Proteases
Table 2. Proteases commonly used for tag removal
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An integral part of the choice of a fusion tag is the choice of the method for removing the tag after purification. This
step almost always involves using a protease to cleave a specific peptide bond between the tag and the protein of interest.
A small number of highly specific proteases are routinely used for this purpose and are listed in Table 2. These include the
tobacco etch virus (TEV) protease; thrombin (factor IIa, fIIa) and factor Xa (fXa) from the blood coagulation cascade; an
enzyme involved in the cleavage or activation of trypsin in the mammalian intestinal tract, enterokinase (EK); proteases involved
in the maturation and deconjugation of SUMO, SUMO proteases (Ulp1, Senp2, and SUMOstar); and a relative newcomer to the field,
a mutated form of the Bacillus subtilis protease, subtilisin BPN' (Bio-Rad's Profinity eXact system). Many of these enzymes have been genetically engineered to enhance
their stability (e.g., AcTEV, ProTEV) or their specificity, (e.g. SUMOstar, Profinity). With the exception of the SUMO proteases,
all of these enzymes have the potential to cleave within the protein of interest.12–13 The SUMO proteases recognize not only their specific cleavage site, xaa-Gly-Gly/yaa, but also the tertiary structure of
SUMO itself, giving them a very high degree of specificity. Bryan, et al., have attempted to introduce the same level of
specificity into the Profinity system by mutating both the subtilisin prodomain as well as the active site of subtilisin to
increase the affinity of the enzyme for the prodomain and to decrease the likelihood of digestion within the protein of interest.14 One interesting consequence of this is that the affinity for the prodomain is so high that these researchers observed product
inhibition of the enzyme. Essentially, the enzyme carries out one catalytic cycle and is then inhibited by the prodomain,
which is retained in the active site, thus preventing further cleavage by this otherwise promiscuous enzyme. Because capture
on the immobilized, mutant subtilisin matrix is an integral part of the system, the column must have a capacity (in moles
of subtilisin) equimolar with the fusion protein. Although this is not problematic on the research scale, it could become
prohibitively expensive at the multigram scale.
The principle concerns with using a protease for removing a tag are 1) removing the protease following digestion, and 2) non-specific
digestion of the target protein by the protease. Resolving the first concern is relatively straightforward, although in most
cases it involves an additional chromatography step. Recombinant forms of TEV and its variants and of the SUMO proteases are
all produced with a hexahistidine (His6) tag, allowing easy removal of the enzyme by metal chelate chromatography. Alternatively,
some of these enzymes have been immobilized on solid supports, allowing their removal by simple filtration or centrifugation
steps. Thrombin, fXa, and EK, which generally are produced from natural sources, can be removed by affinity chromatography,
for instance, on benzamidine-agarose. With the Profinity system, cleavage and separation from the enzyme are combined in a
single step.
The second concern is more difficult to resolve. Non-specific cleavage is influenced by a number of parameters, such as the
enzyme-to-substrate ratio (lower is better), temperature, pH, salt concentration, and length of exposure. TEV protease, thrombin,
fXa, and EK all have well defined recognition sequences, but all of them have been found to cause "nicking" of the target
protein in some instances. TEV protease has been re-engineered to try to increase its specificity (and stability), resulting
in AcTEV (Invitrogen, Carlsbad, CA) and ProTEV (Promega, Madison, WI). Whether or not such engineering has reduced non-specific
proteolysis remains to be seen. In addition, other tricks must be used with the native enzymes. For instance, one supplier
recommends using fXa at pH 6.5, well below its pH optimum, to minimize non-specific cleavage. Of course, this requires the
use of higher enzyme-to-substrate ratios and longer digestion times to achieve complete cleavage. Two of the enzymes listed
(SUMO proteases and the Profinity enzyme) seem to be immune to this problem. SUMO proteases have evolved to recognize both
the tertiary structure of SUMO as well as the cleavage sequence, xaa-Gly-Gly/yaa. The Profinity enzyme has been extensively
mutated to derive a version that has very high affinity for the prodomain of the original enzyme. Thus, it also recognizes
the tertiary structure of the prodomain as well as the cleavage sequence Phe-Met-Ala-Lys/yaa. On the other hand, SUMO proteases
act catalytically (i.e., with a low enzyme-to-substrate ratio) whereas the Profinity enzyme requires equimolar concentrations
of enzyme and substrate.
One final consideration should be mentioned. Although one would ideally have a protein that is fully soluble in phosphate
buffered saline at neutral pH, the reality is that for many proteins to be soluble at useful concentrations, they require
more acidic or more basic pH levels, high or low salt levels, or the presence of chaotropes or detergents. It is therefore
essential that the protease of choice retain substantial activity under adverse conditions. The most robust of the enzymes
cited appear to be the SUMO proteases, the Profinity enzyme, and the TEV protease. Thrombin, fXa, and EK are much more sensitive
to high salt concentrations or to the presence of chaotropes or reducing agents.
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