title: Refolding and structural characterization of the human p53 tumor suppressor protein.
abstract: The human tumor suppressor p53 is a conformationally flexible and functionally complex protein that is only partially understood on a structural level. We expressed full-length p53 in the cytosol of Escherichia coli as inclusion bodies. To obtain active, recombinant p53, we varied renaturation conditions using DNA binding activity and oligomeric state as criteria for successful refolding. The optimized renaturation protocol allows the refolding of active, DNA binding p53 with correct quaternary structure and domain contact interfaces. The purified protein could be allosterically activated for DNA binding by addition of a C-terminally binding antibody. Analytical gelfiltration and chemical cross-linking confirmed the tetrameric quaternary structure and the spectroscopic analysis of renatured p53 by fluorescence and circular dichroism, suggested that native p53 is partially unstructured.
authors.affiliation: Lehrstuhl für Biotechnologie, Institut für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Bell
authors.forename: Silke
authors.initials: S
authors.lastname: Hansen
authors.forename: Johannes
authors.initials: J
authors.lastname: Buchner
mesh_terms: D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D002479:Inclusion Bodies; D020673:Protein Renaturation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013329:Structure-Activity Relationship; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins