Molecular evolution of the thermosensitive PAb1620 epitope of human p53 by DNA shuffling.
Conformational stability of the p53 protein is an absolute necessity for its physiological function as a tumor suppressor. Recent in vitro studies have shown that wild-type p53 is a highly temperature-sensitive protein at the structural and functional levels. Upon heat treatment at 37 degrees C, p53 loses its wild-type (PAb1620(+)) conformation and its ability to bind DNA, but can be stabilized by different classes of ligands. To further investigate the thermal instability of p53, we isolated p53 mutants resistant to heat denaturation. For this purpose, we applied a recently developed random mutagenesis technique called DNA shuffling and screened for p53 variants that could retain reactivity to the native conformation-specific anti-p53 antibody PAb1620 upon thermal treatment. After three rounds of mutagenesis and screening, mutants were isolated with the desired phenotype. The isolated mutants were translated in vitro in either Escherichia coli or rabbit reticulocyte lysate and characterized biochemically. Mutational analysis identified 20 amino acid residues in the core domain of p53 (amino acids 101-120) responsible for the thermostable phenotype. Furthermore, the thermostable mutants could partially protect the PAb1620(+) conformation of tumor-derived p53 mutants from thermal unfolding, providing a novel approach for restoration of wild-type structure and possibly function to a subset of p53 mutants in tumor cells.
Xirodimas
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Cancer Research Campaign Cell Transformation Group, Department of Biochemistry, Medical Science Institute, University of Dundee, Dundee DD1 4HN, Scotland.
Lane
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D000818:Animals; D004247:DNA / Q000235:genetics; D000939:Epitopes / Q000235:genetics; D004926:Escherichia coli; D019143:Evolution, Molecular; D016158:Genes, p53; D005821:Genetic Techniques; D006358:Hot Temperature; D006801:Humans; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013816:Thermodynamics; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics*
D000939:Epitopes; D016159:Tumor Suppressor Protein p53; D004247:DNA