HaloTag

It is a 297 residue protein (33 kDa) derived from a bacterial enzyme, designed to covalently bind to a synthetic ligand.

[2] The HaloTag is composed of two covalently bound segments including a haloalkane dehalogenase and a synthetic ligand of choice.

[3] Functional groups can either be biotin (can be used as an affinity tag) or can be chosen from five available fluorescent dyes including Coumarin, Oregon Green, Alexa Fluor 488, diAcFAM, and TMR.

[6] In the aforementioned reaction, nucleophilic attack of the chloroalkane reactive linker causes displacement of the halogen with an amino acid residue, which results in the formation of a covalent alkyl-enzyme intermediate.

[12] Recently, HaloTag has been engineered to create hybrid protein + small molecule biosensors of neuronal activity.

HaloTag protein coding region can be inserted near the gene of interest. An ampicillin resistance gene is also inserted for purification purposes.
Five available fluorescent dyes that can be bound to the reactive linker. In addition, the terminal chlorine of the reactive chloroalkane can be visualized.
Different parts of the fusion protein. As illustrated in the figure, HaloTag protein consists of the synthetic ligand and reactive chloroalkane linker parts. The HaloTag protein is attached to the protein of interest.