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Expression and purification of dynamin II domains and initial studies on structure and function

Item Type:Article
Title:Expression and purification of dynamin II domains and initial studies on structure and function
Creators Name:Dong, J. and Misselwitz, R. and Welfle, H. and Westermann, P.
Abstract:Dynamin II, a large GTP-binding protein, is involved in endocytosis and in vesicle formation at the trans-Golgi network. To further elucidate functions of dynamin II, the pleckstrin homology domain (PHD), the proline-rich domain (PRD), and the C-terminal part of dynamin II (dynamin(500-870)) were expressed in Escherichia coli. The PHD, tagged C-terminally by a (His) 6 peptide, was expressed to 15% of cellular proteins and could be purified on nickel-chelating agarose. On the contrary, the PRD and dynamin(500-870) had to be tagged with a (His) 6 peptide at the N-terminus to bind to nickel-chelating agarose. Additional tagging with the S-peptide, which forms a stable complex with immobilized S-protein, allowed removal of strongly interacting E. coli proteins. Circular dichroic spectra indicate a structured recombinant PHD with a secondary structure content similar to that of the known PHD from dynamin I. The N-terminally tagged, recombinant PRD is unfolded but nevertheless binds specifically to the SH3 domain of amphiphysin II as well as to proteins extracted from rat brain. The described methods are suitable to isolate functionally active domains of dynamin II in sufficient amount and purity for further studies.
Keywords:Affinity Chromatography, Circular Dichroism, Dynamin II, Peptide Tagging, Pleckstrin Homology Domain, Proline-Rich Domain, Animals, Rats
Source:Protein Expression and Purification
Publisher:Academic Press
Page Range:314-323
Date:1 January 2000
Official Publication:https://doi.org/10.1006/prep.2000.1305
PubMed:View item in PubMed

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