title: Gateway vectors for the production of combinatorially-tagged His6-MBP fusion proteins in the cytoplasm and periplasm of Escherichia coli.
abstract: Many proteins that accumulate in the form of insoluble aggregates when they are overproduced in Escherichia coli can be rendered soluble by fusing them to E. coli maltose binding protein (MBP), and this will often enable them to fold in to their biologically active conformations. Yet, although it is an excellent solubility enhancer, MBP is not a particularly good affinity tag for protein purification. To compensate for this shortcoming, we have engineered and successfully tested Gateway destination vectors for the production of dual His6MBP-tagged fusion proteins in the cytoplasm and periplasm of E. coli. The MBP moiety improves the yield and solubility of its fusion partners while the hexahistidine tag (His-tag) serves to facilitate their purification. The availability of a vector that targets His6MBP fusion proteins to the periplasm expands the utility of this dual tagging approach to include proteins that contain disulfide bonds or are toxic in the bacterial cytoplasm.
authors.affiliation: National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702, USA.
authors.forename: Sreedevi
authors.initials: S
authors.lastname: Nallamsetty
authors.forename: Brian P
authors.initials: BP
authors.lastname: Austin
authors.forename: Kerri J
authors.initials: KJ
authors.lastname: Penrose
authors.forename: David S
authors.initials: DS
authors.lastname: Waugh
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D002352:Carrier Proteins; C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D012260:Ribonucleases