Inhibition of tobacco etch virus protease activity by detergents.
Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.
Mohanty
Arun K
AK
Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22908-0736, USA.
Simmons
Chad R
CR
Wiener
Michael C
MC
D003902:Detergents / Q000494:pharmacology*; D010450:Endopeptidases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010942:Plant Viruses / Q000201:enzymology*; D011480:Protease Inhibitors / Q000494:pharmacology*; D012995:Solubility; D013379:Substrate Specificity
D003902:Detergents; D010446:Peptide Fragments; D011480:Protease Inhibitors; D010450:Endopeptidases; C099488:TEV protease