Protocol

Microtubule Dynamics

Volume 777 of the series Methods in Molecular Biology pp 29-55

Date:

Purification of Tubulin from the Fission Yeast Schizosaccharomyces pombe

  • Douglas R. DrummondAffiliated withCentre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick Email author 
  • , Susan KainAffiliated withLister Hospital
  • , Anthony NewcombeAffiliated withPfizer Biotechnology Ireland
  • , Christina HoeyAffiliated withInstitute of Structural Molecular Biology, Birkbeck College
  • , Miho KatsukiAffiliated withCentre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick
  • , Robert A. CrossAffiliated withCentre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick

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Abstract

The fission yeast Schizosaccharomyces pombe is an attractive source of tubulin for biochemical experiments as it contains few tubulin isoforms and is amenable to genetic manipulation. We describe the preparation of milligram quantities of highly purified native tubulin from S. pombe suitable for use in microtubule dynamics assays as well as structural and other biochemical studies. S. pombe cells are grown in bulk in a fermenter and then lysed using a bead mill. The soluble protein fraction is bound to anion-exchange chromatography resin by batch binding, packed in a ­chromatography column and eluted by a salt gradient. The tubulin-containing fraction is ammonium sulphate precipitated to further concentrate and purify the protein. A round of high-resolution anion-exchange chromatography is carried out before a cycle of polymerisation and depolymerisation to select functional tubulin. Gel filtration is used to remove residual contaminants before a final desalting step. The purified tubulin is concentrated, and then frozen and stored in liquid nitrogen.

Key words

S. pombe Yeast Tubulin Purification Microtubule dynamics