| Autor: |
Jang MH; Department of Chemistry, Hanyang University, Seoul 133-791, Korea., Kim J, Kalme S, Han JW, Yoo HS, Kim J, Koo BS, Kim SK, Yoon MY |
| Jazyk: |
angličtina |
| Zdroj: |
Bioscience, biotechnology, and biochemistry [Biosci Biotechnol Biochem] 2008 Apr; Vol. 72 (4), pp. 1048-55. Date of Electronic Publication: 2008 Apr 07. |
| DOI: |
10.1271/bbb.70794 |
| Abstrakt: |
Alpha and beta 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 alpha/beta-tubulin (CAnm alpha/beta-TUB) encodes a protein of 450 amino acids (aa) each. The recombinant alpha/beta tubulin was overexpressed mainly as an inclusion body in Escherichia coli BL21 (DE3), upon induction with 0.2 mM isopropyl-beta-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 alpha and beta 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). |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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