Molecular evolution of the thermosensitive PAb1620 epitope of human p53 by DNA shuffling
Molecular evolution of the thermosensitive PAb1620 epitope of human p53 by DNA shuffling
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DOI：
10.1074/jbc.274.39.28042
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发表时间：
1999-09-24
影响因子：
4.8
通讯作者：
Lane, DP
中科院分区：
文献类型：
--
作者：
Xirodimas, DP;Lane, DP
p53蛋白的构象稳定性对于其作为肿瘤抑制剂的生理功能是绝对必要的。最近的体外研究表明，野生型p53在结构和功能水平上是一种高度温度敏感的蛋白质。在37摄氏度的热处理后，p53失去了其野生型（PAb 1620（+））构象和结合DNA的能力，但可以通过不同类型的配体稳定。为了进一步研究p53的热不稳定性，我们分离了耐热变性的p53突变体。为此，我们应用了最近开发的随机诱变技术，称为DNA改组和筛选p53的变体，可以保留反应性的天然构象特异性抗p53抗体PAb 1620热处理后。在三轮诱变和筛选后，分离具有所需表型的突变体。分离的突变体在大肠杆菌或兔网织红细胞裂解液中进行体外翻译，并进行生物化学表征。突变分析鉴定了p53核心结构域中负责热稳定表型的20个氨基酸残基（氨基酸101-120）。此外，热稳定突变体可以部分保护肿瘤来源的p53突变体的PAb 1620（+）构象免受热解折叠，为恢复野生型结构和可能对肿瘤细胞中的p53突变体亚群起作用提供了新的方法。
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.