TAGZyme Qcyclase/pGAPase Enzymes

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.

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

* 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.

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...

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 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 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").

 

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