2. Academic Validation
  3. Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag

Kinetic and Structural Characterization of the Self-Labeling Protein Tags HaloTag7, SNAP-tag, and CLIP-tag

  • Biochemistry. 2021 Aug 24;60(33):2560-2575. doi: 10.1021/acs.biochem.1c00258.
Jonas Wilhelm 1,  Stefanie Kühn 1,  Miroslaw Tarnawski 2,  Guillaume Gotthard 3,  Jana Tünnermann 1,  Timo Tänzer 4,  Julie Karpenko 4,  Nicole Mertes 1,  Lin Xue 1,  Ulrike Uhrig 5,  Jochen Reinstein 6,  Julien Hiblot 1 4,  Kai Johnsson 1 4
Affiliations

Affiliations

  • 1 Department of Chemical Biology, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
  • 2 Protein Expression and Characterization Facility, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
  • 3 Structural Biology Group, European Synchrotron Radiation Facility (ESRF), 38043 Grenoble, France.
  • 4 Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland.
  • 5 Chemical Biology Core Facility, European Molecular Biology Laboratory, 69117 Heidelberg, Germany.
  • 6 Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany.
PMID: 34339177 DOI: 10.1021/acs.biochem.1c00258
Abstract

The self-labeling protein tags (SLPs) HaloTag7, SNAP-tag, and CLIP-tag allow the covalent labeling 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 substrates, we report a systematic and comparative study of the labeling kinetics and substrate specificities of HaloTag7, SNAP-tag, and CLIP-tag. HaloTag7 reaches almost diffusion-limited labeling rate constants with certain rhodamine substrates, which are more than 2 orders of magnitude higher than those of SNAP-tag for the corresponding substrates. SNAP-tag labeling rate constants, however, are less affected by the structure of the label than those of HaloTag7, which vary over 6 orders of magnitude for commonly employed substrates. Determining the crystal structures of HaloTag7 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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