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Cloning, Purification, and Polymerization of Capsicum annuum Recombinant α and β Tubulin

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Authors
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
2008-04-07
Publisher
Japan Society for Bioscience, Biotechnology, and Agrochemistry
Citation
Biosci. Biotechnol. Biochem., 72, 1048-1055 (2008)
Keywords
Capsicum annuumdimerizationmicrotubulepepper
Abstract
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).
ISSN
0916-8451 (print)
1347-6947 (online)
Language
English
URI
http://hdl.handle.net/10371/7872
DOI
https://doi.org/10.1271/bbb.70794
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College of Veterinary Medicine (수의과대학)Dept. of Veterinary Medicine (수의학과)Journal Papers (저널논문_수의학과)
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