Applied Microbiology and Biotechnology

, 82:513

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

  • Bon-Sung Koo
  • Haechul Park
  • Satish Kalme
  • Hye-Yeon Park
  • Jin Wook Han
  • Yun-Soo Yeo
  • Sang-Hong Yoon
  • Soo-Jin Kim
  • Chang-Muk Lee
  • Moon-Young Yoon
Applied Genetics and Molecular Biotechnology

DOI: 10.1007/s00253-008-1821-7

Cite this article as:
Koo, BS., Park, H., Kalme, S. et al. Appl Microbiol Biotechnol (2009) 82: 513. doi:10.1007/s00253-008-1821-7

Abstract

Internal fragments of α- and β-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 α- and β-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant α- and β-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38–4.5 mg of α-tubulin and 2.89–4.0 mg of β-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 μM. 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 μM 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.

Keywords

Phytophthora capsiciRecombinant tubulinBenomylDTNBFRET

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Bon-Sung Koo
    • 1
  • Haechul Park
    • 2
  • Satish Kalme
    • 2
    • 3
  • Hye-Yeon Park
    • 2
  • Jin Wook Han
    • 2
  • Yun-Soo Yeo
    • 1
  • Sang-Hong Yoon
    • 1
  • Soo-Jin Kim
    • 1
  • Chang-Muk Lee
    • 1
  • Moon-Young Yoon
    • 2
  1. 1.Microbial Genetics DivisionNational Institute of Agricultural Biotechnology, RDASuwonRepublic of Korea
  2. 2.Department of ChemistryHanyang UniversitySeoulRepublic of Korea
  3. 3.Research Institute for Natural SciencesHanyang UniversitySeoulRepublic of Korea