The HAT (histidine affinity tag) Protein Expression and Purification System provides a convenient and efficient way to express and purify proteins. HAT vectors encode a novel polyhistidine epitope tag that allows proteins expressed in bacteria to be purified at neutral or physiological pH and under native or denaturing conditions.
The HAT (histidine affinity tag) Protein Expression and Purification System provides a convenient and efficient way to express and purify proteins. HAT vectors encode a novel polyhistidine epitope tag that allows proteins expressed in bacteria to be purified at neutral or physiological pH and under native or denaturing conditions.
The HAT system
The HAT (histidine affinity tag) epitope is a naturally-occurring sequence of non-adjacent histidine residues that has a lower overall charge than tags with consecutive His residues.
HAT-protein fusions exhibit solubility that more closely resembles that of wild-type proteins, while still possessing strong affinity for immobilized metal ions. Using the HAT system to purify proteins offers two major advantages:
- The protein is soluble, therefore it does not form aggregates or reside within inclusion bodies.
- The protein can be eluted under mild conditions, such as neutral pH or low imidazole concentration.
Proteins expressed from pHAT vectors are ideal for purification with TALON resin using batch or gravity-flow protocols, or with TALON Superflow Resin using medium pressure FPLC. The cobalt metal ion in the TALON resin has a high affinity for HAT and other polyhistidine-tagged proteins, and a very low affinity for other proteins. The exceptional selectivity of TALON resin reduces background and requires fewer washes with milder conditions that preserve protein integrity.
pHAT vectors
The HAT system includes three pHAT vectors—pHAT10/11/12, which are the same construct but have multiple cloning sites (MCS) in each of the three reading frames. An enterokinase (EK) cleavage site has been incorporated into each of the vectors, allowing removal of the HAT sequence from the purified protein. Restriction sites are present to allow the HAT sequence to be excised, with or without the EK site, for cloning into other vectors.
The pHAT20 Vector, available separately, provides all the advantages of the pHAT10/11/12 vectors along with the convenience of additional cloning sites, making it easier to clone your cDNA in-frame for fusion to the HAT sequence.
