A Trypanosoma cruzi polyantigen obtained by gene fusion: its expression in Staphylococcus aureus and rapid purification.
In order to simplify the large-scale production of three different Trypanosoma cruzi antigens with significant diagnosis value, their coding DNA fragments were fused to produce a single molecule. This tripartite protein was expressed using a shuttle Staphylococcal protein A (SPA) gene fusion vector. The resulting fusion protein location was intracellular when synthesized in Escherichia coli but was also proteolytically degraded during its purification. When the same construct was expressed using the Staphylococcus aureus secretion system, a nondegraded expression product was obtained from the culture medium. A "size effect" seemed to take place in the final yield. The SPA tripartite antigen fusion protein was purified in one step using IgG-Sepharose affinity chromatography. The SPA affinity tail was removed by specific proteolysis with enterokinase and further chromatography on IgG Sepharose. Specific antibodies against individual antigens reacted equally well with the purified tripartite antigen. These results suggest that the simultaneous production of several antigens in a single molecule using the S. aureus secretion system could be a good alternative, when a mixture of cloned antigens is necessary for a strict diagnosis or for immunization experiments.
Moreno
J I
JI
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 66160-7410, USA.
D000818:Animals; D000953:Antigens, Protozoan / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D013205:Staphylococcal Protein A / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000235:genetics / Q000276:immunology* / Q000378:metabolism
D000953:Antigens, Protozoan; D016054:DNA, Protozoan; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A