title: Self-cleavage of fusion protein in vivo using TEV protease to yield native protein.
abstract: Overproduction of proteins from cloned genes using fusion protein expression vectors in Escherichia coli and eukaryotic cells has increased the quantity of protein produced. This approach has been widely used in producing soluble recombinant proteins for structural and functional analysis. One major disadvantage, however, of applying this approach for clinical or bioindustrial uses is that proteolytic removal of the fusion carrier is tedious, expensive, and often results in products with additional amino acid residues than the native proteins. Here we describe a new method for productions of native proteins with original amino termini in vivo via intracellular self-cleavage of the fusion protein using tobacco etch virus (TEV) protease. Our design allows one to simultaneously clone any gene into multiple fusion protein vectors using two unique cloning sites (i.e., SnaBI and XhoI) without restriction digestion, and then rapidly identifies those constructs producing soluble native proteins. This method will make the fusion protein approach more feasible for protein drug research.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Shih
authors.forename: Hui-Chung
authors.initials: HC
authors.lastname: Wu
authors.forename: Su-Ming
authors.initials: SM
authors.lastname: Hu
authors.forename: Ting-Fang
authors.initials: TF
authors.lastname: Wang
authors.forename: Andrew H-J
authors.initials: AH
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D058619:Maltose-Binding Proteins; D009842:Oligopeptides / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease
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