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

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概要

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• 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).

• 社団法人 日本農芸化学会の論文

著者

• YOON Moon-Young

  Department of Chemistry, Hanyang University

• Yoo Han-sang

  Department Of Infectious Diseases College Of Veterinary Medicine Seoul National University

• JANG Myung-Hyun

  Department of Chemistry, Hanyang University

• KIM Jungmok

  Department of Chemistry, Hanyang University

• KALME Satish

  Department of Chemistry, Hanyang University

• HAN Jin-Wook

  Department of Chemistry, Hanyang University

• KIM Jinheung

  Department of Chemistry, Ewha Women’s University

• KOO Bon-sung

  Microbial Function Team, National Institute of Agricultural Biotechnology

• KIM Sung-Kun

  Department of Chemistry and Biochemistry, Baylor University

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

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