Individual Expression of Recombinant - and -Tubulin from Haemonchus contortus: Polymerization and Drug Effects 1 Megan E. Oxberry,* Timothy G. Geary,† Christal A. Winterrowd,† and Roger K. Prichard‡ *Molecular Immunology Laboratory, School of Biomedical Sciences, Curtin University, Perth, Western Australia, 6000, Australia; †Animal Health Discovery Research, Pharmacia and Upjohn, Kalamazoo, Michigan 49007; and ‡Institute of Parasitology, Macdonald Campus, McGill University, 21111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9 Received May 15, 2000, and in revised form September 5, 2000 Three tubulin isotypes from the parasitic nematode Haemonchus contortus were individually expressed in Escherichia coli, purified, and induced to polymerize into microtubules in the absence of microtubule-asso- ciated proteins. The effect of different conditions on the rate of polymerization of pure tubulin was as- sessed. This is the first time that recombinant -tubu- lin has been shown to be capable of polymerization into microtubule-like structures when incubated with recombinant -tubulin. In addition, the present study has shown that: (1) microtubule-associated proteins are not required for tubulin polymerization; and (2) pure -tubulin isotype, 12-16, alone was capable of forming microtubule-like structures in the absence of -tubulin. Polymerization of the recombinant inverte- brate tubulin, as measured by a spectrophotometric assay, was found to be enhanced by a concentration of tubulin > 0.25 mg/mL; temperature >20°C; 2 mM GTP; glycerol; EGTA; and Mg 2 . Polymerization was inhib- ited by GTP (> 2 mM) and albendazole. Calcium ions and a pH range of 6 to 8.5 had no measurable effect on polymerization. Individual isotypes of tubulin poly- merized to approximately the same extent as an -/- tubulin mixture. Samples of tubulin assembled under the above conditions for 60 min were also examined under a transmission electron microscope. Although the spectrophotometric assay indicated polymeriza- tion, it did not predict the structure of the polymer. In many cases tubulin sheets, folded sheets, and rings were observed in addition to, or instead of, microtu- bule-like structures. © 2001 Academic Press Microtubules are composed of soluble tubulin sub- units comprising closely related, but distinct, - and -tubulins (each of which has many isoforms) which can vary between species and even among tissues of a single organism (15). If the tubulin subunits reach a threshold concentration, they can assemble into micro- tubules under certain conditions. Microtubule assem- bly is believed to begin with the formation of a sheet of - and -tubulin subunits arranged head-to-tail, which eventually closes to form a tube. Subunits then con- tinue to bind to each end of a growing microtubule at different rates (5). During the elongation phase, the growing ends of a microtubule are believed to be sta- bilized from depolymerization by a cap of GTP mole- cules bound to the tubulin subunits. Removal of this cap is thought to expose GDP–tubulin and lead to rapid depolymerization. As polymerization proceeds, GTP bound to -tubulin is hydrolyzed to GDP and remains in the microtubule lattice, while GTP bound to -tubu- lin is stable. After a period of elongation, an event causes removal of the GTP cap and exposure of GDP bound to underlying -tubulin which causes the micro- tubule ends to independently cease growth. The micro- tubule shortens at a rapid rate until -tubulin subunits that have dissociated from the microtubule exchange GDP for GTP and resume polymerization (2, 5, 13). Certain conditions, in addition to a critical concen- tration of tubulin subunits (21), are believed to be required for, or to strongly promote, microtubule as- sembly from tubulin partially purified from eukaryotic tissues or cells. These include warm temperatures; a pH of 6.4 – 6.7; hydrolysis of GTP; millimolar levels of Mg 2+ ; glycerol; chelating agents such as EGTA or EDTA; microtubule-stabilizing buffers; and microtu- 1 The work contained in this article was performed at the Institute of Parasitology (address as above). Protein Expression and Purification 21, 30 –39 (2001) doi:10.1006/prep.2000.1347, available online at http://www.idealibrary.com on 30 1046-5928/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.