α- and β-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening

摘要: Internal fragments of α- and β-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), the termini isolated 5′- 3′-rapid amplification cDNA ends. Phytophthora capsici specific primers then used to generate full-length by RT-PCR. The recombinant expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, average yields 3.38–4.5 mg α-tubulin 2.89–4.0 mg β-tubulin, each from 1-l culture. Optimum conditions obtained for formation microtubule-like structures. A value 0.12 mg/ml was as critical concentration polymerization P. tubulin. Benomyl inhibited with half-maximal inhibition (IC50) = 468 ± 20 μM. Approximately 18.66 ± 0.13 cysteine residues per tubulin dimer accessible 5,5′-dithiobis-(2-nitrobenzoic acid), a quantification reagent sulfhydryl 12.43 ± 0.12 presence 200 μM benomyl. order preference accessibility cysteines benomyl > colchicine GTP taxol, changes conformed that binding sites these ligands folding correctly. Fluorescence resonance energy transfer technique high throughput screening chemical library search antimitotic agent. There significant difference relative fluorescence 210-O-2 210-O-14 compared colchicine.