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 Toxoplasma gondii rhoptry protein ROP2 (recROP2(t), 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 recROP2(t) 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-recROP2(t) being readily expressed in a soluble form. Moreover, the insoluble form of MBP-recROP2(t) could be correctly refolded with a recovery of more than 80%. Both forms of MBP-recROP2(t) were purified 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 gel filtration. Subsequent proteolytic cleavages of purified TRX-recROP2(t) and of MBP-recROP2(t) led respectively to the complete degradation or to the truncation of the recROP2(t) moiety. However, recROP2(t), 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.
Jacquet
A
A
Department of Applied Genetics, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, Gosselies, B-6041, Belgium. ajacquet@sga.ulb.ac.be
Daminet
V
V
Haumont
M
M
Garcia
L
L
Chaudoir
S
S
Bollen
A
A
Biemans
R
R
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D000906:Antibodies / Q000097:blood; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D002352:Carrier Proteins / Q000235:genetics; D002455:Cell Division / Q000276:immunology; D004765:Enteropeptidase / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D015951:Factor Xa / Q000737:chemistry; D005260:Female; D005822:Genetic Vectors; D006801:Humans; D008214:Lymphocytes / Q000166:cytology / Q000276:immunology; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D020539:Sequence Analysis, Protein; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013879:Thioredoxins / Q000235:genetics; D014122:Toxoplasma / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D000906:Antibodies; D000953:Antigens, Protozoan; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D010446:Peptide Fragments; D015800:Protozoan Proteins; C090555:ROP 2 protein, Toxoplasma gondii; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D013879:Thioredoxins; D015951:Factor Xa; D004765:Enteropeptidase