A Single Common Protocol for the Expression and Purification of Soluble Mammalian DSPs from Escherichia coli

  • Natalia Stepanyants
  • Patrick J. Macdonald
  • Pooja Madan Mohan
  • Rajesh RamachandranEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2159)


Mammalian DSPs have been historically isolated either from native tissue sources or from transfected insect cell cultures via time-consuming and cumbersome protocols often yielding protein of variable quality and quantity. A facile and highly reproducible alternative methodology involving the heterologous expression and purification of soluble mammalian DSPs from E. coli, which yields highly active and functional protein of a uniform quality and quantity, free of spurious posttranslational modifications inherent to mammalian and insect cell expression systems, is described in this chapter.

Key words

DSP Purification Escherichia coli Polyhistidine Tag Dynamin Drp1 OPA1 



This work was supported by National Institutes of Health grant R01GM121583 awarded to R. R.


  1. 1.
    Stowell MH, Marks B, Wigge P, McMahon HT (1999) Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring. Nat Cell Biol 1:27–32CrossRefGoogle Scholar
  2. 2.
    Wigge P, Kohler K, Vallis Y, Doyle CA, Owen D, Hunt SP, McMahon HT (1997) Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis. Mol Biol Cell 8:2003–2015CrossRefGoogle Scholar
  3. 3.
    Warnock DE, Hinshaw JE, Schmid SL (1996) Dynamin self-assembly stimulates its GTPase activity. J Biol Chem 271:22310–22314CrossRefGoogle Scholar
  4. 4.
    Warnock DE, Terlecky LJ, Schmid SL (1995) Dynamin GTPase is stimulated by crosslinking through the C-terminal proline-rich domain. EMBO J 14:1322–1328CrossRefGoogle Scholar
  5. 5.
    Damke H, Muhlberg AB, Sever S, Sholly S, Warnock DE, Schmid SL (2001) Expression, purification, and functional assays for self-association of dynamin-1. Methods Enzymol 329:447–457CrossRefGoogle Scholar
  6. 6.
    Koirala S, Guo Q, Kalia R, Bui HT, Eckert DM, Frost A, Shaw JM (2013) Interchangeable adaptors regulate mitochondrial dynamin assembly for membrane scission. Proc Natl Acad Sci U S A 110:E1342–E1351CrossRefGoogle Scholar
  7. 7.
    Anggono V, Robinson PJ (2007) Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis. J Neurochem 102:931–943CrossRefGoogle Scholar
  8. 8.
    Cribbs JT, Strack S (2009) Functional characterization of phosphorylation sites in dynamin-related protein 1. Methods Enzymol 457:231–253CrossRefGoogle Scholar
  9. 9.
    Mehrotra N, Nichols J, Ramachandran R (2014) Alternate pleckstrin homology domain orientations regulate dynamin-catalyzed membrane fission. Mol Biol Cell 25:879–890CrossRefGoogle Scholar
  10. 10.
    Macdonald PJ, Stepanyants N, Mehrotra N, Mears JA, Qi X, Sesaki H, Ramachandran R (2014) A dimeric equilibrium intermediate nucleates Drp1 reassembly on mitochondrial membranes for fission. Mol Biol Cell 25:1905–1915CrossRefGoogle Scholar
  11. 11.
    Ban T, Heymann JA, Song Z, Hinshaw JE, Chan DC (2010) OPA1 disease alleles causing dominant optic atrophy have defects in cardiolipin-stimulated GTP hydrolysis and membrane tubulation. Hum Mol Genet 19:2113–2122CrossRefGoogle Scholar
  12. 12.
    Cleland WW (1964) Dithiothreitol, a New Protective Reagent for SH Groups. Biochemistry 3:480–482CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Natalia Stepanyants
    • 1
  • Patrick J. Macdonald
    • 1
  • Pooja Madan Mohan
    • 2
  • Rajesh Ramachandran
    • 1
    • 3
    Email author
  1. 1.Department of Physiology and BiophysicsCase Western Reserve University School of MedicineClevelandUSA
  2. 2.Department of BiochemistryCase Western Reserve University School of MedicineClevelandUSA
  3. 3.Cleveland Center for Membrane and Structural BiologyCase Western Reserve University School of MedicineClevelandUSA

Personalised recommendations