A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor
A new tagging system for production of recombinant proteins in Drosophila S2 cells using the third domain of the urokinase receptor
复制标题
DOI：
10.1016/j.pep.2006.11.013
复制
发表时间：
2007-04-01
影响因子：
1.6
通讯作者：
Ploug, Michael
中科院分区：
文献类型：
--
作者：
Gardsvoll, Henrik;Hansen, Line V.;Ploug, Michael
蛋白质融合标签技术的使用极大地促进了重组蛋白的检测、表达和纯化，因此对新的和更有效的系统的需求正在扩大。我们已经使用了一种可溶性截断形式的尿激酶受体的第三个结构域作为一个方便的C-末端融合伴侣的各种重组细胞外的人蛋白质用于基础癌症研究。这种富含半胱氨酸的结构域的稳定性，其结构采用三指折叠，为其作为用于表达重组蛋白的融合标签的适用性提供了重要的资产。使用这种策略，每升培养物中稳定转染的果蝇S2细胞表达高达20 mg的完整融合蛋白。从S2细胞的条件无血清培养基中纯化这些分泌的融合蛋白，伴随着使用固定化抗uPAR单克隆抗体R2的有效的一步免疫亲和层析程序。在各种重组蛋白和标签的接头区之间包括任选的肠激酶切割位点，这使得能够产生无标签的重组蛋白的高纯度制剂。使用该系统，我们成功地产生了可溶性和完整的重组形式的细胞外蛋白，如CD59，C4.4A和玻连蛋白，以及这些蛋白质的一些截短的结构域构建体。总之，本标记系统提供了用于多种重组蛋白的稳健表达和有效纯化的方便的通用方法。(c)2006年爱思唯尔公司All rights reserved.
The use of protein fusion tag technology greatly facilitates detection, expression and purification of recombinant proteins, and the demands for new and more effective systems are therefore expanding. We have used a soluble truncated form of the third domain of the urokinase receptor as a convenient C-terminal fusion partner for various recombinant extracellular human proteins used in basic cancer research. The stability of this cystein-rich domain, which structure adopts a three-finger fold, provides an important asset for its applicability as a fusion tag for expression of recombinant proteins. Up to 20 mg of intact fusion protein were expressed by stably transfected Drosophila S2 cells per liter of culture using this strategy. Purification of these secreted fusion proteins from the conditioned serum free medium of S2 cells was accompanied by an efficient one-step immunoaffinity chromatography procedure using the immobilized anti-uPAR monoclonal antibody R2. An optional enterokinase cleavage site is included between the various recombinant proteins and the linker region of the tag, which enables generation of highly pure preparations of tag-free recombinant proteins. Using this system we successfully produced soluble and intact recombinant forms of extracellular proteins such as CD59, C4.4A and vitronectin, as well as a number of truncated domain constructs of these proteins. In conclusion, the present tagging system offers a convenient general method for the robust expression and efficient purification of a variety of recombinant proteins. (c) 2006 Elsevier Inc. All rights reserved.