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Expression, Purification, and Structure Determination of Human PTCH1–HH-N Complexes

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Hedgehog Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2374))

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Abstract

Patched-1 (PTCH1), a tumor suppressor, serves as the receptor of Hedgehog (HH) ligand and negatively regulates the HH signaling pathway. Mutations of PTCH1 are implicated in many human cancers. Structural investigation revealed the mechanism of PTCH1-mediated HH signal regulation, further facilitating the therapeutic development of cancers. Here, we describe the expression and purification of a nearly full-length functional PTCH1 variant, PTCH1*. With purified PTCH1* protein, two forms of PTCH1*–Sonic Hedgehog (SHH) complexes were assembled, and their structures subsequently determined by cryo-electron microscope (cryo-EM).

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Acknowledgments

This work was supported by the Endowed Scholars Program in Medical Science of UT Southwestern Medical Center and NIH grant R01 GM135343 (to X.L.). X.Q. is the recipient of a DDBrown Fellow of the Life Sciences Research Foundation. P.S. is supported by NIH grant T32 GM131963. X.L. is a Damon Runyon-Rachleff Innovator supported by the Damon Runyon Cancer Research Foundation (DRR-53-19) and a Rita C. and William P. Clements, Jr. Scholar in Biomedical Research at UT Southwestern Medical Center.

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Authors and Affiliations

  1. Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    **aofeng Qi, Philip Schmiege & Leticia Esparza

  2. Department of Molecular Genetics and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    **aochun Li

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  1. **aofeng Qi
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  2. Philip Schmiege
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  3. Leticia Esparza
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  4. **aochun Li
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Corresponding authors

Correspondence to **aofeng Qi or **aochun Li .

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Editors and Affiliations

  1. Department of Molecular Genetics and Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    **aochun Li

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Qi, X., Schmiege, P., Esparza, L., Li, X. (2022). Expression, Purification, and Structure Determination of Human PTCH1–HH-N Complexes. In: Li, X. (eds) Hedgehog Signaling. Methods in Molecular Biology, vol 2374. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1701-4_10

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  • DOI: https://doi.org/10.1007/978-1-0716-1701-4_10

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1700-7

  • Online ISBN: 978-1-0716-1701-4

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