric proteins to be properly expressed and assembled in transgenic chloroplasts.

Figure 1

Transformed chloroplast genome and PCR analysis of control and chloroplast transformants. (a) Chloroplast genome after homologous recombination with the pLD-LH-CTB vector. Flanking sequences involved in recombination are indicated in white. Primer 3P lands on the native chloroplast genome and 3M lands on the aadA gene, generating a 1.65 kb fragment. Primer 5P lands on the aadA gene and 2M lands on the trnA flanking sequence, generating a 1.9 kb fragment. (b) PCR analysis: primers 3P 3M generated PCR products using total plant DNA as template. Lane 1, molecular mass marker; lane 2, untransformed plant; lanes 3–12, PCR products with DNA template from independent transgenic lines 1–10. (c) PCR analysis: primers 5P 2M generated PCR products using total plant DNA as template. Lane 1, untransformed plant; lane 2, molecular mass marker; lanes 3–5, PCR products with DNA template from independent transgenic lines 1–3 and 5–10, lanes 3–11.

Figure 2

Western blot analysis of CTB expression in E. coli and transgenic chloroplasts. Blots were detected using rabbit anti-cholera serum as the primary antibody and alkaline phosphatase labeled mouse anti-rabbit IgG as the secondary antibody. (a) E. coli protein analysis: purified bacterial CTB (100 ng), boiled (lane 1); unboiled 24 hours and 48 hours transformed (lanes 2 and 4) and untransformed (lanes 3 and 5) E. coli cell extracts. (b) Plant protein analysis: color development detection. Boiled, untransformed plant proteins (lane 1); boiled, purified CTB antigen (100 ng, lane 2); boiled, proteins from four different transgenic lines (lanes 3–6); molecular mass markers (lane 7). (c) Chemiluminescent detection: Plant proteins: untransformed, unboiled (lane 1); untransformed, boiled (lane 2); transgenic lines 3 and 7, boiled (lanes 3 and 5); transgenic line 3, unboiled (lane 4); purified CTB antigen (100 ng), boiled (lane 6); purified CTB antigen (100 ng), unboiled (lane 7).

Figure 3

Southern blot analysis of T₀ and T₁ transgenic plants. (a) A representation of untransformed and transformed chloroplast genomes. Plant DNA was digested with BglII and hybridized with the 0.81 kb probe, which contained the chloroplast flanking sequences used for homologous recombination. Untransformed control plant chloroplast genomes should generate a 4.47 kb fragment and transformed plant chloroplast genomes should generate a 6.17 kb fragment. Southern blot results of (b) T₀ lines untransformed Petit Havana (lane 1); transgenic plants (lanes 2–4); and (c) T₁ lines transgenic plants (lanes 1–4) and untransformed Petit Havana (lane 5). P is the 16 S rRNA promotor and T is the psbA 3′ UTR.

Figure 4

(a) Plant phenotypes: 1, confirmed transgenic line 7; 2, untransformed Petit Havana. (b) Seedlings (ten day-old) of T₁ transgenic (1, 2 and 3) and untransformed plant (4) plated on 500 mg/l spectinomycin selection medium.

Figure 5

(a) CTB ELISA quantification: absorbance of CTB-antibody complex in known concentrations of total soluble plant protein was compared to absorbance of known concentration of bacterial CTB-antibody complex and the amount of CTB was expressed as a percentage of the total soluble plant protein. Total soluble plant protein from young, mature and old leaves of transgenic lines 3 and 7 was quantified. (b) CTB-GM1-ganglioside binding ELISA assays. Plates, coated first with GM1-gangliosides and BSA, respectively, were plated with total soluble plant protein from transgenic lines 3 and 7, untransformed plant total soluble protein and 300 ng of purified bacterial CTB. The absorbance of the GM1-ganglioside-CTB-antibody complex in each case was measured.