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Removal of N-Terminal His-Tags 233
After removal of DAPase and Qcyclase, the processed protein is treated
with pGAPase, a pyroglutamyl aminopeptidase that removes the N-terminal
pyroglutamyl residue rendering a purified tag-free protein with the native N
terminus. This step can be performed in batch mode or on-column where
pGAPase is immobilized. The yield for the complete tag removal process using
TAGZyme is typically over 90%.
A number of potentially therapeutic proteins contain a natural stop position
for DAPase at their N terminus (e.g., R or K at position 1) in the mature or active
form found in vivo. To produce and purify these proteins using recombinant
DNA technology, a His-tag without the additional Gln can be added to the
N terminus, and a process that only requires DAPase for tag removal can be
developed. DAPase cleavage will proceed until the stop position is reached.
Removal of DAPase and elution of the tag-free, purified protein can be achieved
in a single step. This type of process is not explained further in this chapter
but information can be found elsewhere (5).
The precise amino acid sequence of a His-tag and the nucleotide sequence
selected to encode it are of great importance for the overall performance of the
resulting construct during expression, post-translational processing, purification
and tag removal. For these reasons, vectors have been optimized for use with
TAGZyme ((5), see Fig. 2). Other vectors may be used following the guidelines
for TAGZyme tag design and gene construction strategy (see Subheading 2.1).
Additionally, custom-optimized His-tag sequences for expression in E. coli
or in other hosts and for tag removal can also be generated via mutagenesis
of UZ-HT15. For expression in eukaryotic hosts, a signal peptide may be
placed upstream of the His-tag to facilitate secretion. It is important to use a
well-characterized signal peptide with known a cleavage site to ensure that the
correct number of amino acid residues in the secreted protein will be suitable
for TAGZyme removal of the tag (7).
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Fig. 1. (see Subheading 3.2.) that acts when an N-terminal Q is found resulting
in the formation of a pyroglutamyl. DAPase cleavage is blocked when a pyroglutamyl
is present at the N terminus. After DAPase/Qcyclase treatment and subtractive
Immobilized Metal Affinity Chromatography (IMAC) for enzyme removal, the protein
is treated with pGAPase to remove the pyroglutamyl residue (see Subheading 3.3.).
This step can also be performed using pGAPase bound to an IMAC to simplify the
process (see Subheading 3.4.). Finally, a DAPase test (see Subheading 3.5.) can be
performed on the purified tag-free protein to ensure that the final product does not
contain tag residues. A DAPase stop position is found at the N terminus (P) that
results in a truncated TNF where the first six amino acids (VR SS SR) are cleaved.
This can be confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis
(see Fig. 4).