第七章色谱分离技术5——固定金属离子亲和层析.pptVIP

第七章 层析分离技术（5） 本章内容 7.1 层析技术导论 7.2 凝胶过滤层析 7.3 离子交换层析 7.4 亲和层析 7.5 固定金属离子亲和层析 7.6 疏水作用层析 7.7 反相层析 7.5 固定金属离子亲和层析(参见P154~158) （Immobilized Metal Ion Affinity Chromatography，IMAC） 一、基本原理 二、固定金属离子亲和树脂的种类 三、带有HIS-Tag重组蛋白的表达和纯化 四、工艺条件 7.5 固定金属离子亲和层析一、基本原理 亚氨二乙酸（iminodiacetic acid，IDA）与环氧活化的琼脂糖偶合,可以形成一种带有双羧甲基氨基琼脂糖,进而与过渡金属离子(Cu2+, Zn2+ Ni2+ ) 螯合,形成吸附活性中心 。 吸附机理：金属螯合介质能与蛋白质中的组氨酸上的咪唑基及半胱氨酸上的巯基结合 7.5 固定金属离子亲和层析二、固定金属离子亲和树脂的种类 Immobilized-metal affinity chromatography (IMAC) was first used to purify proteins in1975 (Porath et al. 1975) using the chelating ligand iminodiacetic acid (IDA, Figure 4). IDA was charged with metal ions such as Zn2+, Cu2+, or Ni2+, and then used to purify a variety of different proteins and peptides (Sulkowski 1985). IDA has only 3 metal-chelatingsites and cannot tightly bind metal ions. Weak binding leads to ion leaching upon loading with strongly chelating proteins and peptides or during wash steps. This results in low yields, impure products, and metal-ion contamination of isolated proteins. Nitrilotriacetic acid (NTA) is a tetradentate chelating adsorbent that overcomes these problems. NTA固定金属离子亲和树脂的特点 ①NTA occupies four of the six ligand binding sites in the coordination sphere of the nickel ion,leaving two sites free to interact with the 6xHis tag (Figure 5). ②NTA binds metal ions far more stably than IDA and retains the ions under a wide variety of conditions, especially under stringent wash conditions. ③The unique,patented NTA matrices can therefore bind 6xHis-tagged proteins more tightly than IDA matrices, allowing the purification of proteins from less than 1% of the total proteinpreparation to more than 95% homogeneity in just one step (Janknecht et al. 1991) ④The high surface concentration of the NTA ligand is sufficient for the binding of approximately 5–10 mg of 6xHis-taggedprotein per milliliter of resin. 7.5 固定金属离子亲和层析三、带有HIS-Tag重组蛋白的表达和纯化 7.5 固定金属离子亲和层析三、带有HIS-Tag重组蛋白的表达和纯化 1. 常用的表达载体 2. 外源基因片段克隆到表达载体上 3. 表达方式的鉴定 4.