Effect of codon optimization and subcellular targeting on Toxoplasma gondii antigen SAG1 expression in tobacco leaves to use in subcutaneous and oral immunization in mice

Abstract

BackgroundCodon optimization and subcellular targeting were studied with the aim to increase the expression levels of the SAG178-322 antigen of Toxoplasma gondii in tobacco leaves. The expression of the tobacco-optimized and native versions of the SAG1 gene was explored by transient expression from the Agrobacterium tumefaciens binary expression vector, which allows targeting the recombinant protein to the endoplasmic reticulum (ER) and the apoplast. Finally, mice were subcutaneously and orally immunized with leaf extracts-SAG1 and the strategy of prime boost with rSAG1 expressed in Escherichia coli was used to optimize the oral immunization with leaf extracts-SAG1.ResultsLeaves agroinfiltrated with an unmodified SAG1 gene accumulated 5- to 10-fold more than leaves agroinfiltrated with a codon-optimized SAG1 gene. ER localization allowed the accumulation of higher levels of native SAG1. However, no significant differences were observed between the mRNA accumulations of the different versions of SAG1. Subcutaneous immunization with leaf extracts-SAG1 (SAG1) protected mice against an oral challenge with a non-lethal cyst dose, and this effect could be associated with the secretion of significant levels of IFN-γ. The protection was increased when mice were ID boosted with rSAG1 (SAG1+boost). This group elicited a significant Th1 humoral and cellular immune response characterized by high levels of IFN-γ. In an oral immunization assay, the SAG1+boost group showed a significantly lower brain cyst burden compared to the rest of the groups.ConclusionTransient agroinfiltration was useful for the expression of all of the recombinant proteins tested. Our results support the usefulness of endoplasmic reticulum signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The results showed that this plant-produced protein has potential for use as vaccine and provides a potential means for protecting humans and animals against toxoplasmosis.

Expression of Antibody Fragments with a ControlledN-Glycosylation Pattern and Induction of Endoplasmic Reticulum-Derived Vesicles in Seeds of Arabidopsis

“…Bars = 200 nm (A, B, D, and E) and 1,000 nm (C). C-terminal KDEL tag, which has also been reported to additionally increase the accumulation level (Wandelt et al, 1992;Schouten et al, 1996;Fiedler et al, 1997;Ko et al, 2005;Petruccelli et al, 2006;Laguía-Becher et al, 2010). In our study, the addition of a KDEL tag increased the stability of the protein (especially for the HA78 constructs); however, the accumulation level actually decreased.…”

“…In this respect, a frequently reported approach is KDELmediated retention within the endoplasmic reticulum (ER), which seems to allow for increased protein accumulation (Wandelt et al, 1992;Fiedler et al, 1997;Ko et al, 2005;Petruccelli et al, 2006;Laguía-Becher et al, 2010).…”

Canonical histone H2Ba and H2A.X dimerize in an opposite genomic localization to H2A.Z/H2B.Z dimers in Toxoplasma gondii

“…Samples were boiled for 5 min and 20 µl was loaded per well in a 15% SDS-PAGE gel for immunoblotting. The absence of contaminating proteins was corroborated by Western blot with murine anti-SAG1antibody [22]. …”

Molecular farming on rescue of pharma industry for next generations

“…Codon optimization, however, may play positive or negative roles in the transcription and translation of heterologous proteins (Lico et al, 2012). For example, the codon optimization of the surface antigen SAG178-322, from Toxoplasma gondii, demonstrates that the codon optimization does not always result in an improved expression level, where the optimized codon of SAG1 resulted in an accumulation level 5-to 10-fold lower than a non-optimized codon (Laguia-Becher et al, 2010).…”