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).
Bibliographical noteFunding Information:
This study was carried out with the support of the On-Site Cooperative Agriculture Research Project (20070201080024) and the National Institute of Agricultural Biotechnology, RDA, of the Republic of Korea.
- Capsicum annuum
- Rapid amplification of cDNA ends (RACE)-PCR