Individual expression of recombinant alpha- and beta-tubulin from Haemonchus contortus: polymerization and drug effects.
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-associated proteins. The effect of different conditions on the rate of polymerization of pure tubulin was assessed. This is the first time that recombinant alpha-tubulin has been shown to be capable of polymerization into microtubule-like structures when incubated with recombinant beta-tubulin. In addition, the present study has shown that: (1) microtubule-associated proteins are not required for tubulin polymerization; and (2) pure beta-tubulin isotype, beta12-16, alone was capable of forming microtubule-like structures in the absence of alpha-tubulin. Polymerization of the recombinant invertebrate tubulin, as measured by a spectrophotometric assay, was found to be enhanced by a concentration of tubulin >0.25 mg/mL; temperature > or =20 degrees C; 2 mM GTP; glycerol; EGTA; and Mg(2+). Polymerization was inhibited 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 polymerized to approximately the same extent as an alpha-/beta-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 polymerization, 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, microtubule-like structures.
Oxberry
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ME
Molecular Immunology Laboratory, School of Biomedical Sciences, Perth, Western Australia, 6000, Australia.
Geary
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TG
Winterrowd
C A
CA
Prichard
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RK
D000818:Animals; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006189:Haemonchus / Q000235:genetics*; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure*; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*
D018076:DNA, Complementary; D046911:Macromolecular Substances; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014404:Tubulin