Expression and purification of TOG domains
Gene fragments encoding the first (residues 1–306) and both (residues 1–590) of the tandem N-terminal TOG domains found in S. cerevisiae
Stu2 were subcloned into pGEX-6P-1 for expression of GST fusions in E. coli
(pGEX-6P-1 Stu2 1-306; pGEX-6P-1 Stu2 1-590). Fresh transformants of the expression plasmids in BL21(DE3) T1 pRARE were used to inoculate an overnight culture in MDAG-135 medium (Studier, 2005
) containing antibiotics (100 μg/ml ampicillin and 15 μg/ml chloramphenicol). This culture was diluted 500-fold into 1 l of Terrific Broth (1.2% tryptone, DIFCO 211705; 2.4% yeast extract, DIFCO 2212750; 0.4% glycerol; 17 mM KH2
; 72 mM K2
) with the same antibiotics and shaken at 37°C until the OD600
reached 0.5. After shifting the cultures to 18°C for 1 h, expression was induced by adding 0.2 mM isopropyl β-d
-1-thiogalactopyranoside and shaking for 18 h. Induced cultures were pelleted (5000 rpm, 10 min at 4°C, JLA 8.1000; Beckman Coulter, Brea, CA) and then resuspended in an equal volume of 2× phosphate-buffered saline (PBS) made from a 10× stock (10× PBS: 27 mM KCl, 15 mM KH2
, 81 mM Na2
, 1.37 M NaCl) with 1 mM dithiothreitol (DTT). After addition of benzonase (Novagen, Gibbstown, NJ) and protease inhibitors, the cells were lysed using two passes through an ice-cold Emulsiflex C5 microfluidizer (Avestin, Ottawa, Canada) at 1500 bar. The lysate was clarified by two sequential centrifugation steps (12,000 rpm, 30 min at 4°C; Beckman JA-12) and then applied to a GSTrap (5 ml = 1 column volume [CV]; GE Healthcare, Buckinghamshire, United Kingdom) preequilibrated in 2× PBS with 1 mM DTT (wash buffer) at 0.5 CV/min. The column was then washed with 10 CV of wash buffer with 0.1% Tween 20 and then 2 CV of 5 mM ATP with 10 mM MgCl2
in 2× PBS. The column was incubated for 20 min and then washed with 5 CV of 6× PBS, followed by 5 CV of wash buffer. The GST fusion was eluted using 5 mM reduced glutathione in wash buffer at pH 8.0. The pool of eluted protein was desalted or dialyzed against three changes of 100 mM NaHCO3
with 100 mM NaCl at pH 8.2 (coupling buffer). The concentration of the GST fusion was determined by measuring the absorbance at 280 nm using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE) and the calculated extinction coefficient. One liter of bacterial culture yielded approximately 70 mg of GST-TOG1/2 fusion.
Preparation of the tubulin affinity resin
Tubulin affinity columns were prepared by coupling GST-TOG1/2 fusions to HiTrap NHS-activated HP columns (1 and 5 ml; GE Healthcare) following the manufacturer's instructions with modifications. Before coupling, 80 mM MgCl2 was added to the dialyzed GST-fusion preparation in coupling buffer. Immediately after washing the HiTrap NHS-activated HP columns with 3 CV of ice-cold 1 mM HCl at 1.5 CV/min, coupling was performed by continuously recirculating the GST-TOG1/2 fusion (1.5 CV/min, 4 CV of a 4 mg/ml preparation) for 30 min at room temperature. Unreacted NHS groups were blocked by washing with 6 CV of 0.5 M ethanolamine and 0.5 M NaCl at pH 8.3 and then incubated for 30 min at room temperature. The column was then washed with 6× PBS, followed by 1× PBS with 50% glycerol (storage buffer) and stored at −20°C. The coupling efficiency was >75% as assessed by comparing protein levels on SDS–PAGE before and after incubation with the resin, meaning that at least 12 mg of GST-TOG12 fusion was coupled to a 1-ml column. The columns retained the capacity to bind tubulin for at least 4 mo when stored at −20°C and could be used repetitively if washed between runs with 10 CV of 10× PBS, followed by 5 CV of 1× PBS and then 5 CV of storage buffer.
