PotM: Substrates for Fusion (Halo/Snap/Clip)-Tagged Proteins

Published on 25/01/2022

Discover our selection of substrates for Halo-, Snap-, and Clip-tagged proteins bearing different terminal groups suitable for further functionalization, e.g. biotinylation and Click chemistry.

PotM: Substrates for Fusion (Halo/Snap/Clip)-Tagged Proteins

Site-specific protein labeling is a versatile tool for studying protein function and interaction in living cells. Various peptide-based protein tags have been developed. Herein, we are focusing on HaloTag®, SNAP-Tag® and CLIP-Tag^TM, as well as corresponding substrates offered by Iris Biotech. Those labeling systems enable the specific, covalent attachment of in principle any molecule of choice to a protein of interest.

The HaloTag® is a 33 kDa self-labeling protein tag derived from the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous. Its active site reacts in a nucleophilic attack with chloroalkane linker substrates to form an irreversible bond in the case of the mutated enzyme. The chloroalkane linker can easily be functionalized with a label of choice, e.g. fluorophore, biotin. For the wild-type enzyme, this intermediate would be hydrolyzed, leading to the regeneration of the enzyme.

Schematic illustration of the HaloTag® reaction

The SNAP-tag® is a 20 kDa self-labeling protein tag based on a modified form of the human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair enzyme. A cysteine residue within the SNAP-tag® undergoes an irreversible reaction with synthetic O6-benzylguanine (BG) derivatives resulting in a covalent thioether bond. The BG moiety can easily be further functionalized with a label of choice, e.g. fluorophore, biotin, generally without affecting the reaction of the substrate with the SNAP-tag®.

Schematic illustration of the SNAP-tag® reaction

The CLIP-tag^TM (20 kDa) is a modified version of the SNAP-tag, engineered to react with benzylcytosine (BC) instead of benzylguanine (BG). Thus, properties are similar. CLIP-tag^TM- and SNAP-tag®-fused proteins can be labeled simultaneously in the same cells.

Schematic illustration of the CLIP-tag^TM reaction

Table summarizing main properties of HaloTag®, SNAP-tag® and CLIP-tag^TM.

	HaloTag	SNAP-tag	CLIP-tag
Origin	Haloalkane dehalogenase (Rhodococcus rhodochrous)	Human O6-alkylguanine-DNA-alkyltransferase	Human O6-alkylguanine-DNA-alkyltransferase
Reactivity	Chloroalkane derivatives	O6-benzylguanine derivatives	Benzylcytosine derivatives
Length	297 amino acids	182 amino acids	182 amino acids
Molecular Weight	33.6 kDa	19.4 kDa	19.4 kDa

Iris Biotech offers a Biotin- (RL-3860) as well as an ICG-functionalized (RL-3830) SNAP-tag® substrate, as well as the corresponding CLIP-tag^TM suitable (RL-3840, RL-3870) derivatives. Biotinylated proteins can for example be selectively isolated based on the high affinity towards avidin representing a useful tool for purification, immobilization, and labeling. Indocyanine green (ICG) is a near-infrared fluorescence imaging dye (absorption maximum 800 nm + slight absorption in the visible range; emission maximum 810 nm) approved by the FDA.

As substrates for the HaloTag® various products are offered (see related products), e.g. suitable for further functionalization via Click Chemistry.

You could not find the derivative you are looking for? Please inquire for a Custom Synthesis!

SNAP-tag® is a registered trademark and CLIP-tag^TM a trademark of New England Biolabs, Inc. HaloTag® is a registered trademark to Promega Corporation. HaloTag® Technology is proprietary to Promega Corporation.

References:

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Site-specific, Covalent Labeling of Recombinant Antibody Fragments via Fusion to an Engineered Version of 6-O-Alkylguanine DNA Alkyltransferase; F. Kampmeier, M. Ribbert, T. Nachreiner, S. Dembski, F. Beaufils, A. Brecht, S. Barth; Bioconjugate Chem. 2009; 20(5): 1010-1015. https://doi.org/10.1021/bc9000257.

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HaloTag: a novel protein labeling technology for cell imaging and protein analysis; G. V. Los, L. P. Encell, M. G. McDougall, D. D. Hartzell, N. Karassina, C. Zimprich, M. G. Wood, R. Learish, R. F. Ohana, M. Urh, D. Simpson, J. Mendez, K. Zimmerman, P. Otto, G. Vidugiris, J. Zhu, A. Darzins, D. H. Klaubert, R. F. Bulleit and K. V. Wood; ACS Chem Biol 2008; 3: 373-382. https://doi.org/10.1021/cb800025k

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