Expression, purification, and characterization of anuran saxiphilins using thermofluor, fluorescence polarization, and isothermal titration calorimetry

Zhou Chen; Sandra Zakrzewska; Holly S Hajare; J Du Bois; Daniel L Minor Jr

doi:10.1016/j.xpro.2023.102792

Expression, purification, and characterization of anuran saxiphilins using thermofluor, fluorescence polarization, and isothermal titration calorimetry

STAR Protoc. 2024 Mar 15;5(1):102792. doi: 10.1016/j.xpro.2023.102792. Epub 2023 Dec 20.

Authors

Zhou Chen¹, Sandra Zakrzewska¹, Holly S Hajare², J Du Bois³, Daniel L Minor Jr⁴

Affiliations

¹ Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158-2330, USA.
² Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
³ Department of Chemistry, Stanford University, Stanford, CA 94305, USA. Electronic address: jdubois@stanford.edu.
⁴ Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158-2330, USA; Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158-2330, USA; California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, CA 94158-2330, USA; Kavli Institute for Fundamental Neuroscience, University of California, San Francisco, San Francisco, CA 94158-2330, USA; Molecular Biophysics and Integrated Bio-imaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. Electronic address: daniel.minor@ucsf.edu.

Abstract

Anuran saxiphilins (Sxphs) are "toxin sponge" proteins thought to prevent the lethal effects of small-molecule neurotoxins through sequestration. Here, we present a protocol for the expression, purification, and characterization of Sxphs. We describe steps for using thermofluor, fluorescence polarization, and isothermal titration calorimetry assays that probe Sxph:saxitoxin interactions using a range of sample quantities. These assays are generalizable and can be used for other paralytic shellfish poisoning toxin-binding proteins. For complete details on the use and execution of this protocol, please refer to Chen et al. (2022).¹.

Keywords: Protein Biochemistry; Protein expression and purification; Structural Biology.

MeSH terms

Calorimetry
Fluorescence Polarization
Neurotoxins*
Saxitoxin* / metabolism

Substances

Saxitoxin
Neurotoxins

Grants and funding

R01 GM117263/GM/NIGMS NIH HHS/United States