Products

 

TAGZyme

TAGZyme is an enzymatic system for the affinity purification of recombinant proteins using his-tags and tag removal. It combines a dipeptidase (DAPase) for exoproteolytic cleavage from the N-terminus and -where required- two accessory aminopeptidases (Qcyclase and pGAPase) for the complete removal of the his-tag. All three enzymes display a non-cleavable his-tag for removal

 

If the protein you need to produce contains a suitable stop position for DAPase cleavage at its N-terminus, then tag cleavage is performed in a single step after an initial IMAC purification of the his-tag protein (see TAGZyme for more information). In these cases a his-tag containing an even number of residues (e.g., our optimized tag sequence MKHQHQHQHQHQHQ) is used. Alternatively, the commonly used 6xHis tag present in some general purpose vectors can also be used (e.g, MKHHHHHH). Care should be used to avoid using vectors that include 'bad sequences' that will affect the cleavage of the tag (e.g., GS). In these cases, we have recently described a straightforward method for the modification of the sequence for use with TAGZyme (Arnau et al. 2006).

 

For all other proteins and for HTS projects devoted to production of numerous proteins or sequence variants of a protein (e.g., molecular evolution), tag cleavage is done with DAPase and Qcyclase using a his-tag containing an uneven number of amino acids where the C-terminal is Q (e.g., MKHQHQHQHQHQHQQ). Under these conditions when Q is at the N-terminus, it is converted to pyroglutamyl by Qcyclase preventing further cleavage. After enzyme removal, the pyroglutamyl is removed using pGAPase.

 

DAPase

DAPase is a recombinant dipeptidyl peptidase I. It cleaves dipeptides sequentially from the N-terminus of peptides and proteins. Not all dipeptide combinations can be cleaved by DAPase. This is used to design the his-tag and the fusion to the protein of interest in order to enable affinity tag (e.g., his-tag) removal without affecting the desired sequence of the purified protein. Interestingly, many human therapeutic proteins and more than 25 % of all proteins do contain a suitable DAPase stop position at the N-terminus and can therefore be produced using a his-tag and DAPase cleavage. Typical yields are above 90 %.

 

Only reduced amounts of DAPase are required for effective tag removal (1:2000 to 1:5000 depending on the target protein). And the purification process can be typically run in 5-8 h. DAPase is produced to cGMP in animal free medium to comply with regulatory issues.

 

If you are interested in testing your his-tag protein construction for its suitability for tag removal using DAPase alone or in combination with Qcyclase and pGAPase, please send us an email here.

 

Qcyclase

Qcyclase is a recombinant plant glutamine cyclotransferase. It converts N-terminal Q to pyroglutamyl. Qcyclase is used in combination with DAPase for the cleavage of uneven-numbered tags where Q is present as the last residue of the tag. Qcyclase can also be used to protect the N-terminus of proteins to prevent aggregation.

 

pGAPase

pGAPse is a recombinant bacterial pyroglutamyl aminopeptidase. It catalyzes the removal of N-terminal pyroglutamyl residues. It is used as the last step in tag removal for proteins without a suitable DAPase stop position and for HTS projects. It is also applicable for the deblocking of N-termini prior to amino acid sequencing of peptides and proteins.