Expression of a Recombinant Toxoplasma gondii ROP2 Fragment as a Fusion Protein in Bacteria Circumvents Insolubility and Proteolytic Degradation

• Published in: Protein expression and purification 1999-12, Vol.17 (3), p.392-400
• Main Authors: Jacquet, Alain, Daminet, Véronique, Haumont, Michèle, Garcia, Lida, Chaudoir, Sylvie, Bollen, Alex, Biemans, Ralph
• Format: Article
• Language: English
• Subjects: Animals; Antibodies - blood; Antigens, Protozoan - biosynthesis; Antigens, Protozoan - chemistry; Antigens, Protozoan - genetics; Antigens, Protozoan - immunology; ATP-Binding Cassette Transporters; Carrier Proteins - genetics; Cell Division - immunology; Enteropeptidase - chemistry; Enzyme-Linked Immunosorbent Assay; Escherichia coli - metabolism; Escherichia coli Proteins; Factor Xa - chemistry; Female; Genetic Vectors; Humans; Lymphocytes - cytology; Lymphocytes - immunology; Maltose-Binding Proteins; Membrane Proteins - biosynthesis; Membrane Proteins - chemistry; Membrane Proteins - genetics; Membrane Proteins - immunology; Mice; Mice, Inbred BALB C; Monosaccharide Transport Proteins; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Protozoan Proteins - biosynthesis; Protozoan Proteins - chemistry; Protozoan Proteins - genetics; Protozoan Proteins - immunology; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - immunology; Sequence Analysis, Protein; Spleen - cytology; Spleen - immunology; Thioredoxins - genetics; Toxoplasma - metabolism
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1006/prep.1999.1150

A 268-amino-acid-residue carboxy-terminal antigenic fragment of the Toxoplasma gondii rhoptry 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 recROP2t production 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–recROP2t being readily expressed in a soluble form. Moreover, the insoluble form of MBP–recROP2t could be correctly refolded with a recovery of more than 80%. Both forms of MBP–recROP2t were purified to homogeneity by amylose chromatography. In contrast, the refolding of TRX–recROP2t promoted 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–recROP2t and of MBP–recROP2t led respectively to the complete degradation or to the truncation of the recROP2t moiety. 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. gondii proteins which are difficult to produce because of insolubility and degradation.