Synthesis and assembly of cholera holotoxin in potato plants

DNA fragments encoding the cholera toxin (CTX) A subunit (CTA) and B subunit (CTB) were transferred into Solanum tuberosum leaf cells under control of the bi-directional mannopine synthase P1P2 promoters by Agrobacterium tumefaciens-mediated leaf disc transformation methods. The presence of both CTA and CTB genes in the transformed plant genome was confirmed by PCR methods. Recombinant CTA and CTB proteins were detected in transformed tuber tissue extracts by immunoblot analysis. Plant synthesized CTA migrated on SDS-PAGE gels identically to purified bacterial CTA. In contrast to bacterial CTA, plant synthesized CTA was resistant to thiol-reduction. The oligomeric form of recombinant CTB was detected on SDS-PAGE gels by immunoblotting. A large protein band with a molecular mass (MW ∼ 80 kDa) equivalent to the sum of CTA and pentameric CTB molecules was detected in transformed plant tuber extracts by both anti-CTX and anti-CTA antibodies. In contrast to bacterial CTX, this protein complex was relatively stable in the presence of SDS. Similar to bacterial CTX, the 80 kDa oligomeric protein was not detected in the presence of β-mercaptoethanol. Taken together the estimated molecular mass of the ologomeric protein, the strong immunological reaction with both CTA and CTB specific antibodies, and disassociation of the ologomer in the presence of reducing agents indicate that the 80 kDa protein synthesized in transformed potato cells is homologous to cholera holotoxin.