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Home › Products › Protein research › Expression vectors & systems › E. coli expression systems › HAT-tag expression system

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Histidine affinity tag—HAT Protein Expression and Purification System

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.

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Cat. # Product Size License Quantity Details
631205 HAT™ Protein Expression & Purification System Each USD $744.00

This is a complete system for bacterial expression and IMAC purification of proteins. The system is based on the pHAT Vectors, which contain a novel histidine affinity tag (HAT) that allows one-step purification of tagged proteins from crude cell lysates. The HAT sequence in these vectors is followed by an enterokinase (EK) cleavage site and an MCS. The HAT
sequence and EK site are in frame with the lacZ initiation codon. The vector construct is provided in all three reading frames.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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FPLC purification of HAT-tagged protein with TALON Superflow

FPLC purification of HAT-tagged protein with TALON Superflow
FPLC purification of HAT-tagged protein with TALON Superflow. E. coli cells were extracted in 50 mM sodium phosphate, 0.3 M NaCl, 5 mM imidazole, pH 7.0 and eluted in 150 mM imidazole (1). Panel A. Cell lysate was purified with a TALON Superflow Column. Panel B. SDS-PAGE analysis of the procedure. M: molecular weight marker.

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pHAT10/11/12 and pHAT20 Vector maps

pHAT10/11/12 and pHAT20 Vector maps
pHAT10/11/12 and pHAT20 Vector maps.

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Amino acid sequences of 6xHis, 6xHN, and HAT polyhistidine tags

Amino acid sequences of 6xHis, 6xHN, and HAT polyhistidine tags
Amino acid sequences of 6xHis, 6xHN, and HAT polyhistidine tags.

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Features and benefits of the HAT (histidine affinity tag) Protein Expression & Purification System, which allows proteins expressed in bacteria to be purified at neutral or physiological pH and in native or denaturing conditions

Features and benefits of the HAT (histidine affinity tag) Protein Expression & Purification System, which allows proteins expressed in bacteria to be purified at neutral or physiological pH and in native or denaturing conditions
Features and benefits of the HAT (histidine affinity tag) Protein Expression & Purification System, which allows proteins expressed in bacteria to be purified at neutral or physiological pH and in native or denaturing conditions.

Overview

  • Purify proteins at neutral pH
  • Use under native or denaturing conditions
  • Higher protein solubility
  • Obtain optimum purification with TALON IMAC resin

More Information

Applications

Easy protein expression and purification in bacterial systems, for higher protein solubility

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.

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