Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags
Cloning strategy, production and purification of proteins with exopeptidase-cleavable His-tags
复制标题
DOI：
10.1038/nprot.2006.388
复制
发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Pedersen, John
中科院分区：
文献类型：
--
作者：
Arnau, Jose;Lauritzen, Conni;Pedersen, John
在这里，我们提出了一种克隆策略，用于生产带有多组氨酸序列标记的重组蛋白，该序列可以被外肽酶（DAPase）切割。该方法可以与最常用的载体一起使用，并且可以表达his标签蛋白，无论其自然序列如何，都可以以其天然形式纯化。这种方法利用TAGZyme系统去除氨基末端亲和标签。标记去除可以单独使用DAPase（重组二肽基肽酶）或与两种辅助酶（Qcyclase和pGAPase）联合完成。该系统已用于大肠杆菌胞内蛋白的生产，并可应用于其他表达宿主，用于生产分泌蛋白或需要翻译后修饰的蛋白。以大肠杆菌中人类白细胞介素1 β的产生为例说明了这种方法。从最初的PCR到产生具有真实N端的分离蛋白的完整程序可以在5天内完成。
Here, we present a cloning strategy for the production of recombinant proteins tagged with a polyhistidine sequence that can be cleaved by the exopeptidase, DAPase. The method can be used with most commonly available vectors and results in the expression of a His-tag protein that can be purified in its native form regardless of its natural sequence. This approach takes advantage of the TAGZyme system for the removal of amino-terminal affinity tags. Tag removal is accomplished either with DAPase (a recombinant dipeptidyl peptidase) alone or in combination with two accessory enzymes, Qcyclase and pGAPase. The system has been used for the production of intracellular proteins in Escherichia coli and can be applied to other expression hosts for the production of secreted proteins or proteins that require post-translational modification. The production of human interleukin 1 beta in E. coli is used as an example to illustrate this method. The complete protocol from initial PCR to the production of a detagged protein with its authentic N terminus can be performed within 5 days.