TAGZyme System

For expression of His-tagged proteins and removal of His tags

Features

  • Expressed His-tag optimized for removal by TAGZyme enzymes
  • Efficient His-tag removal: >95% in just 30 minutes at 37°C
  • High-level expression of N-terminally His-tagged proteins
  • High-purity end products
  • Complete removal of contaminants by Ni-NTA method
TAGZyme DAPase Enzyme (2.5 U)

Cat. No. / ID: 34362

For processing of approximately 50 mg tagged protein: 2.5 units DAPase Enzyme, 20 mM Cysteamine-HCl (1 ml)
$493.00
EnzymeVector
TAGZyme Enzyme
TAGZyme pQE Vector Set
Quantity
2.5 U (1 ml)
50 U (25 ml)
Add to cart
The TAGZyme System is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

The TAGZyme system includes TAGZyme Enzymes, reagents and Ni-NTA Agarose sufficient for highly specific and efficient His-tag removal from up to 10 mg His-tagged protein. The TAGZyme system can be used for His-tag removal from proteins containing an intrinsic DAPase stop point (expressed using the TAGZyme pQE-2 vector, and processed using TAGZyme DAPase Enzyme), or from proteins that contain an engineered glutamine stop point (expressed using the TAGZyme pQE-1 vector, and processed using TAGZyme DAPase, Qcyclase and pGAPase Enzymes). To choose the right vector for your application, contact QIAGEN Technical Services or your local distributor.

TAGZyme Qcyclase/pGAPase Enzymes (150 U) (Cat.no 34342) and the TAGZyme Kit (Cat.no 34300) will be discontinued during May 2021. They remain available as long as stocks last. The successor product recommended for proteins with intrinsic stop point is TAGZyme DAPase Enzyme (2.5 U) (Cat.no 34362).

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"). When a stop point has be introduced as a glutamine residue, this is efficiently removed by the combined action of DAPase-Qcyclase and pGAPase. Authentic target protein is recovered from the reaction solution by subtractive IMAC. As TAGZyme DAPase, Qcyclase and pGAPase Enzymes all carry an uncleavable His tag, they are efficiently removed together with unprocessed His-tagged recombinant protein.

See figures

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.


Both TAGZyme pQE vectors contain multiple cloning sites for the expression of N-terminal 6xHis-tagged proteins that possesses a sequence optimized for DAPase digestion (see figure  "Features of the TAGZyme pQE-1 and pQE-2 vectors"). High levels of expression are obtained in E. coli based on the T5-promoter transcription-translation system. The TAGZyme pQE-2 vector is suitable for proteins containing an intrinsic DAPase stop point. For proteins lacking such a stop point, the TAGZyme pQE-1 vector encodes for a glutamine residue between the His-tag sequence and the protein sequence, which serves as a DAPase stop point when converted to pyroglutamate by the action of excess Qcyclase enzyme.


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 acid DAPase 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...
See figures

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 a C-terminal 6xHis 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 6xHis-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 6xHis-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 6xHis-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").

See figures

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

The TAGZyme System is intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Supporting data and figures

Resources

Selection Guides (1)
Vector Sequences & Maps (2)
For the pQE-2 vector
For the pQE-1 vector
Package Insert (1)
Kit Handbooks (1)
For Exoproteolytic cleavage of N-terminal His tags

Publications

Production and comprehensive quality control of recombinant human Interleukin-1beta: a case study for a process development strategy.
Block H; Kubicek J; Labahn J; Roth U; Schäfer F;
Protein Expr Purif; 2007; 57 (2):244-54 2007 Oct 17 PMID:18053740

FAQ

Is it possible to cleave the 6xHis-tag from an expressed protein?

Yes. In order to cleave off an N-terminal 6xHis tag, a protease cleavage site must be inserted between the coding sequences of the tag and the N-terminus of the protein. Factor Xa Protease recognizes the amino acid sequence Ile-Glu-Gly-Arg and cleaves the peptide bond C-terminal of the arginine residue. The expression vector pQE-30 Xa encodes a Factor Xa Protease recognition site between the N-terminal 6xHis-tag sequence and the multiple cloning site.

If the gene of interest is cloned blunt ended at the 5´-end using the StuI restriction site of the vector, Factor Xa Protease cleavage of the purified recombinant protein results in a protein product without any vector-derived amino acids at the N-terminus.

Tags can also be removed exoproteolytically using the TAGZyme System. This system is an efficient and specific solution for the complete removal of small N-terminal His tags and other amino acid tags by the use of exopeptidases. For detailed information on the procedure please review the TAGZyme handbook.

Please note that both tag removal options work on N-terminal 6xHis tags only.

 

 

FAQ ID -140
Does endogenously expressed DAPase in human cells degrade proteins that are expressed in cell culture?
DAPase (dipeptidyl aminopeptidase I = Cathepsin C), even though endogenously expressed in human tissues and cells, will have only a negligible effect on the degradation of proteins expressed in cell culture. The reason for this is the low level of endogenous DAPase compared to the much higher level of typically overexpressed recombinant protein. However, it is always worthwhile to run a time-course expression experiment to check for possible protein degradation, since proteases and peptidases tend to degrade proteins over time.
FAQ ID -527
How complete is the removal of DAPase in the TAGZyme process?
The removal of DAPase is >99%.
FAQ ID -319
What are the calculated molecular weights of the TAGZyme enzymes?
DAPase: heterodimer, 24 kDa and 6 kDa subunit; Qcyclase: 35 kDa; pGAPase: 28 kDa
FAQ ID -329
Is it possible to store TAGzyme enzymes at°C?
Yes. The TAGzyme enzymes do not experience any loss in function after several freeze and thaw cycles. However, storage at -80°C is not necessary as storage at -20°C is adequate.
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