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 (see figure "Features of the TAGZyme pQE-1 and pQE-2 vectors"). 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 (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
|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... |