Expression of a Recombinant Toxoplasma gondiiROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

Autor: Jacquet, Alain, Daminet, Véronique, Haumont, Michèle, Garcia, Lida, Chaudoir, Sylvie, Bollen, Alex, Biemans, Ralph
Zdroj: Protein Expression and Purification; December 1999, Vol. 17 Issue: 3 p392-400, 9p
Abstrakt: A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondiirhoptry protein ROP2 (recROP2t, residues 196–464) was expressed in Escherichia coli.This recombinant fragment was produced at low concentration and in a highly insoluble form. By contrast, the level of recROP2tproduction was drastically greater when the same coding sequence was fused to the C-terminus of thioredoxin (TRX) or to the maltose-binding protein (MBP) gene. While both fusion proteins were found to be mainly insoluble, solubilization could be achieved without significant degradation. MBP was more efficient than TRX in increasing the recovery of soluble protein with more than 10% of total MBP–recROP2tbeing readily expressed in a soluble form. Moreover, the insoluble form of MBP–recROP2tcould be correctly refolded with a recovery of more than 80%. Both forms of MBP–recROP2twere purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX–recROP2tpromoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by gel filtration. Subsequent proteolytic cleavages of purified TRX–recROP2tand of MBP–recROP2tled respectively to the complete degradation or to the truncation of the recROP2tmoiety. However, recROP2t, despite the presence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Finally, both fusion proteins were able to induce specific humoral and cell-mediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondiiproteins which are difficult to produce because of insolubility and degradation.
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