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 pe...
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2001
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_10465928_v22_n2_p225_JuriAyub http://hdl.handle.net/20.500.12110/paper_10465928_v22_n2_p225_JuriAyub |
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paper:paper_10465928_v22_n2_p225_JuriAyub2023-06-08T16:01:13Z Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex 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 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. 2001 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_10465928_v22_n2_p225_JuriAyub http://hdl.handle.net/20.500.12110/paper_10465928_v22_n2_p225_JuriAyub |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex |
| topic_facet |
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 |
| description |
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. |
| title |
Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex |
| title_short |
Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex |
| title_full |
Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex |
| title_fullStr |
Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex |
| title_full_unstemmed |
Overexpression and refolding of the hydrophobic ribosomal P0 protein from Trypanosoma cruzi: A component of the P1/P2/P0 complex |
| title_sort |
overexpression and refolding of the hydrophobic ribosomal p0 protein from trypanosoma cruzi: a component of the p1/p2/p0 complex |
| publishDate |
2001 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_10465928_v22_n2_p225_JuriAyub http://hdl.handle.net/20.500.12110/paper_10465928_v22_n2_p225_JuriAyub |
| _version_ |
1768541762809233408 |