Cat. No. / ID: 34362
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.
TAGZyme DAPase Enzyme efficiently removes dipeptides sequentially from N-terminal His tags up to the "stop point" expressed using TAGZyme pQE-2 vector.
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...|
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.
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:
Schematic summary of the overall cleavage strategy using TAGZyme enzymes. DAPase enzyme cleavage of a N-terminal His tag from a protein containing a natural stop point to obtain the mature target 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.