a- and b-tubulin from Phytophthora capsici KACC40483: molecular cloning, biochemical characterization, and antimicrotubule screening
Kim. S. J Yoon. S. -H Park. H. -Y Kalme. S Park. H Yeo. Y. -S Han. J. W Koo. B. -S Lee. C. -M Yoon. M. -Y
Applied Microbiology and Biotechnology ; 2009 [Vol.82] Pages:513-524
Abstract
Internal fragments of a- and b-tubulin genes were generated using reverse transcription polymerase chain reaction (RT-PCR), and the termini were isolated using 5- and 3-rapid amplification of cDNA ends. Phytophthora capsici a- and b-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant a- and b-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38–4.5 mg of a-tubulin and 2.89–4.0 mg of b-tubulin, each from 1-l culture. Optimum conditions were obtained for formation of microtubule-like structures. A value of 0.12 mg/ml was obtained as the critical concentration of polymerization of P. capsici tubulin. Benomyl inhibited polymerization with half-maximal inhibition (IC50) =468±20 uM. Approximately 18.66±0.13 cysteine residues per tubulin dimer were accessible to 5,5'-dithiobis-(2-nitrobenzoic acid), a quantification reagent of sulfhydryl and 12.43±0.12 residues were accessible in the presence of 200 uM benomyl. The order of preference for accessibility to cysteines was benomyl > colchicine > GTP > taxol, and cysteine accessibility changes conformed that binding sites of these ligands in tubulin were folding correctly. Fluorescence resonance energy transfer technique was used for high throughput screening of chemical library in search of antimitotic agent. There was significant difference in relative fluorescence by 210-O-2 and 210-O-14 as compared to colchicine.
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