Alpha- and beta-tubulin from Phytophthora capsici KACC 40483: molecular cloning, biochemical characterization, and antimicrotubule screening.
Internal fragments of alpha- and beta-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 alpha- and beta-tubulin specific primers were then used to generate full-length cDNA by RT-PCR. The recombinant alpha- and beta-tubulin genes were expressed in Escherichia coli BL21 (DE3), purified under denaturing conditions, and average yields were 3.38-4.5 mg of alpha-tubulin and 2.89-4.0 mg of beta-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 (IC(50)) = 468 +/- 20 microM. 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 microM 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.
Koo
Bon-Sung
BS
Microbial Genetics Division, National Institute of Agricultural Biotechnology, RDA, Suwon, 441-707, Republic of Korea.
Park
Haechul
H
Kalme
Satish
S
Park
Hye-Yeon
HY
Han
Jin Wook
JW
Yeo
Yun-Soo
YS
Yoon
Sang-Hong
SH
Kim
Soo-Jin
SJ
Lee
Chang-Muk
CM
Yoon
Moon-Young
MY
D020418:Algal Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects*; D008969:Molecular Sequence Data; D010838:Phytophthora / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D016415:Sequence Alignment; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology*
D020418:Algal Proteins; D014404:Tubulin; D050257:Tubulin Modulators