Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: a component of the P1/P2/P0 complex.
The P0 protein is part of the ribosomal eukaryotic stalk, which is an elongated lateral protuberance of the large ribosomal subunit involved in the translocation step of protein synthesis. P0 is the minimal portion of the stalk that is able to support accurate protein synthesis. The P0 C-terminal peptide is highly antigenic and a major target of the antibody response in patients with systemic lupus erythematosus and patients suffering chronic heart disease produced by the Trypanosoma cruzi parasite. The T. cruzi P0 (TcP0) protein was cloned into the pRSET A vector and expressed in Escherichia coli fused to a His-tag. The identity of the protein was confirmed by immunoblotting. Due to the formation of inclusion bodies the protein was purified using the following steps: (i) differential centrifugation to separate the inclusion bodies from soluble proteins and (ii) affinity chromatography under denaturing conditions. TcP0 showed high tendency to aggregation during refolding assays. However, TcP0 could be efficiently folded in the presence of a low concentration of SDS. The folding of the protein was confirmed using urea gradient electrophoresis, limited proteolysis, circular dichroism, and tryptophan fluorescence. Native electrophoresis showed that the folded TcP0 (and not a folding intermediate) was the cause of aggregation in the absence of SDS. The protocol described here permitted us to obtain large amounts (up to 30 mg per culture liter) of pure and folded TcP0, a very hydrophobic protein with a high tendency to aggregation.
Juri Ayub
M
M
Laboratorio de Biología Molecular Estructural, Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de San Luis, Ejército de los Andes 950, San Luis, 5700, Argentina.
Levin
M J
MJ
Aguilar
C F
CF
D000818:Animals; D002942:Circular Dichroism; D005822:Genetic Vectors; D046911:Macromolecular Substances; D010750:Phosphoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D014349:Trypanosoma cruzi / Q000737:chemistry*
D046911:Macromolecular Substances; D010750:Phosphoproteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C031937:phosphoprotein P2, ribosomal; C084989:ribosomal PO protein, Trypanosoma cruzi; C035690:ribosomal phosphoprotein P1; C054675:ribosomal protein P0