Construction and use of new cloning vectors for the rapid isolation of recombinant proteins from Escherichia coli.
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.
Rocco
C J
CJ
Department of Bacteriology, University of Wisconsin, 1550 Linden Drive, Madison, WI 53706, USA.
Dennison
K L
KL
Klenchin
Vadim A
VA
Rayment
I
I
Escalante-Semerena
J C
JC
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
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