Tobacco etch virus cleavage site

The proteinase responsible for most of these cleavages is a viral-encoded kDa protein. By using cell-free systems to manipulate and express cloned cDNA sequences, a amino acid segment containing a putative proteolytic cleavage site of the TEV polyprotein has been introduced into the TEV capsid protein sequence. This recombinant protein is cleaved by the kDa proteinase at the introduced cleavage site, thus demonstrating portability of a functional cleavage site.

The role of the conserved amino acid sequence in determining substrate activity was tested by construction of engineered proteins that contained part or all of this motif. 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.

Gov't, Non-P. Research Support, U. Gov't, P. Substances Endopeptidases TEV protease.