HaloTag protein coding region can be inserted near the gene of interest. An ampicillin resistance gene is also inserted for purification purposes.
HaloTag is a self-labeling protein tag. It is a 297 residue protein (33 kDa) derived from a bacterial enzyme, designed to covalently bind to a synthetic ligand. The bacterial enzyme can be fused to various proteins of interest.[1] The synthetic ligand is chosen from a number of available ligands in accordance with the type of experiments to be performed. This bacterial enzyme is a haloalkane dehalogenase, which acts as a hydrolase and is designed to facilitate visualization of the subcellular localization of a protein of interest, immobilization of a protein of interest, or capture of the binding partners of a protein of interest within its biochemical environment.[2] The HaloTag is composed of two covalently bound segments including a haloalkane dehalogenase and a synthetic ligand of choice. These synthetic ligands consist of a reactive chloroalkane linker bound to a functional group.[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. These fluorescent dyes can be used in the visualization of either living or chemically fixed cells.[4]
^Los GV, Encell LP, McDougall MG, Hartzell DD, Karassina N, Zimprich C, et al. (June 2008). "HaloTag: a novel protein labeling technology for cell imaging and protein analysis". ACS Chemical Biology. 3 (6): 373–82. doi:10.1021/cb800025k. PMID 18533659.
^Giepmans BN, Adams SR, Ellisman MH, Tsien RY (April 2006). "The fluorescent toolbox for assessing protein location and function". Science. 312 (5771): 217–24. Bibcode:2006Sci...312..217G. doi:10.1126/science.1124618. PMID 16614209. S2CID 1288600.
^Kogure T, Karasawa S, Araki T, Saito K, Kinjo M, Miyawaki A (May 2006). "A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy". Nature Biotechnology. 24 (5): 577–81. doi:10.1038/nbt1207. PMID 16648840. S2CID 19616823.
^Miller LW, Cornish VW (February 2005). "Selective chemical labeling of proteins in living cells". Current Opinion in Chemical Biology. 9 (1): 56–61. doi:10.1016/j.cbpa.2004.12.007. PMID 15701454.
HaloTag is a self-labeling protein tag. It is a 297 residue protein (33 kDa) derived from a bacterial enzyme, designed to covalently bind to a synthetic...
ISBN 9780124201385. PMC 4198327. PMID 24974023. N Peterson S, Kwon K (2012). "The HaloTag: Improving Soluble Expression and Applications in Protein Functional Analysis"...
bound by nanobodies HaloTag, a mutated bacterial haloalkane dehalogenase that covalently attaches to haloalkane substrates SNAP-tag, a mutated eukaryotic...
Harling JD, Crews CM (August 2015). "HaloPROTACS: Use of Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins". ACS Chemical Biology...
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