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).

 

Fig. 1. Schematic representation of TAGZyme application for Histagged proteins using A) DAPase alone or B) DAPase in combination with Qcyclase and pGAPase
 

TAGZyme processing of his-tags
A key feature of the TAGZyme enzymes manufactured by Unizyme Laboratories is their affinity for metal-chelate matrices due to their his-tags (HT). This property makes it possible to design an efficient purification and de-tagging process based on the exclusive use of immobilized metal-chelate affinity chromatography (IMAC) and desalting.

 

*) Excision of his-tags from proteins with natural DPPI stop-points is partly covered by US Patent 5,619,169 owned by Novo Nordisk.