From the Department of Pharmacology, Hamamatsu University School of Medicine, Japan.
From the Department of Pharmacology, Hamamatsu University School of Medicine, Japan.
Protein Expr Purif. 2020 Jun;170:105607. doi: 10.1016/j.pep.2020.105607. Epub 2020 Feb 13.
It is well known that camelids (camels and llamas) have fully functional antibodies with only a heavy chain consisting of a single variable domain and two constant domains. This single variable domain is called a "nanobody" and many nanobodies are synthesized in the cytosol of Escherichia coli, however, most of the nanobodies become inclusion bodies without tags to enhance their solubility. We generated a vector system to enable the secretary expression of nanobodies in Escherichia coli. In this system, several NBs were secreted into the culture supernatant. Since the vector contained 6xHis tag and AviTAG, biotinylation (even fluorescent-labeled) of AviTAG was achieved during cell culture, and purification of the supernatant was a step by immobilized metal ion adsorption chromatography. The procedure described in this study is believed to be as simple as regular plasmid minipreps. Therefore, many laboratories can use this method.
众所周知，骆驼（骆驼和羊驼）具有完全功能的抗体，其重链仅由一个单可变域和两个恒定域组成。这个单可变域被称为“纳米抗体”，许多纳米抗体在大肠杆菌的细胞质中合成，然而，大多数纳米抗体没有标签来提高其溶解度，因此成为包涵体。我们生成了一个载体系统，使纳米抗体能够在大肠杆菌中分泌表达。在这个系统中，有几个纳米抗体被分泌到培养上清液中。由于载体含有 6xHis 标签和 AviTAG，因此在细胞培养过程中可以对 AviTAG 进行生物素化（甚至荧光标记），而上清液的纯化是通过固定化金属离子吸附层析来完成的。本研究中描述的步骤被认为与常规质粒小量抽提一样简单。因此，许多实验室都可以使用这种方法。
