EXPRESSION OF CLONED BETA-TUBULIN GENES OF HAEMONCHUS-CONTORTUS IN ESCHERICHIA-COLI - INTERACTION OF RECOMBINANT BETA-TUBULIN WITH NATIVE TUBULIN AND MEBENDAZOLE

Two distinct beta-tubulin cDNA isotypes (beta 8-9 and beta 12-16) from Haemonchus contortus were expressed for the first time in Escherichia coli and characterised by their specific mebendazole (MBZ) binding an d polymerization properties. Beta-tubulin was expressed without transl ational fusion to an E. coli sequence under the regulation of the tryp tophan promoter in the pTrp2 vector. Beta-tubulin was produced in larg e amounts in insoluble 'inclusion bodies'. The inclusion bodies were p urified and solubilised and the beta-tubulin renatured by treatment wi th urea followed by dilution with alkaline buffer and a shift to physi ological pH. The yield was more than 10 mg of beta-tubulin per litre o f cell culture. The recombinant tubulin produced was recognized in Wes tern blot by specific anti-beta-tubulin antibodies. Tritiated MBZ bind ing to the recombinant H. contortus beta-tubulin was measured in the p resence or absence of whole, tubulin-free or tubulin-rich extracts of H. contortus. Some [H-3]MBZ high-affinity binding (HB) to 'pure' (no o ther eukaryotic protein present) beta 8-9 or beta 12-16 was observed. Enhanced high-affinity binding was observed when recombinant beta 8-9 or beta 12-16 were mixed and pre-incubated with whole supernatants or tubulin-enriched extracts from H. contortus. The enhancement was more than additive. beta 12-16 bound more MBZ and caused a greater enhancem ent than beta 8-9 Mixing recombinant beta 8-9 or beta 12-16 with whole supernatants or tubulin-enriched fractions From H. contortus promoted polymerization at 37 degrees C. Use of S-35-labelled protein showed t hat the polymer contained recombinant tubulin. Western blot using spec ific anti-alpha-tubulin monoclonal antibodies showed that the polymer contained alpha-tubulin. Similarly the recombinant nematode beta-tubul in co-polymerized with tubulin from chicken brain. Our data suggest th at the recombinant beta-tubulin can interact and copolymerize with par asite or chicken tubulin. Furthermore the interaction of recombinant n ematode beta-tubulin with native tubulin and/or microtubule associated proteins (MAPs) resulted in the formation of high-affinity MBZ-bindin g sites. However, interaction of recombinant beta-tubulin with microtu bule proteins from chicken brain did not result in the formation of hi gh-affinity MBZ-binding sites.