High level of expression of the Toxoplasma gondii-recombinant Rop2 protein in Escherichia coli as a soluble form for optimal use in diagnosis.
The rhoptry 2 protein (Rop2) is an interesting protein of Toxoplasma gondii that is involved in the parasite invasion of host cell, it has three T-cell epitopes and high antigenic value. However, the expression of Rop2 as a recombinant protein in Escherichia coli is not an easy task, showing low levels of expression or degradation and solubility problems. Using a recombinant Rop2(196-561) fused to 6 histidine residues, we showed high levels of expression in bacteria growing in terrific broth. rRop2(196-561) was purified mainly as a soluble product and in high concentrations (approx 1 mg/mL) under native conditions (40 mM imidazol in phosphate buffer). However, after a cycle of freezing-thawing rRop2(196-561) became insoluble. When glycerol was added to 26%, immediately after purification, the protein stayed soluble after cycles of freezing-thawing. Finally, it was demonstrated that under these conditions soluble rRop2(196-561) keeps its diagnostic value in contrast with the insoluble protein.
Nigro
M
M
Departamento de Parasitología Sanitaria, Instituto Nacional de Parasitología Dr. M. Fatala Chaben, ANLIS Dr. Carlos G. Malbrán, Av. Velez Sarsfield 563, 1281-Buenos Aires, Argentina.
Martin
V
V
Kaufer
F
F
Carral
L
L
Angel
S O
SO
Pszenny
V
V
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics*; D004926:Escherichia coli; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012995:Solubility; D014122:Toxoplasma / Q000235:genetics*; D014123:Toxoplasmosis / Q000175:diagnosis
D000953:Antigens, Protozoan; D008565:Membrane Proteins; D015800:Protozoan Proteins; C090555:ROP 2 protein, Toxoplasma gondii; D011993:Recombinant Fusion Proteins