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Cloning, purification, and polymerization of Capsicum annuum recombinant α and β tubulin

Title
Cloning, purification, and polymerization of Capsicum annuum recombinant α and β tubulin
Authors
Jang M.-H.Kim J.Kalme S.Han J.-W.Yoo H.-S.Koo B.-S.Kim S.-K.Yoon M.-Y.
Ewha Authors
김진흥
SCOPUS Author ID
김진흥scopus
Issue Date
2008
Journal Title
Bioscience, Biotechnology and Biochemistry
ISSN
0916-8451JCR Link
Citation
vol. 72, no. 4, pp. 1048 - 1055
Indexed
SCI; SCIE; SCOPUS WOS scopus
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).
DOI
10.1271/bbb.70794
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자연과학대학 > 화학·나노과학전공 > Journal papers
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