Expression and purification of soluble His(6)-tagged TEV protease.
This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. The protease is initially produced as a fusion to the C-terminus of E. coli maltose binding protein (MBP), which causes it to accumulate in a soluble and active form rather than in inclusion bodies. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. An S219V mutation in the protease reduces its rate of autolysis by approximately 100-fold and also gives rise to an enzyme with greater catalytic efficiency than the wild-type protease.
Tropea
Joseph E
JE
National Cancer Institute at Frederick, Macromolecular Crystallography Laboratory, Center for Cancer Research, Frederick, MD, USA.
Cherry
Scott
S
Waugh
David S
DS
D000818:Animals; D002846:Chromatography, Affinity; D010450:Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D006639:Histidine / Q000737:chemistry*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry*; D033902:Periplasmic Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D029968:Escherichia coli Proteins; C471213:His-His-His-His-His-His; C461619:MalE protein, E coli; D050505:Mutant Proteins; D009842:Oligopeptides; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D006639:Histidine; D010450:Endopeptidases; C099488:TEV protease