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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. © 2001 Academic Press.


Documento: Artículo
Título:Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex
Autor:Juri Ayub, M.; Levin, M.J.; Aguilar, C.F.
Filiación:Laboratorio De Biología Molecular Estructural, Facultad De Química, Universidad Nacional De San Luis, Ejército de los Andes 950, 5700, San Luis, Argentina
Instituto De Investigaciones En Ingeniería Genética, Biología Molecular (INGEBI), Vuelta de Obligado 2490, 1428, Buenos Aires, Argentina
Palabras clave:antigenicity; carboxy terminal sequence; cell inclusion; chronic disease; cloning; expression vector; gene overexpression; heart disease; histidine; hybrid protein; protein aggregation; protein folding; protein function; protein purification; protein subunit; ribosome protein; systemic lupus erythematosus; Escherichia coli; Escherichia coli; Eukaryota; Trypanosoma; Trypanosoma cruzi; Trypanosoma cruzi
Página de inicio:225
Página de fin:233
Título revista:Protein Expression and Purification
Título revista abreviado:Protein Expr. Purif.


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---------- APA ----------
Juri Ayub, M., Levin, M.J. & Aguilar, C.F. (2001) . Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex. Protein Expression and Purification, 22(2), 225-233.
---------- CHICAGO ----------
Juri Ayub, M., Levin, M.J., Aguilar, C.F. "Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex" . Protein Expression and Purification 22, no. 2 (2001) : 225-233.
---------- MLA ----------
Juri Ayub, M., Levin, M.J., Aguilar, C.F. "Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex" . Protein Expression and Purification, vol. 22, no. 2, 2001, pp. 225-233.
---------- VANCOUVER ----------
Juri Ayub, M., Levin, M.J., Aguilar, C.F. Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex. Protein Expr. Purif. 2001;22(2):225-233.