Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA.
Methods Mol Biol. 2020;2133:163-181. doi: 10.1007/978-1-0716-0434-2_8.
The split inteins from the DnaE cyanobacterial family are efficient and versatile tools for protein engineering and chemical biology applications. Their ultrafast splicing kinetics allow for the efficient production of native proteins from two separate polypeptides both in vitro and in cells. They can also be used to generate proteins with C-terminal thioesters for downstream applications. In this chapter, we describe a method based on a genetically fused version of the DnaE intein Npu for the preparation of doubly modified proteins through recombinant expression. In particular, we provide protocols for the recombinant production of modified ubiquitin through amber suppression where fused Npu is used (1) as a traceless purification tag or (2) as a protein engineering tool to introduce C-terminal modifications for subsequent attachment to other proteins of interest. Our purification protocol allows for quick and facile separation of truncated products and eliminates the need for engineering protease cleavage sites. Our approach can be easily adapted to different proteins and applications where the simultaneous presence of internal and C-terminal modifications is desirable.
来自 DnaE 蓝藻家族的分裂内含肽是蛋白质工程和化学生物学应用的有效且通用的工具。它们超快的剪接动力学允许在体外和细胞内从两个单独的多肽高效生产天然蛋白质。它们还可用于生成带有 C 末端硫酯的蛋白质，用于下游应用。在本章中，我们描述了一种基于 DnaE 内含肽 Npu 的基因融合版本的方法，用于通过重组表达制备双重修饰的蛋白质。特别地，我们提供了通过琥珀终止制备修饰的泛素的重组生产方案，其中融合的 Npu 被用作（1）无痕迹的纯化标签或（2）作为蛋白质工程工具，用于引入 C 末端修饰，以便随后连接到其他感兴趣的蛋白质。我们的纯化方案允许快速方便地分离截断产物，并消除了对工程蛋白酶切割位点的需求。我们的方法可以很容易地适应不同的蛋白质和应用，其中需要同时存在内部和 C 末端修饰。
