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Affinity Purification and Functional Characterization of Dynamin-Related Protein 1

  • Ryan W. Clinton
  • Brianna L. Bauer
  • Jason A. MearsEmail author
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Part of the Methods in Molecular Biology book series (MIMB, volume 2159)

Abstract

Purification of dynamin-related proteins is complicated by their oligomeric tendencies. In this chapter, we describe an established purification regime to isolate the mitochondrial fission protein Drp1 using bacterial expression. Key attributes of dynamins include their ability to hydrolyze GTP and self-assemble into larger polymers under specific conditions. Therefore, the GTPase activity of Drp1 should be examined to confirm isolation of functional protein, and we describe a conventional colorimetric assay to assess enzyme activity. To determine the ability of Drp1 to self-assemble, we induce Drp1 polymerization through addition of a non-hydrolyzable GTP analogue. A sedimentation assay provides a quantitative measure of polymerization that complements a qualitative assessment through visualization of Drp1 oligomers using negative-stain electron microscopy (EM). Importantly, we highlight the caveats of affinity tags and the influence that these peptide sequences can have on Drp1 function given their proximity to functional domains.

Key words

Dynamin-related protein Mitochondrial fission GTPase Protein oligomerization Electron microscopy 

Notes

Acknowledgement

The authors would like to acknowledge the Ramachandran lab for providing GST-tagged Drp1. Protein purification and characterization studies were performed by RWC and BLB. RWC was supported by the American Heart Association (16GRNT30950012). BLB is supported by the Molecular Therapeutics Training Grant (NIH, T32 GM008803-15). This work and JAM are supported by the NIH (R01 GM125844-01 and R01 CA208516-01A1).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Ryan W. Clinton
    • 1
  • Brianna L. Bauer
    • 1
  • Jason A. Mears
    • 1
    Email author
  1. 1.Department of PharmacologyCenter for Mitochondrial Diseases and Cleveland Center for Membrane and Structural Biology, Case Western Reserve University School of MedicineClevelandUSA

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