title: Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli.
abstract: We describe the construction and use of two sets of vectors for the over-expression and purification of protein from Escherichia coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His(6)) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His(6) and maltose-binding protein tag in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.
authors.affiliation: Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, USA.
authors.forename: C J
authors.initials: CJ
authors.lastname: Rocco
authors.forename: K L
authors.initials: KL
authors.lastname: Dennison
authors.forename: Vadim A
authors.initials: VA
authors.lastname: Klenchin
authors.forename: I
authors.initials: I
authors.lastname: Rayment
authors.forename: J C
authors.initials: JC
authors.lastname: Escalante-Semerena
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D002951:Citrates / Q000737:chemistry; D003001:Cloning, Molecular; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005821:Genetic Techniques; D005822:Genetic Vectors; D006639:Histidine / Q000737:chemistry; D006836:Hydro-Lyases / Q000235:genetics; D008957:Models, Genetic; D009842:Oligopeptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D019779:Salmonella enterica / Q000201:enzymology; D020592:Shewanella / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D002951:Citrates; D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D006639:Histidine; C031605:2-methylcitric acid; D010450:Endopeptidases; C099488:TEV protease; D006836:Hydro-Lyases; C471418:methylcitrate dehydratase, E coli