TAGZyme System

For expression of 6xHis-tagged recombinant proteins with subsequent exoproteolytic tag removal and purification

• High specificity — exoproteolytic digest means no nonspecific cleavage takes place within the body of the protein
• A complete solution for His-tag removal — optimized vectors, enzymes, and Ni-NTA resin
• Efficient tag removal — >95% in just 30 minutes at 37°C
• High-purity end products — TAGZyme Enzymes, residual uncleaved protein, and tag fragments are completely removed by subtractive Ni-NTA chromatography
• Recombinant enzymes available in GMP-grade quality — important for the manufacture of therapeutics

The TAGZyme System utilizes recombinant exoproteases, which allow the efficient and precise removal of N-terminal His tags from proteins. In combination with Ni-NTA technology, the 6xHis-tagged TAGZyme Enzymes provide high-purity proteins free from vector-encoded amino-acid sequences. TAGZyme pQE vectors are available that facilitate affinity-tag removal using the TAGZyme System.

Principle

His-tagged recombinant proteins have become a valuable tool 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, there are some applications, such as structure-determination studies by X-ray or NMR, or the production of therapeutics, where a protein product free from vector-derived amino acids may be preferred.

The TAGZyme System removes N-terminal His tags from recombinant proteins with high specificityand efficiency. DAPase Enzyme is used to sequentially cleave off dipeptides from the N-terminus of a purified, His-tagged protein (see figure "His-tag Removal Using TAGZyme Enzymes A", steps 1 and 2). 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").

The removal of affinity tags can be quickly and efficiently analyzed by SDS-PAGE or using the Agilent BioAnalyzer and Lab-on-a-Chip technology.

New! You can now easily check the TAGZyme compatibility of your N-terminal sequences (including His tags) with the TAGDesigner.

DAPase stop points

* 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.

In the case of recombinant proteins that contain intrinsic stop points, 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 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 figure "Purification of Detagged Proteins"). For proteins that contain an intrinsic DAPase stop point, 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 TAGZyme pQE Vector Set ).

His-Tag Removal Using TAGZyme Enzymes

Schematic summary of the overall cleavage strategy.  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.

A DAPase stop point can be introduced into a protein sequence by inserting a glutamine codon into the expression construct. 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 DAPase Enzyme, the glutamine residue appears at the N-terminus (see figure "His-tag Removal Using TAGZyme Enzymes 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 Using TAGZyme Enzymes B", step 3). Dipeptides containing pyroglutamate in the N-terminal position cannot serve as DAPase substrates and further cleavage is therefore halted.

The reaction mixture is then subjected to a round of subtractive IMAC in which the 6xHis-tagged 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 Using TAGZyme Enzymes 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").

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 (see TAGZyme pQE Vector Set ).

Procedure

Recombinant proteins containing His tags appropriate for removal using the TAGZyme System are incubated with the appropriate TAGZyme Enzyme(s) which are removed along with tag fragments and unprocessed protein by a round of Ni-NTA subtractive IMAC. The mature protein free of vector-encoded amino acids is collected in the flow-through fraction.

Applications

The TAGZyme System offers an absence of non 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

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.

Efficient His-Tag Removal Using the TAGZyme System

Removal of the tag from 6xHis-tagged human tumor necrosis factor α(hTNF α) using the TAGZyme System. 1: Purified 6xHis-tagged hTNF α. 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 hTNF α was recovered by subtractive IMAC, subjected to pGAPase digestion, and mature hTNF αwas 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 hTNF α) for 2 hours in order to analyze the efficiency of the pGAPase-catalyzed removal of pyroglutamate. The two subunits of 6xHis-tagged DAPase Enzyme are visible. All samples were subjected to SDS-PAGE and the gel stained by Coomassie Blue. M: markers.

6x His-tag/ Ni-NTA and TAGZyme Patent and Licensing information

QIAGEN is the exclusive license holder for His-tag technology and offers a complete range of fully licensed products for expression, purification, detection, and assay of 6xHis-tagged proteins, which can be used for research purposes without infringing any claims of the US patent no. 4,569,794 and its foreign equivalents. For further details click here.

DAPase is not to be used for the elucidation of any form of crystal structure of DPPI.
DAPase is not to be used for drug discovery, i.e., design, test or construction of DPPI ligands including DPPI inhibitors.