The diagnostic performance of recombinant Trypanosoma cruzi ribosomal P2beta protein is influenced by its expression system.
In the present work, we have determined the effect of expression vectors and their corresponding host bacteria on the antigenic performance of Trypanosoma cruzi P2beta (TcP2beta) full-length recombinant protein. The gene encoding the TcP2beta ribosomal protein was cloned in pMAL-c2 and pET-32a vectors that allow the expression of high levels of soluble fusion proteins. A panel of 32 positive and 32 negative sera was assayed with the purified proteins expressed using pMal-c2 (TcP2beta-MBP) and pET-32a (TcP2beta-TRX) vectors and with MBP and TRX purified from pMAL-c2 and pET-32a vectors, respectively. The antigenic behavior of each TcP2beta recombinant protein differed in the diagnostic performance in terms of DI(+) (93.7 for TcP2beta-MBP vs 100% for TcP2beta-TRX), in DI(-) (90.5 for TcP2beta-MBP vs 100% for TcP2beta-TRX) and in cross-reaction with negative sera. To determine if the higher reactivity of expressed pMAL-c2 protein was due to folding during protein expression or to a steric effect related to the protein adsorption at the titration plate, the reactivity of sera against soluble proteins was assessed by ELISA inhibition assays. As each soluble protein preserved its level of reactivity, we concluded that differences in reactivity were due to intrinsic characteristics of the proteins and not to differences in patterns of adsorption to the plates.
Marcipar
Iván S
IS
Instituto de Tecnología Biológica (INTEBIO), Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Santa Fe, Argentina. imarcipr@fbcb.unl.edu.ar
Olivares
María Laura
ML
Robles
Lucía
L
Dekanty
Andrés
A
Marcipar
Alberto
A
Silber
Ariel M
AM
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics; D014355:Chagas Disease / Q000175:diagnosis*; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D058619:Maltose-Binding Proteins; D010750:Phosphoproteins / Q000235:genetics / Q000276:immunology; D016133:Polymerase Chain Reaction; D015800:Protozoan Proteins / Q000235:genetics / Q000276:immunology; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012269:Ribosomal Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D012698:Serologic Tests; D013879:Thioredoxins / Q000235:genetics; D014349:Trypanosoma cruzi / Q000737:chemistry / Q000235:genetics* / Q000276:immunology
D002352:Carrier Proteins; D018076:DNA, Complementary; D058619:Maltose-Binding Proteins; D010750:Phosphoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C031937:phosphoprotein P2, ribosomal; C033223:polyhistidine; D006639:Histidine; D013879:Thioredoxins