text.skipToContent text.skipToNavigation

TAGZyme DAPase Enzyme

For the removal of N-terminal His tags from proteins expressed using TAGZyme pQE vectors

  • Highly specific exoproteolytic activity prevents internal cleavage
  • Efficient tag removal: >95% in just 30 minutes at 37°C
  • High-purity end products
  • Completely removal of contaminants by Ni-NTA method

TAGZyme DAPase Enzyme removes dipeptides from N-terminal His tags expressed using TAGZyme pQE vectors with high efficiency and processivity. The TAGZyme DAPase Enzyme — a recombinant exopeptidase carrying a His tag at its C-terminus — is removed from reaction mixtures by subtractive immobilized-metal affinity chromatography (IMAC), enabling pure, detagged target protein to be recovered in the flow-through fraction.

Cat No./ID: 34362
TAGZyme DAPase Enzyme (2.5 U)
$500.00
Add To Cart
For processing of approximately 50 mg tagged protein: 2.5 units DAPase Enzyme, 20 mM Cysteamine-HCl (1 ml)
Cat No./ID: 34366
TAGZyme DAPase Enzyme (50 U)
$7,986.00
Add To Cart
For processing of approximately 1 g tagged protein: 50 units DAPase Enzyme, 20 mM Cysteamine-HCl (25 ml)
The TAGZyme DAPase Enzyme is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
0
Purification of detagged proteins.
Scheme of the combined cleavage and purification strategy. [A] Procedure for proteins having a natural DAPase stop point. [B] Procedure for proteins with an introduced glutamine DAPase stop point.
1
Efficient His-tag removal.
Removal of the tag from 6xHis-tagged human tumor necrosis factor α (hTNFα) using the TAGZyme system. 1: Purified 6xHis-tagged hTNFα. 2: After incubation for 10 minutes with DAPase and Qcyclase enzymes at 37°C. 3: After incubation for 20 minutes with DAPase and Qcyclase enzymes at 37°C. 4: After incubation for 30 minutes with DAPase and Qcyclase enzymes at 37°C. 5: After completion of the reaction detagged, pyroglutamyl-extended hTNFα was recovered by subtractive IMAC, subjected to pGAPase digestion, and mature hTNFα was recovered in the flow-through fraction of a second round of subtractive IMAC. 6: An aliquot of the processed protein was incubated with excess DAPase (0.125 U/mg hTNFα) for 2 hours in order to analyze the efficiency of the pGAPase-catalyzed removal of pyroglutamate. The two subunits of His-tagged DAPase enzyme are visible. All samples were subjected to SDS-PAGE and the gel stained by Coomassie Blue. M: markers.
2
His-tag removal.
Schematic summary of the overall cleavage strategy using TAGZyme enzymes. [A] DAPase enzyme cleavage of a N-terminal His tag from a protein containing a natural stop point to obtain the mature target protein. [B] Cleavage of an N-terminal His tag from a protein making use of a glutamine stop point. Following dipeptide cleavage by DAPase enzyme, an N-terminal glutamine residue is converted to pyroglutamate that in turn is removed by pGAPase enzyme action.
Performance
TAGZyme DAPase Enzyme efficiently removes dipeptides sequentially from N-terminal His tags up to the "stop point" expressed using TAGZyme pQE vectors (see figure "Efficient His-tag removal"). Authentic target protein is recovered from the reaction solution by subtractive IMAC. As TAGZyme DAPase Enzyme itself carries an uncleavable His tag at its C-terminus, it is efficiently removed together with unprocessed His-tagged recombinant protein.
Principle

His-tagged recombinant proteins have become valuable tools in studying protein structure and function. The small size and low immunogenicity of the His tag means that its removal is not usually required. However, a protein product free from vector-derived amino acids is preferred for some applications, such as structure-determination studies by X-ray or NMR, or the production of therapeutics.

The TAGZyme system removes N-terminal His tags from recombinant proteins with high specificity and efficiency. DAPase enzyme is used to sequentially cleave off dipeptides from the N-terminus of the purified, His-tagged protein (see figure "His-tag removal": A). Digestion is halted when the enzyme reaches a “stop point”, an amino acid motif that cannot serve as a substrate (see table "DAPase stop points"). If a recombinant protein does not contain an intrinsic DAPase stop point, one can be introduced by inserting a glutamine codon into the expression construct. In the expressed protein, this glutamine is converted to pyroglutamate, a stop point for the DAPase enzme (see figure "His-tag removal": B).

