title: A versatile ligation-independent cloning method suitable for high-throughput expression screening applications.
abstract: This article describes the construction of a set of versatile expression vectors based on the In-Fusion cloning enzyme and their use for high-throughput cloning and expression screening. Modifications to commonly used vectors rendering them compatible with In-Fusion has produced a ligation-independent cloning system that is (1) insert sequence independent (2) capable of cloning large PCR fragments (3) efficient over a wide (20-fold) insert concentration range and (4) applicable to expression in multiple hosts. The system enables the precise engineering of (His(6)-) tagged constructs with no undesirable vector or restriction-site-derived amino acids added to the expressed protein. The use of a multiple host-enabled vector allows rapid screening in both E. coli and eukaryotic hosts (HEK293T cells and insect cell hosts, e.g. Sf9 cells). These high-throughput screening activities have prompted the development and validation of automated protocols for transfection of mammalian cells and Ni-NTA protein purification.
authors.affiliation: The Oxford Protein Production Facility, Henry Wellcome Building for Genomic Medicine, University of Oxford, Oxford, UK.
authors.forename: Nick S
authors.initials: NS
authors.lastname: Berrow
authors.forename: David
authors.initials: D
authors.lastname: Alderton
authors.forename: Sarah
authors.initials: S
authors.lastname: Sainsbury
authors.forename: Joanne
authors.initials: J
authors.lastname: Nettleship
authors.forename: Rene
authors.initials: R
authors.lastname: Assenberg
authors.forename: Nahid
authors.initials: N
authors.lastname: Rahman
authors.forename: David I
authors.initials: DI
authors.lastname: Stuart
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Owens
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral; D005822:Genetic Vectors / Q000737:chemistry; D006801:Humans; D009343:Neisseria / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C500377:KIAA0319 protein, human; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors