title: A vector design that allows fast and convenient production of differently tagged proteins.
abstract: Recombinant-tagged proteins have a widespread use in experimental research as well as in clinical diagnostic and therapeutic approaches. Well-stocked sets of differently tagged variants of a same protein would be of great help. However, the construction of differently tagging vectors is a demanding task since cloning procedures need several tailored DNA inserts. In this study, we describe a novel vector system that allows a cost- and time-effective production of differently tagged variants of a same protein by using the same DNA fragment and a set of vectors each carrying a different tag. The design of these expression vectors is based on an intronic region that becomes functional upon cloning the insert sequence, splicing of which attaches a certain tag to the protein termini. This strategy allows for the cloning of the fragment that codes for the protein of interest, without any further modification, into different vectors, previously built and ready-to-use, each carrying a tag that will be joined to the protein. Proof of principle for our expression system, presented here, is shown through the production of a functional anti-GD2 Fab fragment tagged with biotin or polyhistidine, or a combination of both, followed by the demonstration of the functional competencies of both the protein and the tags.
authors.affiliation: Department of Experimental Medicine, University of Genoa, Via De Toni 14, 16132 Genoa, Italy.
authors.forename: Omar
authors.initials: O
authors.lastname: Scapolan
authors.forename: Andrea N
authors.initials: AN
authors.lastname: Mazzarello
authors.forename: Maria
authors.initials: M
authors.lastname: Bono
authors.forename: Marzia
authors.initials: M
authors.lastname: Occhino
authors.forename: Vasily
authors.initials: V
authors.lastname: Ogryzko
authors.forename: Marco
authors.initials: M
authors.lastname: Bestagno
authors.forename: Paolo
authors.initials: P
authors.lastname: Scartezzini
authors.forename: Silvia
authors.initials: S
authors.lastname: Bruno
authors.forename: Franco
authors.initials: F
authors.lastname: Fais
authors.forename: Fabio
authors.initials: F
authors.lastname: Ghiotto
mesh_terms: D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000378:metabolism; D042541:Intracellular Space / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D015203:Reproducibility of Results; D013194:Staining and Labeling
chemical_list: D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D001710:Biotin
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