کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5914700 | 1162751 | 2011 | 7 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
زیست شناسی مولکولی
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چکیده انگلیسی
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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Structural Biology - Volume 175, Issue 2, August 2011, Pages 113-119
Journal: Journal of Structural Biology - Volume 175, Issue 2, August 2011, Pages 113-119
نویسندگان
Mark P.A. Luna-Vargas, Evangelos Christodoulou, Andrea Alfieri, Willem J. van Dijk, Magda Stadnik, Richard G. Hibbert, Danny D. Sahtoe, Marcello Clerici, Valeria De Marco, Dene Littler, Patrick H.N. Celie, Titia K. Sixma, Anastassis Perrakis,