Native tubulin-folding cofactor E purified from baculovirus-infected Sf9 cells dissociates tubulin dimers.
Tubulin-folding cofactor E (TBCE) is an alpha-tubulin-binding protein involved in the formation of the tubulin dimer and in microtubule dynamics, through the regulation of tubulin heterodimer dissociation. TBCE has also been implicated in two important related human disorders, the Kenny-Caffey and Sanjad-Sakati syndromes. The expression of TBCE as a recombinant protein in bacteria results in the formation of insoluble inclusion bodies in the absence of denaturing agents. Although the active protein can be obtained from mammalian tissues, biochemical studies of TBCE present a special challenge. To express and purify native TBCE, a recombinant baculovirus expression system was used. Native wild-type TBCE purified from Sf9 extracts was sequentially purified chromatographically through cation exchange, hydrophobic interaction, and high-resolution gel-filtration columns. Mass spectrometric analysis identified 30% of the sequence of human TBCE. A stoichiometric excess of purified TBCE dissociated tubulin heterodimers. This reaction produced a highly unstable TBCE-alpha-tubulin complex, which formed aggregates. To distinguish between the aggregation of tubulin dimers induced by TBCE and tubulin dissociation, TBCE and tubulin were incubated with tubulin-folding cofactor A (TBCA). This cofactor captures the beta-tubulin released from the heterodimer with a stoichiometry of 1:1, as previously demonstrated. The beta-tubulin polypeptide was recovered as TBCA-beta-tubulin complexes, as demonstrated by non-denaturing gel electrophoresis and specific antibodies directed against beta-tubulin and TBCA.
Kortazar
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Departamentos de Biología Molecular--Unidad Asociada al Centro de Investigaciones (CSIC) and Anatomía y Biología Celular, Universidad de Cantabria, Cardenal Herrera Oria s/n. 39011, Santander, Spain.
Carranza
G
G
Bellido
J
J
Villegas
J C
JC
Fanarraga
M L
ML
Zabala
J C
JC
D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D002853:Chromatography, Liquid; D019281:Dimerization; D030342:Genetic Diseases, Inborn / Q000235:genetics; D006801:Humans; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D018411:Spodoptera / Q000166:cytology; D013577:Syndrome; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D018832:Molecular Chaperones; D011994:Recombinant Proteins; C469270:TBCE protein, human; D014404:Tubulin