DAPase stop points
Amino acidDAPase stop point (↓) sequence*
 Lysine (Lys, K)  Xaa-Xaa...Xaa-Xaa Lys-Xaa...
 Arginine (Arg, R)  Xaa-Xaa...Xaa-Xaa ↓ Arg-Xaa...
 Proline (Pro, P)  Xaa-Xaa...Xaa-Xaa ↓ Xaa-Xaa-Pro-Xaa...
 Proline (Pro, P)  Xaa-Xaa...Xaa-Xaa ↓ Xaa-Pro-Xaa-Xaa...
 Glutamine (Gln, Q)  Xaa-Xaa...Xaa-Xaa ↓ Gln-Xaa...
 Isoleucine (Ile, I)  Xaa-Xaa...Xaa-Xaa ↓ Xaa-Ile-Xaa-Xaa...
* Natural DAPase stop points (↓) are the following amino acids in the given position within a dipeptide (dipeptides that are cleaved off are underlined).
In the presence of excess Qcyclase. Enzyme, which converts the glutamine residue to pyroglutamate.

Procedure

With recombinant proteins that contain intrinsic stop points, expression using the TAGZyme pQE-2 vector allows complete and efficient removal of the N-terminal His tag irrespective of the cloning site of the DNA insert (see figure "His-tag removal": A). After incubation with DAPase enzyme, the reaction mixture is subjected to subtractive immobilized-metal affinity chromatography (IMAC) using a Ni-NTA matrix (see figure "Purification of detagged proteins"). His-tag fragments and TAGZyme DAPase Enzyme (which carries an uncleavable C-terminal His tag) bind to the matrix, and pure, detagged target protein is recovered in the flow-through fraction.

When an intrinsic DAPase stop point is lacking, one can be introduced into a protein sequence by inserting a glutamine codon into the expression construct. TAGZyme pQE-1 vector encodes for a glutamine residue between the His-tag sequence and the protein sequence, and its use is recommended in conjunction with the TAGZyme system. The glutamine residue is introduced at an odd-numbered position directly behind the His tag and directly before the first amino acid of the native protein. His-tag removal is carried out using both DAPase enzyme and excess Qcyclase enzyme. After removal of His-tag dipeptides by the DAPase enzyme, the glutamine residue appears at the N-terminus (see figure "His-tag removal": B, step 2). Excess Qcyclase enzyme in the reaction catalyzes the formation of a pyroglutamate residue from the glutamine residue at the N-terminus (see figure "His-tag removal": B, step 3). Dipeptides containing pyroglutamate in the N-terminal position cannot serve as DAPase substrates and further cleavage is halted. The reaction mixture is subjected to a round of subtractive IMAC in which the His-tagged TAGZyme DAPase and Qcyclase Enzymes are removed. The target protein is collected in the flow-through fraction. The pyroglutamate residue at the N-terminus of the target protein is then removed by treatment with His-tagged pGAPase enzyme (see figure "His-tag removal": B, step 4), which is itself removed by a second round of subtractive IMAC, leaving pure, detagged target protein in the flow-through fraction (see figure "Purification of detagged proteins").

 

Applications

The TAGZyme system offers specific cleavage, the use of recombinant reagents, and the complete removal of all contaminants, making it the method of choice for the production of His-tag-free proteins for applications including:

  • Protein structure determination by NMR or X-ray crystallography
  • Production of therapeutic proteins
Features
Specifications
Applications Production of therapeutic proteins, protein structure determination using NMR or X-ray crystallography
Efficiency of Tag removal >95%
Protease recognition site Various signals (please see handbook)
Special feature Highly specific exoproteolytic activity
Tag removal Exoproteolytic
Time 30 minutes

You are not authorized to download the resource

Handbooks & Protocols (1)
For Exoproteolytic cleavage of N-terminal His tags
Show details
Kit Handbooks (1)
For Exoproteolytic cleavage of N-terminal His tags
Show details
References
0
fragment fix placeholder

Customers who bought these products also bought