High-level expression of the HIV-1 Pr55gag polyprotein in transgenic tobacco chloroplasts.
Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency virus Gag (Pr55(gag)) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55(gag) expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments, the native Pr55(gag) sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the 5'-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7-8% TSP, equivalent to 312-363 mg/kg FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55(gag) polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55(gag) polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.
Scotti
Nunzia
N
CNR-IGV, Institute of Plant Genetics, National Research Council, Via Università 133, 80055 Portici, Italy. nscotti@unina.it
Alagna
Fiammetta
F
Ferraiolo
Enrico
E
Formisano
Gelsomina
G
Sannino
Lorenza
L
Buonaguro
Luigi
L
De Stradis
Angelo
A
Vitale
Alessandro
A
Monti
Luigi
L
Grillo
Stefania
S
Buonaguro
Franco M
FM
Cardi
Teodoro
T
D002736:Chloroplasts / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D018506:Gene Expression Regulation, Plant; D005822:Genetic Vectors / Q000235:genetics; D015497:HIV-1 / Q000378:metabolism*; D010641:Phenotype; D018515:Plant Leaves / Q000378:metabolism / Q000648:ultrastructure; D030821:Plants, Genetically Modified; D020815:Polyproteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000254:growth & development; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D019076:Transgenes; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
D020815:Polyproteins; D011994:Recombinant Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus