High yield expression and single-step purification of Toxoplasma gondii SAG1, GRA1, and GRA7 antigens in Escherichia coli
Section snippets
Bacterial strains, plasmids, enzymes, and reagent
The E. coli TOP10F′ strain (Invitrogen, Carlsbad, CA) was used for the preparation of plasmids and cloning, and E. coli BL21(DE3)pLysS (Promega, Madison, WI) was applied to expression of recombinant antigens. The pUET1 (DNA-Gdańsk II s.c. Poland) plasmid was used for the construction of the expression system. The E. coli cells with plasmids were cultured aerobically at 37 °C in LB medium supplemented with tetracycline or ampicillin for the TOP10F′ strain and with
Expression and purification of the recombinant antigens
The recombinant plasmids pUETΔSAG1, pUETΔGRA1, and pUETΔGRA7 were transformed into E. coli BL21(DE3) pLysS. The pLysS plasmid, which expresses T7 lysozyme in the bacterial cytoplasm, strongly represses protein expression from the pET vectors in the absence of induction, thus, enabling expression of very toxic proteins. We found that colonies could easily be obtained at 37 °C. However, the growth of cells containing these expression plasmids was slightly slower in LB medium supplemented with the
Discussion
In this study, the DNA sequences encoding fragments of the T. gondii SAG1, GRA1, and GRA7 antigens were cloned in T7 promoter-based expression vector and protocols for high-level expression were optimized. The recombinant antigens could easily be purified using one-step chromatography procedure. The expression systems used provide an easy means to produce a large amount of immunologically active recombinant T. gondii proteins.
There have been several reports of expression and purification of the
Acknowledgements
The work was supported by the State Committee for Scientific Research Grants 4 PO5A 099 17 to J.K. and 4 PO5A 103 18 to H.P.
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