Expression of a recombinant Toxoplasma gondii ROP2 fragment as a fusion protein in bacteria circumvents insolubility and proteolytic degradation

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxopla sma gondii rhoptry protein ROP2 (recROP2(t), residues 196-464) was expresse d in Escherichia coli. This recombinant; fragment was produced at low conce ntration and in a highly insoluble form. By contrast, the level of recROP2( t) production was drastically greater when the same coding sequence was fus ed 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 m ore efffcient than TRX in increasing the recovery of soluble protein with m ore than 10% of total MBP-recROP2(t) being readily expressed in a soluble f orm. Moreover, the insoluble form of MBP-recROP2(t) could be correctly refo lded with a recovery of more than 80%, Both forms of MBP-recROP2(t) were pu rified to homogeneity by amylose chromatography. In contrast, the refolding of TRX-recROP2(t) promoted aggregation of the protein, which was prevented by the use of zwitterionic detergent during the one-step purification by g el filtration. Subsequent proteolytic cleavages of purified TRX-recROP2(t) and of MBP-recROP2(t) led respectively to the complete degradation or to th e truncation of the recROP2(t) moiety. However, recROP2(t), despite the pre sence of the fusion partners, adopted a suitable conformation recognized by human serum-derived antibodies from T. gondii-seropositive individuals. Fi nally, both fusion proteins were able to induce specific humoral and cell-m ediated immune response to the ROP2 fragment. Such fusions could represent an alternative to study the immunogenicity of T. gondii proteins which are difficult to produce because of insolubility and degradation. (C) 1999 Acad emic Press.