title: Cloning, purification, and polymerization of Capsicum annuum recombinant alpha and beta tubulin.
abstract: 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).
authors.affiliation: Department of Chemistry, Hanyang University, Seoul 133-791, Korea.
authors.forename: Myung-Hyun
authors.initials: MH
authors.lastname: Jang
authors.forename: Jungmok
authors.initials: J
authors.lastname: Kim
authors.forename: Satish
authors.initials: S
authors.lastname: Kalme
authors.forename: Jin-Wook
authors.initials: JW
authors.lastname: Han
authors.forename: Han-Sang
authors.initials: HS
authors.lastname: Yoo
authors.forename: Jinheung
authors.initials: J
authors.lastname: Kim
authors.forename: Bon-sung
authors.initials: BS
authors.lastname: Koo
authors.forename: Sung-Kun
authors.initials: SK
authors.lastname: Kim
authors.forename: Moon-Young
authors.initials: MY
authors.lastname: Yoon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002212:Capsicum / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D019281:Dimerization; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D014404:Tubulin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D001704:Biopolymers; D011994:Recombinant Proteins; D014404:Tubulin