X-ray crystallography of the transcriptional activator PhoB from Escherichia coli

大肠杆菌转录激活剂 PhoB 的 X 射线晶体学

• 批准号: 09680662
• 负责人: AKIBA Toshihiko
• 金额: $ 0.38万
• 依托单位: National Institute of Genetics
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680662/
• 关键词: PhoB protein; transcriptional activator; DNA-binding protein; crystallization; X-ray crystallography; protein over-expression; column chromatography; protein purification; リン酸化; 転写制御; 二成分制御系

1. The C-terminal DNA-binding domain of PhoB has been overexpressed and purified to homogeneity as judged by overloaded silver-stained SDS-PAGE gels by an improved method including (1) precipitation of nucleic acid by protamine sulfate, (2) heparin affinity chromatography, and (3) cation exchange chromatography operated in a pH gradient mode.The purified protein fragment was crystallized in the same condition as reported previously.Trigonal crystals appeared after one month of incubation.This growth speed is three-times faster than that of crystals grown from protein solutions prepared by the older methods.The crystals were soaked in K_2PtCl_4 solution to produce heavy atom derivatives, from one of which X-ray diffraction patterns were collected.2. Induction of overexpression of intact PhoB protein was frequently failed, presumably due to loss of protein-expressing cells.To circumvent this problem, we have devised two procedures ; (1) protein production by leaky cells in the absence of an inducer, and (2) use of solid medium for preculture and use of more stringent antibiotics.Yield of the protein was 10 mg/L in the best case.3. Intact PhoB protein has been purified to homogeneity as judged by overloaded CBB-stained SDS-PAGE gels by the newly developed method, which includes (1) precipitation of nucleic acid by protamine sulfate, (2) anion exchange chromatography, (3) heparin affinity chromatography, and (4) cation exchange chromatography operated in a pH gradient mode.4. Crystallization conditions of intact PhoB protein have been searched by a micro-dialysis method and a micro-sitting drop method.The latter method was more useful in the present case where supply of the protein was limited.The best result obtained so far is a microcrystal-like granular precipitation produced by ammonium sulfate.Spherulites were obtained in a few examples of crystallization in the presence of acetylphosphate and Mg^<++> which induce in vitro phosphorylation of the protein.

1. PhoB的C-末端DNA结合结构域已被过表达并通过改进的方法纯化至均一，所述方法包括（1）用硫酸鱼精蛋白沉淀核酸，（2）肝素亲和层析，以及（3）阳离子交换层析在pH梯度模式下操作。纯化的蛋白片段在与先前报道的相同条件下结晶。用K_2PtCl_4溶液浸泡晶体，制备重原子衍生物，并收集其中一个重原子衍生物的X射线衍射图.诱导完整PhoB蛋白过表达常常失败，可能是由于蛋白表达细胞的丢失，为了解决这个问题，我们设计了两种方法：（1）在没有诱导剂的情况下通过渗漏细胞生产蛋白;（2）使用固体培养基进行预培养并使用更严格的抗生素，在最佳情况下蛋白产量为10 mg/L.完整的PhoB蛋白已被纯化至均一，如通过超载CBB染色的SDS-PAGE凝胶通过新开发的方法所判断的，所述方法包括（1）通过硫酸鱼精蛋白沉淀核酸，（2）阴离子交换层析，（3）肝素亲和层析，和（4）在pH梯度模式下操作的阳离子交换层析.用微透析法和微滴法对完整PhoB蛋白的结晶条件进行了研究。微滴法在目前蛋白质供应有限的情况下更有用。迄今为止获得的最好结果是用硫酸铵产生的微晶状颗粒沉淀。在乙酰磷酸盐和Mg^++存在下的几个结晶实例中获得了球晶。其诱导蛋白质的体外磷酸化。