TEV protease is a highly sequence-specific cysteine protease from
Tobacco Etch Virus. It is a member of the PA clan of chymotrypsin-like
proteases. Due to its high sequence specificity it is frequently used for
the controlled cleavage of fusion proteins in vitro and in vivo.
Origin The tobacco etch virus encodes its
entire genome as a single massive polyprotein. This is cleaved into
functional units by the three proteases: P1 protease, helper-component protease
and TEV protease. The native protease also contains an internal self-cleavage
site. This site is slowly cleaved to inactivate the enzyme.
Structure and Function The structure of TEV protease has been
solved by X-ray crystallography. It is composed of two β-barrels and a flexible
C-terminal tail and displays structural homology to the chymotrypsin superfamily
of proteases. Although homologous to cellular serine proteases, TEV protease
uses a cysteine as its catalytic nucleophile.
Covalent catalysis is performed with an Asp-His-Cys triad, split between the two
barrels. The substrate is held as a β-sheet, forming an antiparallel
interaction with the cleft between the barrels and a parallel interaction with
the C-terminal tail. The enzyme therefore forms a binding tunnel around
the substrate and side chain interactions control specificity.
Specificity The preferred, native cleavage sequence
was first identified by examining the cut sites in the native polyprotein
substrate for recurring sequence. The consensus for these native cut sites is
ENLYFQ\S where ‘\’ denotes the cleaved peptide bond. Residues of the substrate
are labelled P6 to P1 before the cut site and P1’ after the cut site. Early
works also measured cleavage of an array of similar substrates to characterise
how specific the protease was for the native sequence.
Studies have subsequently used sequencing of cleaved substrates from a
pool of randomised sequences to determine preference patterns. Although
ENLYFQ\S is the optimal sequence, the protease is active to a greater or
lesser extent on a range of substrates. The highest cleavage is of sequences
closest to the consensus EXLYΦQ\φ where X is any residue, Φ is any large or
medium hydrophobe and φ is any small hydrophobic or polar residue.
Specificity is endowed by the large contact area between enzyme and
substrate. Proteases such as trypsin have specificity for one residue before
and after the cleaved bond due to a shallow binding cleft with only one or
two pockets that bind the substrate side chains. Conversely, viral proteases such
as TEV protease have a long C-terminal tail which completely covers the
substrate to create a binding tunnel. This tunnel contains a set of tight
binding pockets such that each side chain of the substrate peptide is bound
in a complementary site. In particular, peptide side chain P6-Glu
contacts a network of three hydrogen bonds; P5-Asn points into the solvent,
making no specific interactions; P4-Leu is buried in a hydrophobic pocket;
P3-Tyr is held in a hydrophobic pocket with a short hydrogen bond at the end;
P2-Phe is also surrounded by hydrophobes including the face of the triad
histidine; P1-Gln forms four hydrogen bonds; and P1’-Ser is only partly
enclosed in a shallow hydrophobic groove.
As a biochemical tool One of the main uses of this protein is
for removing affinity tags from purified proteins. The reason for the use of TEV
protease as a biochemical tool is its high sequence specificity. This
specificity allows for the controlled cleavage of proteins when the preference
sequence is inserted into flexible loops. It also makes it relatively
non-toxic in vivo as the recognized sequence scarcely occurs in proteins.
Although rational design has had limited success in changing protease
specificity, directed evolution has been used to change the preferred residue
either before or after the cleavage site.
However, TEV protease does have limitations as a biochemical tool. It is
prone to deactivation by self-cleavage, though this can be abolished through a
single S219V mutation in the internal cleavage site. The protease expressed
alone is also poorly soluble, however several attempts have been made to
improve its solubility through directed evolution and computational design. It
has also been shown that expression can be improved by fusion to Maltose Binding
Protein which acts a solubility enhancing partner.
The molecular weight of this enzyme varies between 25 and 27 kDa depending
on the specific construct used. External links
TEV polyprotein on UniProt TEV protease on MEROPS
National Cancer Institute TEV FAQ References