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230 Arnau et al.
downstream processing, in terms of both selectivity and recovery. Using an
affinity tag and a single purification step, it is possible to achieve a yield of over
95% compared to the yields typically obtained using three or more standard
chromatographic steps (40–50%).
Histidine tags (His-tags) are the most widely used affinity tags in research
and protein structural studies (1). Compared to similar approaches, tagging
with a His-tag offers several advantages: low levels of toxicity and immunogenicity,
a smaller size and no net charge at neutral pH. The incorporation of
a His-tag allows for single-step purification using Immobilized Metal Affinity
Chromatography (IMAC) resins.
Purification using His-tag proteins relies on the high affinity displayed by
short histidine tracks for chelated nickel, cobalt or zinc at neutral or weak
basic pH. Metal ions are immobilized to a chromatographic support such as
nitriloacetate, and metal binding occurs via the imidazole side chain of histidine.
IMAC matrices display high protein-binding capacity and recovery (typically
more than 90%). Importantly, IMAC is chemically stable to the extensive
cleaning-in-place procedures widely used in pharmaceutical production.
For pharmaceutical applications, the affinity tag may need to be removed
before the protein can be used for clinical or structural studies. A common
approach is to include an unusual cleavage site between the His-tag and the
native protein sequence. This tag removal step is then performed by the addition
of the specific endoprotease to the purified tagged protein. In spite of the
specificity, unspecific cleavage can often occur at cryptic sites or during long
treatments (2,3), representing a challenge for the purification process and the
intactness of the protein.
By engineering the specific endoprotease to include the same affinity tag as
the target protein, an efficient removal of the process enzyme(s), the unprocessed
fusion protein and the released tag can be designed. An affinity-tagged
endoproteasecanalsobeusedforon-columncleavage.Furthermore,simultaneous
affinity purification and on-column processing can be achieved. Immobilization
of process enzymes is especially important for large-scale applications, as it may
result in cost reductions, for example, with the use of lower amounts of enzyme.
TAGZyme is an enzymatic system based on engineered aminopeptidases
designed for the efficient and accurate removal of N-terminal affinity tags such
as His-tags. Because TAGZyme is designed for the removal of N-terminal tags
by exopeptidases and not endoproteases, the native protein sequence is not
affected during tag removal. The major enzyme in the TAGZyme system is
DAPase, a recombinant dipeptidyl peptidase I. DAPase cleaves sequentially
dipeptides from the N terminus of virtually any protein, provided the amino
acid sequence does not contain (i) an arginine or lysine at the N terminus
or at an uneven position in the sequence; (ii) a proline anywhere in the tag.