Native tubulin-folding cofactor E purified from baculovirus-infected Sf9 cells dissociates tubulin dimers

Protein Expression and Purification
D KortazarJ C Zabala

Abstract

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 incu...Continue Reading

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Citations

Jun 10, 2011·Cellular and Molecular Life Sciences : CMLS·Ewa Szolajska, Jadwiga Chroboczek
Jan 29, 2010·PloS One·Mónica López FanarragaJuan Carlos Zabala
Mar 6, 2010·Cellular and Molecular Life Sciences : CMLS·Olga VoloshinDina Raveh
Sep 4, 2012·Cellular and Molecular Life Sciences : CMLS·Gerardo CarranzaJuan Carlos Zabala
Dec 23, 2006·Experimental Cell Research·D KortazarJ C Zabala
Dec 1, 2010·Biomolecular Concepts·João GonçalvesHelena Soares
Jun 7, 2017·Zygote : the Biology of Gametes and Early Embryos·Victoria Jiménez-Moreno, Ekaitz Agirregoitia
Apr 25, 2015·Journal of Cell Science·Marina SernaJosé María Valpuesta
May 11, 2021·Frontiers in Cell and Developmental Biology·Sofia NolascoJuan Carlos Zabala

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