Arnau José, Lauritzen Conni, Pedersen John
Unizyme Laboratories, Dr Neergaards vej 17, DK 2970 Hørsholm, Denmark.
Nat Protoc. 2006;1(5):2326-33. doi: 10.1038/nprot.2006.388.
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 1beta 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.
在此，我们提出一种克隆策略，用于生产带有多组氨酸序列标签的重组蛋白，该序列可被外肽酶DAPase切割。该方法可用于大多数常用载体，并能表达出His标签蛋白，无论其天然序列如何，均可将其以天然形式纯化。这种方法利用了TAGZyme系统去除氨基末端亲和标签。标签去除可单独使用DAPase（一种重组二肽基肽酶）或与两种辅助酶Qcyclase和pGAPase联合完成。该系统已用于在大肠杆菌中生产细胞内蛋白，也可应用于其他表达宿主以生产分泌蛋白或需要翻译后修饰的蛋白。以在大肠杆菌中生产人白细胞介素1β为例来说明该方法。从最初的PCR到生产出具有真实N末端的去标签蛋白的完整方案可在5天内完成。
