Expression of His-tagged Shigella IpaC in Arabidopsis.
Although the expression of histidine (His)-tagged proteins in bacteria is routine, few His-tagged proteins have been expressed in plants, and no His-tagged proteins from bacterial pathogens have been expressed in plants, to our knowledge. Here, we demonstrate expression of the Shigella flexneri invasion plasmid antigen, IpaC, in Arabidopsis thaliana. S. flexneri is the causitive trigger for bacillary dysentery, and IpaC is essential for bacterial entry into epithelial cells. IpaC, attached to a 5' leader containing six tandem His codons, was cloned into a pBI121 vector. This clone was introduced into Agrobacterium tumefaciens and Arabidopsis plants were then transformed. T1 and T2 plant generations were obtained. Total plant proteins were extracted from T2 leaves; the Bradford assay was used to determine protein concentrations. A nickel-coated ELISA plate method, using both anti-His and anti-IpaC 1 degrees antibodies, was used to detect and quantify IpaC in transgenic Arabidopsis plants. Between 1.9 and 2.3 microg IpaC/mg total plant protein was obtained; this equals 0.2% of total protein, an amount comparable to other recombinant protein estimates in plants. Expressing His-tagged proteins from bacterial pathogens, in plants, is important because plant material could ultimately be fed or applied intranasally to animals that are "at risk" for infection by such bacterial pathogens, thus causing them to raise antibodies against the pathogens--functioning as a vaccine.
Department of Biology, 128 Macelwane Hall, Saint Louis University, 3507 Laclede Avenue, Saint Louis, MO 63103, USA. amacrae@swbell.net
Amy F
AF
MacRae
Johanna
J
Preiszner
Shirley
S
Ng
Robert I
RI
Bolla
D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000235:genetics*; D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular / Q000379:methods; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D018506:Gene Expression Regulation, Plant / Q000502:physiology; D006639:Histidine / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012760:Shigella / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D000942:Antigens, Bacterial; D011994:Recombinant Proteins; C092342:IpaC protein, Shigella; D006639:Histidine