The world's first wiki where authorship really matters (Nature Genetics, 2008). Due credit and reputation for authors. Imagine a global collaborative knowledge base for original thoughts. Search thousands of articles and collaborate with scientists around the globe.

wikigene or wiki gene protein drug chemical gene disease author authorship tracking collaborative publishing evolutionary knowledge reputation system wiki2.0 global collaboration genes proteins drugs chemicals diseases compound
Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

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 and polymerization properties. Beta-tubulin was expressed without translational fusion to an E. coli sequence under the regulation of the tryptophan promoter in the pTrp2 vector. Beta-tubulin was produced in large amounts in insoluble 'inclusion bodies'. The inclusion bodies were purified and solubilised and the beta-tubulin renatured by treatment with urea followed by dilution with alkaline buffer and a shift to physiological pH. The yield was more than 10 mg of beta-tubulin per litre of cell culture. The recombinant tubulin produced was recognized in Western blot by specific anti-beta-tubulin antibodies. Tritiated MBZ binding to the recombinant H. contortus beta-tubulin was measured in the presence or absence of whole, tubulin-free or tubulin-rich extracts of H. contortus. Some [3H]MBZ high-affinity binding (HB) to 'pure' (no other 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 enhancement than beta 8-9. Mixing recombinant beta 8-9 or beta 12-16 with whole supernatants or tubulin-enriched fractions from H. contortus promoter polymerization at 37 degrees C. Use of 35S-labelled protein showed that the polymer contained recombinant tubulin. Western blot using specific anti-alpha-tubulin monoclonal antibodies showed that the polymer contained alpha-tubulin. Similarly the recombinant nematode beta-tubulin co-polymerized with tubulin from chicken brain. Our data suggest that the recombinant beta-tubulin can interact and copolymerize with parasite or chicken tubulin. Furthermore the interaction of recombinant nematode beta-tubulin with native tubulin and/or microtubule associated proteins (MAPs) resulted in the formation of high-affinity MBZ-binding sites. However, interaction of recombinant beta-tubulin with microtubule proteins from chicken brain did not result in the formation of high-affinity MBZ-binding sites.[1]


WikiGenes - Universities