S-Space College of Veterinary Medicine (수의과대학) Dept. of Veterinary Medicine (수의학과) Journal Papers (저널논문_수의학과)
Cloning, Purification, and Polymerization of Capsicum annuum Recombinant α and β Tubulin
- Jang, Myung-Hyun; Kim, Jungmok; Kalme, Satish; Han, Jin-Wook; Yoo, Han Sang; Kim, Jinheung; Koo, Bon-sung; Kim, Sung-Kun; Yoon, Moon-Young
- Issue Date
- Biosci. Biotechnol. Biochem., 72, 1048-1055 (2008)
- α and β tubulin genes were cloned from the Capsicum annuum leaves using rapid amplification of cDNA ends (RACE)-PCR. Nucleotide sequence analysis revealed that 1,353 bp Capsicum annuum α⁄β-tubulin (CAnm α⁄β-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant α⁄β tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and its content was as high as 50% of the total protein content. Effective fusion protein purification and refolding are described. The average yields of α and β tubulin were 2.0 and 1.3 mg/l of culture respectively. The apparent molecular weight of each tubulin was estimated to be 55 kDa by SDS-polyacrylamide gel electrophoresis (PAGE). The tubulin monomers were found to be assembly competent using a standard dimerization assay, and also retained antigenicity with anti-His/T7 antibodies. The purified tubulins were polymerized to microtubule-like structures in the presence of 2 mM guanosine 5′-triphosphate (GTP).
- 0916-8451 (print)
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