Department of Biochemistry and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
J Struct Biol. 2011 Aug;175(2):113-9. doi: 10.1016/j.jsb.2011.03.017. Epub 2011 Mar 29.
High-throughput methods to produce a large number of soluble recombinant protein variants are particularly important in the process of determining the three-dimensional structure of proteins and their complexes. Here, we describe a collection of protein expression vectors for ligation-independent cloning, which allow co-expression strategies by implementing different affinity tags and antibiotic resistances. Since the same PCR product can be inserted in all but one of the vectors, this allows efficiency in versatility while screening for optimal expression strategies. We first demonstrate the use of these vectors for protein expression in Escherichia coli, on a set of proteins belonging to the ubiquitin specific protease (USP) Family. We have selected 35 USPs, created 145 different expression constructs into the pETNKI-His-3C-LIC-kan vector, and obtained 38 soluble recombinant proteins for 21 different USPs. Finally, we exemplify the use of our vectors for bacterial co-expression and for expression in insect cells, with USP4 and USP7 respectively. We conclude that our ligation-independent cloning strategy allows for high-throughput screening for the expression of soluble proteins in a variety of vectors in E. coli and in insect cells. In addition, the same vectors can be used for co-expression studies, at least for simple binary complexes. Application in the family of ubiquitin specific proteases led to a number of soluble USPs that are used for functional and crystallization studies.
高通量方法可用于大量产生可溶性重组蛋白变体，这在确定蛋白质及其复合物的三维结构的过程中尤为重要。在这里，我们描述了一组用于无连接克隆的蛋白质表达载体，这些载体可通过实施不同的亲和标签和抗生素抗性来实现共表达策略。由于除了一个载体之外，同一个 PCR 产物可以插入所有载体中，因此这在筛选最佳表达策略时提高了通用性和效率。我们首先在一组属于泛素特异性蛋白酶 (USP) 家族的蛋白质中展示了这些载体在大肠杆菌中的蛋白质表达用途。我们选择了 35 个 USP，将 145 种不同的表达构建体创建到 pETNKI-His-3C-LIC-kan 载体中，并获得了 38 种可溶性重组蛋白，其中 21 种不同的 USP 有表达。最后，我们分别以 USP4 和 USP7 为例，说明了我们的载体在细菌共表达和昆虫细胞表达中的用途。我们得出结论，我们的无连接克隆策略允许在大肠杆菌和昆虫细胞中的多种载体中高通量筛选可溶性蛋白的表达。此外，相同的载体可用于共表达研究，至少对于简单的二元复合物而言是如此。在泛素特异性蛋白酶家族中的应用产生了许多可溶性 USP，它们可用于功能和结晶研究。
