High‐yield expression of a viral peptide animal vaccine in transgenic tobacco chloroplasts

Abstract

SummaryThe 2L21 peptide, which confers protection to dogs against challenge with virulent canine parvovirus (CPV ), was expressed in tobacco chloroplasts as a C-terminal translational fusion with the cholera toxin B subunit (CTB) or the green fluorescent protein (GFP). Expression of recombinant proteins was dependent on plant age. A very high-yield production was achieved in mature plants at the time of full flowering (310 mg CTB-2L21 protein per plant).Both young and senescent plants accumulated lower amounts of recombinant proteins than mature plants. This shows the importance of the time of harvest when scaling up the process. The maximum level of CTB-2L21 was 7.49 mg /g fresh weight (equivalent to 31.1% of total soluble protein, TSP) and that of GFP-2L21 was 5.96 mg /g fresh weight (equivalent to 22.6% of TSP). The 2L21 inserted epitope could be detected with a CPV-neutralizing monoclonal antibody, indicating that the epitope is correctly presented at the C-terminus of the fusion proteins. The resulting chimera CTB-2L21 protein retained pentamerization and G M1 -ganglioside binding characteristics of the native CTB and induced antibodies able to recognize VP2 protein from CPV. To our knowledge, this is the first report of an animal vaccine epitope expression in transgenic chloroplasts. The high expression of antigens in chloroplasts would reduce the amount of plant material required for vaccination (∼100 mg for a dose of 500 µg antigen) and would permit encapsulation of freeze-dried material or pill formation.

Controlled Expression of Recombinant Proteins in Physcomitrella patens by a Conditional Heat-shock Promoter: a Tool for Plant Research and Biotechnology

“…Finally, the high rate at which high amounts of hsp17.3B-mediated recombinant proteins can accumulate in moss (2.3% of TSP in half a day) is of significant biotechnological interest. Higher yields have been reported using a chloroplast transgenic system in tobacco (31.1% TSP) (Molina et al, 2004) and a seed specific promoter in A. thaliana (36.5% TSP) (De Jaeger et al, 2002). However, the time required to achieve such amounts was 60 and at least 40 days, respectively.…”

Metabolic Engineering of the Chloroplast Genome Using the Echerichia coli ubiC Gene Reveals That Chorismate Is a Readily Abundant Plant Precursor for p-Hydroxybenzoic Acid Biosynthesis

“…Because of these advantages the chloroplast genome has been engineered to confer several useful agronomic traits, including herbicide resistance (Daniell et al, 1998), insect resistance (McBride et al, 1995;Kota et al, 1999), disease resistance (DeGray et al, 2001), drought tolerance (Lee et al, 2003), salt tolerance , and phytoremediation (Ruiz et al, 2003). The chloroplast genome has also been used in molecular farming to express human therapeutic proteins (Guda et al, 2000;Staub et al, 2000;Fernandez-San Millan et al, 2003;Daniell et al, 2004bDaniell et al, , 2004c, vaccines for human (Daniell et al, 2001a(Daniell et al, , 2004cDaniell, 2004;Watson et al, 2004) or animal use (Molina et al, 2004), and biomaterials (Guda et al, 2000;Lossl et al, 2003). Although most of these studies were done in tobacco, highly efficient stable plastid transformation of major crop species has recently been reported for carrot , cotton (Gossypium hirsutum; Kumar et al, 2004b), and soybean (Glycine max; Dufourmantel et al, 2004).…”

Feasibility Study of Rice Growth in Plant Factories

“…Proteins can achieve high expression levels due to the high copy-number of transgenes per cell. It has been reported that an expression level of up to 30% of total soluble protein for an animal vaccine can be produced in transgenic tobacco plants [18]. Other plants have also been used, including rice [11,19,20], wheat [20], maize [21] and oilseed rape [22][23][24], since it is considered that full-scale commercial production could involve grain and oilseed crops.…”