Purification of Native Proteins from the Cytoplasm and Histidine Tails : A Comparison of Proteins and Protocols
Purification of Native Proteins from the Cytoplasm and Histidine Tails : A Comparison of Proteins and Protocols
复制标题
DOI：
--
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Péter;Lindner;B. Guth;Christoph;Wiilfing;C. Krebber;Boris Steipe;Frank;Muller;
中科院分区：
文献类型：
--
作者：
Péter;Lindner;B. Guth;Christoph;Wiilfing;C. Krebber;Boris Steipe;Frank;Muller;
几种蛋白质纯化在一个单一的步骤从大肠杆菌在天然形式使用融合组氨酸尾和固定化金属亲和层析（IMAC）。报道和比较的程序是从周质中纯化的携带C-末端（His）5尾的抗体片段（单链Fv片段和VL结构域）的程序，E.大肠杆菌二硫键异构酶携带一个C-末端（His）的尾巴纯化从周质，和酵母柠檬酸合成酶携带的N-末端和C-末端（His）的尾巴纯化从细胞质。由于周质折叠过程中的阻断，所研究的鼠单链T细胞受体尽管是可溶的，但只能以变性形式纯化，从而证明了天然纯化对折叠状态的敏感性。柱和金属离子的比较表明，Zn+ -亚氨基二乙酸和Ni + -次氮基三乙酸给出了同样好的V L结构域和Fv片段的一步纯化，但仅在金属和螯合剂的这种组合中。缓冲液是次要的，优选的洗脱方法取决于蛋白质的稳定性。在携带（无金属）组氨酸尾的VL结构域的晶体结构测定中，未检测到超过前两个组氨酸残基的显著电子密度，这意味着未配体亲和尾的无序结构。当在变性条件下使用IMAC时，f.大肠杆菌蛋白酶可以共纯化和重折叠或在天然条件下共纯化，并且敏感蛋白可以在其体外重折叠期间被消化。质粒-en-
Several proteins were purified in a single step from Escherichia coli in native form using fused histidine tails and immobilized metal affinity chromatography (IMAC). The procedures reported and compared are those for antibody fragments (single-chain Fv fragments and VL domains) carrying a C-terminal (His)5 tail purified from the periplasm, the E. coli disulfide isomerase carrying a C-terminal (His)s tail purified from the periplasm, and yeast citrate synthase carrying both a N-terminal and a C-terminal (His)s tail purified from the cytoplasm. Because of blocks in the periplasmic folding process, the investigated murine single-chain T -cell receptor could be purified only in denatured form, despite being soluble, thereby demonstrating the sensitivity of the native purification to the folding state. A comparison of columns and metal ions showed that Zn+ -iminodiacetic acid and Ni + -nitrilotriacetic acid gave equally good one-step purifications of V L domains and Fv fragments, but only in this combination of metal and chelator. The buffer is of secondary importance, and the preferred method of elution depends on the stability of the protein. In a crystal structure determination of the VL domain carrying the (metal-free) histidine tail, no significant electron density beyond the first two histidine residues was detected, implying a disordered structure of the unliganded affinity tail. When IMAC is used under denaturing conditions, an f. coli protease can be co-purified and refolded or co-purified under native conditions, and sensitive proteins can be digested during their in vitro refolding. Plasmid-en-