Kortazar D, Carranza G, Bellido J, Villegas J C, Fanarraga M L, Zabala J C
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.
Protein Expr Purif. 2006 Oct;49(2):196-202. doi: 10.1016/j.pep.2006.03.005. Epub 2006 Mar 30.
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.
微管蛋白折叠辅助因子E（TBCE）是一种α-微管蛋白结合蛋白，通过调节微管蛋白异二聚体的解离，参与微管蛋白二聚体的形成和微管动力学过程。TBCE还与两种重要的相关人类疾病——肯尼-卡菲综合征和桑贾德-萨卡蒂综合征有关。在没有变性剂的情况下，TBCE作为重组蛋白在细菌中表达会形成不溶性包涵体。尽管活性蛋白可以从哺乳动物组织中获得，但对TBCE的生化研究面临特殊挑战。为了表达和纯化天然TBCE，使用了重组杆状病毒表达系统。从Sf9提取物中纯化的天然野生型TBCE依次通过阳离子交换、疏水相互作用和高分辨率凝胶过滤柱进行色谱纯化。质谱分析确定了人类TBCE序列的30%。纯化的TBCE化学计量过量会使微管蛋白异二聚体解离。该反应产生了一种高度不稳定的TBCE-α-微管蛋白复合物，该复合物形成聚集体。为了区分由TBCE诱导的微管蛋白二聚体聚集和微管蛋白解离，将TBCE和微管蛋白与微管蛋白折叠辅助因子A（TBCA）一起孵育。如先前所示，该辅助因子以1:1的化学计量捕获从异二聚体中释放的β-微管蛋白。通过非变性凝胶电泳以及针对β-微管蛋白和TBCA的特异性抗体证明，β-微管蛋白多肽以TBCA-β-微管蛋白复合物的形式回收。
