Cloning, purification, and polymerization of Capsicum annuum recombinant alpha and beta tubulin.
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).
Jang
Myung-Hyun
MH
Department of Chemistry, Hanyang University, Seoul 133-791, Korea.
Kim
Jungmok
J
Kalme
Satish
S
Han
Jin-Wook
JW
Yoo
Han-Sang
HS
Kim
Jinheung
J
Koo
Bon-sung
BS
Kim
Sung-Kun
SK
Yoon
Moon-Young
MY
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*
D001704:Biopolymers; D011994:Recombinant Proteins; D014404:Tubulin