TAGZyme principles
Basic Principles of the TAGZyme System
The TAGZyme System is an enzymatic system for the complete removal of N-terminal polyhistidine tags (his-tags) from recombinant proteins. Generation of untagged, target protein involves the use of either one or three exo-enzymes depending on the sequence of the target-protein In any case, the metal binding polyhistidine sequence is first cleaved off by DAPase which catalyzes a stepwise removal of N-terminal dipeptides except if (A) the amino group of the N-terminus is blocked, (B) the site of cleavage is on either side of a proline, or (C) the N-terminal residue is either lysine or arginine. This ability of certain residues to function as DAPase stop-points is exploited to ensure the integrity of the desired protein by preventing excessive digestion. See the TAGZyme presentation.
Excision of his-tags from proteins with natural DAPase stop-points*)
A range of mature proteins have DAPase stop-points of type B or C. Accordingly, N-terminal his-tags containing an even number of amino acid residues may be completely and specifically removed by treatment with DAPase alone (Fig. 1A).
Excision of his-tags from proteins without natural DAPase stop-points
If the N-terminus of the target-protein does not contain blocking residues, a type A stop-point consisting of an N-terminal pyroglutamyl is generated enzymatically by co-incubation with Qcyclase. Qcyclase catalyzes the cyclization of N-terminal glutamine residues to pyroglutamyl (Fig. 1B, step 1), and the glutamine residue to be converted should be inserted between the sequences of the his-tag and the target protein. An uneven competition between Qcyclase in excess and DAPase ensures immediate cyclization of the inserted glutamine when the his-tag is completely digested and the glutamine appears in the N-terminus. This pyroglutamyl-extended form of the target-protein is protected against further DAPase digestion.Following the co-incubation, Qcyclase and DAPase are removed from the product (pyroglutamyl extended target-protein) and the target sequence is then obtained by cleaving off the pyroglutamyl residue with pGAPase (Fig.1B, step 2).
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