title: Enabling high-throughput ligation-independent cloning and protein expression for the family of ubiquitin specific proteases.
abstract: 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.
authors: Department of Biochemistry and Centre for Biomedical Genetics, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
authors: Mark P A
authors: MP
authors: Luna-Vargas
authors: Evangelos
authors: E
authors: Christodoulou
authors: Andrea
authors: A
authors: Alfieri
authors: Willem J
authors: WJ
authors: van Dijk
authors: Magda
authors: M
authors: Stadnik
authors: Richard G
authors: RG
authors: Hibbert
authors: Danny D
authors: DD
authors: Sahtoe
authors: Marcello
authors: M
authors: Clerici
authors: Valeria De
authors: VD
authors: Marco
authors: Dene
authors: D
authors: Littler
authors: Patrick H N
authors: PH
authors: Celie
authors: Titia K
authors: TK
authors: Sixma
authors: Anastassis
authors: A
authors: Perrakis
mesh_terms: D000818:Animals; D057205:Automation, Laboratory; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D064570:Ubiquitin-Specific Proteases
chemical_list: D011994:Recombinant Proteins; D010450:Endopeptidases; D064570:Ubiquitin-Specific Proteases