Optimization of Expression and Purification of Schistosoma mansoni Antigens in Fusion with Rhizavidin

Abstract

Schistosomiasis causes significant morbidity and mortality. Vaccine efforts to date indicate the need to increase the immunogenicity of Schistosoma antigens. The multiple antigen-presenting system, whereby proteins are genetically fused to rhizavidin and affinity linked to biotinylated templates, enables the generation of robust immune responses. The objective of this work was to express and purify the S. mansoni antigens, SmTSP-2 and SmCD59.2, in fusion with rhizavidin. The fusion with rhizavidin greatly decreased the expression level of rSmTSP-2, but not rSmCD59.2, and both were expressed in the insoluble fraction, requiring optimization of culture conditions. Evaluation of different E. coli strains and media showed that BL21-DE3 cultured in Terrific Broth provided the highest expression levels of both proteins. Investigation of a range of time and temperature of induction showed that E. coli strains expressing rRzv:SmTSP-2 and rRzv:SmCD59.2 showed the highest protein production at 23 °C for 15 h. Recombinant proteins were purified by a single step of affinity chromatography allowing isolation of these proteins in high concentration and purity. The optimization process increased final soluble protein yield of rRzv:SmTSP-2 by fourfold and rRzv:SmCD59.2 by tenfold, providing ~ 20 mg/L of each protein. Optimized fusion protein production will allow antigen use in biotin-rhizavidin affinity platforms.

Keywords: Fusion proteins; Protein expression; Protein purification; Rhizavidin–biotin system; Schistosoma proteins.

Fig. 1

Protein expression of rSmAg with and without fusion to rhizavidin. A SDS-PAGE analysis of the expression of rSmTSP-2, rSmCD59.2, rRzv:SmTSP-2, and rRzv:SmCD59.2 by E. coli BL21 (DE3) in 2YT medium. Proteins were separated in 15% SDS-PAGE and stained with Coomassie Blue. B Comparison between the band area values calculated by densitometry in the ImageJ program. Statistical analysis by “non-parametric t-test” in the Graph Prism program. S soluble fraction, I inclusion bodies fraction, NS no statistical difference; *P value > 0.05. Red arrows show the region of the protein band

Fig. 2

Expression of rRzv:SmTSP-2 and rRzv:SmCD59.2 in E. coli BL21 (DE3), BL21 Star (DE3) pLysS, BL21–SI and BL21-CodonPlus (DE3) RIL as determined by SDS-PAGE. A rRzv:SmTSP-2 and B rRzv:SmCD59.2; cell concentration was normalized by OD₆₀₀, separated in 15% SDS-PAGE and stained by Coomassie Blue. N.I. not induced, IND. induced, S soluble fraction, I inclusion bodies fraction; Red arrows show the region of the protein band

Fig. 3

Analysis of different culture media. A SDS-PAGE analysis of rRzv:SmTSP-2 and rRzv:SmCD59.2 expression by E. coli BL21 (DE3) in 2YT, 2HKII, and TB medium. Proteins were separated in 15% SDS-PAGE and stained by Coomassie Blue. B Western blot analysis of rRzv:SmTSP-2 and rRzv:SmCD59.2 expression by E. coli BL21 (DE3) in TB medium. N.I. not induced, IND. induced, S soluble fraction, I inclusion bodies fraction; Red arrows show the region protein band

Fig. 4

Production of rRzv:SmTSP-2 and rRzv:SmCD59.2 proteins varying the temperature and induction time. A The final OD_600nm values obtained in culture condition varying temperature (16, 23, and 30 °C) and induction time (12, 15, and 18 h). B Productivity as determined by protein expression levels (calculated area of Western blot bands by densitometry). Densitometry was performed using the program ImageJ and the results were normalized in relation to the largest calculated area. h hour, Temp. Temperature, N normalized values

Fig. 5

SDS-PAGE and Western blot of purified rRzv: rSmTSP-2 and rRzv: SmCD59.2 proteins. A Purified rRzv: SmTSP-2 and B Purified rRzv: SmCD59.2; Left box contains SDS-PAGE of the purified proteins, and right box is the Western blot performed with specific antibody against each protein. SDS SDS-PAGE, WB Western blot