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Kinetic and structural characterization of the self-labeling protein tags HaloTag7, SNAP-tag and CLIP-tag

By Jonas Wilhelm, Stefanie Kuehn, Miroslaw Tarnawski, Guillaume Gotthard, Jana Tuennermann, Timo Taenzer, Julie Karpenko, Nicole Mertes, Lin Xue, Ulrike Uhrig, Jochen Reinstein, Julien Hiblot, Kai Johnsson

Posted 13 Apr 2021
bioRxiv DOI: 10.1101/2021.04.13.439540

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag and CLIP-tag allow the covalent label-ing of fusion proteins with synthetic molecules for applications in bioimaging and biotechnology. To guide the selection of an SLP-substrate pair and provide guidelines for the design of sub-strates, we report a systematic and comparative study on the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rates with certain rhodamine substrates, which are more than two orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rates how-ever are less affected by the structure of the label than those of HaloTag7, which vary over six orders of magnitude for commonly employed substrates. Solving the crystal structures of Halo-Tag7 and SNAP-tag labeled with fluorescent substrates allowed us to rationalize their substrate preferences. We also demonstrate how these insights can be exploited to design substrates with improved labeling kinetics.

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