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

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). 

TAGZyme Qcyclase/pGAPase Enzymes (150 U) (Cat.no 34342) and the TAGZyme Kit (Cat.no 34300) have been discontinued. The successor product recommended for proteins with an 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 . 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.

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 . 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 . Digestion is halted when the enzyme reaches a “stop point”, an amino acid motif that cannot serve as a substrate . 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 .

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...

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 . After incubation with DAPase enzyme, the reaction mixture is subjected to subtractive immobilized-metal affinity chromatography (IMAC) using a Ni-NTA matrix. 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 . Excess Qcyclase enzyme in the reaction catalyzes the formation of a pyroglutamate residue from the glutamine residue at the N-terminus . 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 , which is itself removed by a second round of subtractive IMAC, leaving pure, detagged target protein in the flow-through fraction .

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.