p21Waf1/Cip1的表达纯化以及与其靶分子的相互作用

p21Waf1/Cip1，是目前具有最广泛激酶抑制活性的细胞周期抑制蛋白。随着研究的发展，发现它在细胞增殖、分化、凋亡等方面具有越来越多的功能，并且这些功能很大程度上依赖于它能够与各种各样的靶蛋白相互作用。p21与靶分子的作用与它的结构特点密切相关， 因而从分子水平上研究p21蛋白的构象特点、以及其与靶分子的相互作用具有非常重要的意义。 在本文中，我们首先运用分子克隆技术在大肠杆菌中成功地构建了由温度控制的pBV220-p21表达体系。经30℃培养、42℃诱导表达以后，p21蛋白以包涵体形式得到高效表达，通过离子交换和分子筛两步纯化得到电泳纯的、具有生物活性的p21蛋白。利用生物化学、生物物理学等方法和手段我们首次较为系统地表征了p21蛋白的构象特点， 发现其具有典型的无规卷曲的二级结构；色氨酸很大程度上暴露于溶液中，处在正电荷的微环境中；首次发现p21蛋白能够与一些阴离子配合物（如suramin、Bis-ANS等）以近10-7数量级的解离常数直接相互作用，化学计量比为：1：1，进一步研究表明suramin在p21上的结合位点可能是p21的C-末端。在此基础上，我们通过丹磺酰氯标记过的钙调蛋白（CaM）的荧光光谱、p21141-164突变的色氨酸的内源荧光光谱及荧光猝灭方法、圆二色谱、非变性蛋白电泳等方法和手段表征了p21及p21141–164与CaM的作用。首次发现p21与CaM结合的解离常数在10-7数量级，p21与CaM之间除了疏水作用，还有较强的静电作用，以及一定的H键作用。深入研究表明p21141-164在CaM上的结合位点在CaM的Ca2+诱导的疏水区域，p21141–164的150和151位突变的色氨酸在结合后离CaM的疏水区域较近，而159位的色氨酸离的较远。同时发现p21141–164与CaM结合后只诱导出约14%的α螺旋结构，即p21141–164基本上是以无结构的状态与CaM结合的。

Expression, purification and spectral characterization of p21(Waf1/Cip1)

p21(Waf1/Cip1), best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, can interact with various target proteins, and this ability relies on its structural plasticity. Therefore, studies on the structural properties of p(21) are very important to understand its structure-function relationship. However, detailed studies on its secondary structure and biophysical properties have been comparatively sparse. A human p(21) gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109.

Biophysical characterization of the interaction of p21 with calmodulin: A mechanistic study

p21 is a protein with important roles in cell proliferation, cell cycle regulation and apoptosis. Several studies have demonstrated that its intracellular localization plays an important role in the functional regulation and binding of calmodulin favors its nuclear translocation. However, the detail mechanism of the interaction with p21 and calmodulin is not well understood. In this report, peptides derived from the C-terminal of p21 that cover the binding domain of calmodulin were used to investigate the association of p21 with calmodulin.