Cloning and protein preparation of TTL samples has been described by Prota et al. (2013) (link). In brief, chicken TTL containing a C-terminal hexahistidine tag was overexpressed in Escherichia coli BL21 (DE3) cells and purified on a HisTrap affinity column (GE Healthcare). The fractions containing TTL protein were pooled, concentrated to 5 ml using a Centriprep device (Mw cutoff 30,000; Amicon) and loaded onto a Superdex 200 16/60 column for the final purification step in 20 mM Bis-Tris propane, pH 6.5, 200 mM NaCl, 2.5 mM MgCl2, 5 mM β-mercaptoethanol, and 1% glycerol. The protein-containing fractions were collected, concentrated to ∼20 mg/ml, and frozen in aliquots in liquid nitrogen for storage.
The stathmin-like domain clone of RB3 was a gift from A. Sobel (Institut du Fer-à-Moulin, Paris, France). The protein was prepared according to Ravelli et al. (2004) (link). Bovine and pork brain tubulin was prepared according to well established protocols (Andreu, 2007 (link)). The bovine brain tubulin was purchased from the Centro de Investigaciones Biológicas (Microtubule Stabilizing Agents Group), CSIC, Madrid, Spain. Before the reconstitution of the T2R complex, tubulin was subjected to one cycle of polymerization/depolymerization (Dorleans et al., 2007 (link)). The composition of isoforms and post-translationally modified versions of brain αβ-tubulin is as follows: β-tubulin consists of 58% Tub B2, 25% Tub B3, 13% Tub B4 (Banerjee et al., 1988 (link)), out of which Tub B2 is polyglutamylated at Glu435 (Rüdiger et al., 1992 (link)), Tub B3 at Glu438 (Alexander et al., 1991 (link)), and Tub B4 at Glu434 (Mary et al., 1994 (link)). The composition of the α-tubulin pool is less well known; however, the highly homologous members of the Tub A1 family, which are the main components of the brain tubulin pool, are modified at Glu445 (Eddé et al., 1990 (link)). The C terminus of α-tubulin in brain is present as a mixture of tyrosinated tubulin, detyrosinated tubulin, Δ2-tubulin, and perhaps other modification variants that have only recently been discovered (e.g., Δ3-tubulin; Berezniuk et al., 2012 (link)). In adult brain, ∼35–50% of the total tubulin pool cannot be retyrosinated (Paturle et al., 1989 (link)), indicating that this pool is in the Δ2 form or further modified. Moreover, 15–20% of the pool is tyrosinated tubulin, and 35–40% is detyrosinated tubulin (Barra et al., 1988 (link)). Because the aim of our biochemical and biophysical experiments was to qualitatively/semi-quantitatively compare different TTL variants, and not to provide absolute quantitative numbers, the heterogeneity of our tubulin samples is not expected to affect the interpretation of the data.
The stathmin-like domain clone of RB3 was a gift from A. Sobel (Institut du Fer-à-Moulin, Paris, France). The protein was prepared according to Ravelli et al. (2004) (link). Bovine and pork brain tubulin was prepared according to well established protocols (Andreu, 2007 (link)). The bovine brain tubulin was purchased from the Centro de Investigaciones Biológicas (Microtubule Stabilizing Agents Group), CSIC, Madrid, Spain. Before the reconstitution of the T2R complex, tubulin was subjected to one cycle of polymerization/depolymerization (Dorleans et al., 2007 (link)). The composition of isoforms and post-translationally modified versions of brain αβ-tubulin is as follows: β-tubulin consists of 58% Tub B2, 25% Tub B3, 13% Tub B4 (Banerjee et al., 1988 (link)), out of which Tub B2 is polyglutamylated at Glu435 (Rüdiger et al., 1992 (link)), Tub B3 at Glu438 (Alexander et al., 1991 (link)), and Tub B4 at Glu434 (Mary et al., 1994 (link)). The composition of the α-tubulin pool is less well known; however, the highly homologous members of the Tub A1 family, which are the main components of the brain tubulin pool, are modified at Glu445 (Eddé et al., 1990 (link)). The C terminus of α-tubulin in brain is present as a mixture of tyrosinated tubulin, detyrosinated tubulin, Δ2-tubulin, and perhaps other modification variants that have only recently been discovered (e.g., Δ3-tubulin; Berezniuk et al., 2012 (link)). In adult brain, ∼35–50% of the total tubulin pool cannot be retyrosinated (Paturle et al., 1989 (link)), indicating that this pool is in the Δ2 form or further modified. Moreover, 15–20% of the pool is tyrosinated tubulin, and 35–40% is detyrosinated tubulin (Barra et al., 1988 (link)). Because the aim of our biochemical and biophysical experiments was to qualitatively/semi-quantitatively compare different TTL variants, and not to provide absolute quantitative numbers, the heterogeneity of our tubulin samples is not expected to affect the interpretation of the data.
