S_1125_9_TAGZyme_DAPase_Enzyme

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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)
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EnzymeVector
TAGZyme Enzyme
TAGZyme pQE Vector
Quantity
2.5 U (1 mL)
50 U (25 mL)
The TAGZyme System is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Features

  • Expressed His-tag optimized for removal by TAGZyme enzyme
  • 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

Product Details

The TAGZyme Enzyme DAPase includes sufficient enzyme 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.

Performance

TAGZyme DAPase Enzyme efficiently removes dipeptides sequentially from N-terminal His tags up to the "stop point" expressed using TAGZyme pQE-2 vector.

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 pQE-2 vector is suitable for proteins containing an intrinsic DAPase stop point. 

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”). 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").

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"). 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.

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

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)
TAGZyme Handbook
PDF (2MB)
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.
-80