Purification using the TOG affinity column
The protocol for SF+ extracts was developed most extensively. The only notable differences between this primary example and the purifications from other later sources, described later, are in the wash and elution steps. Although GTP, Tween 20, and glycerol were not uniformly included in subsequent purifications, these additives may improve the purity and assembly properties of the tubulins purified with the following methods. The inclusion of Tween 20 and glycerol during wash steps improved the quality of our eluted SF+ tubulin preparation, as judged from electron micrographs of microtubules. Similarly, the substitution of ammonium sulfate for potassium chloride in the elution buffer, based on an earlier report in which this salt was shown to reduce loss of bound nucleotide, may improve the assembly properties of the final preparations (Croom et al., 1986
). In all the preparations, one should take care to limit the length of exposure of tubulin to high salt by rapidly desalting into storage buffer (e.g., BRB80).
Purification of S. frugiperda tubulin
SF+ cells (500 ml) were grown to 4 × 106
cells/ml in SF900II medium (Invitrogen, Carlsbad, CA) and pelleted by centrifugation at 1700 × g
for 15 min. Cells were resuspended in an equal volume of 1× BRB80 containing 3 U of benzonase, 1 mM DTT, and protease inhibitors. Cells were lysed by douncing on ice (20 strokes). The extract was cleared by centrifugation (80,000 rpm, 10 min, 4°C, Sorvall S100 AT6; Sorvall-Hitachi, Thermo Scientific, Waltham, MA) and then filtered through a 0.45-μm Milliex-HV polyvinylidene fluoride membrane (Millipore, Bedford, MA). The purification was performed at 4°C. The extract was loaded at 0.5 CV/min onto a TOG column preequilibrated with 1× BRB80 and followed by 4 CV of 1× BRB80 and 100 μM Mg2+
GTP to clear out most of the extract from the column. The flow rate was then changed to 1 CV/min for the following wash steps: 1) 10 CV of 1× BRB80, 10 μM Mg2+
GTP; 2) 3 CV of 1× BRB80, 100 μM Mg2+
GTP, 10 mM MgCl2
, and 5 mM ATP followed by a 15-min incubation; 3) 5 CV of 1× BRB80 and 10 μM Mg2+
GTP; and 4) 5 CV of 1× BRB80, 0.1% Tween 20 and 10% glycerol. The tubulin was eluted with 3 CV of 1× BRB80, 10 μM Mg2+
GTP, and 500 mM (NH4
. Ammonium sulfate was used because tubulin has been shown to better retain its nucleotide in the presence of this salt (Croom et al., 1986
). After pooling, the amount of tubulin in the peak fractions was determined by Bradford assay (Bio-Rad, Richmond, CA). Care was taken to limit exposure of the tubulin to high salt. The eluate was quickly desalted into 1× BRB80 and 10 μM Mg2+
GTP with a PD10 desalting column. GTP can be omitted or used at lower concentration for subsequent assembly with alternative nucleotides like GMPCPP. The tubulin was concentrated to at least 20 μM with an Amicon Ultra 10K MWCO centrifugal filter (Millipore), and glycerol was added to 5% before snap freezing in liquid nitrogen. The column was then washed with 10× PBS and could then either be reequilibrated or stored in 50% glycerol and 1× PBS at −20°C.
Purification of Homo sapiens HEK293 tubulin
A 500-ml culture at 2 × 106 cells/ml in FreeStyle 293 medium was harvested by centrifugation at 1700 × g for 15 min. The tubulin purification was performed as per the SF+ protocol except that the Tween/glycerol wash step was omitted.
Purification of C. elegans tubulin
Worms were cultured using a modified protocol from that found in the WormBook (Stiernagle, 2006
). A preculture of C600 bacteria was used to inoculate 1 l of Luria–Bertani medium (1% tryptone, DIFCO 211705; 0.5% yeast extract, DIFCO 2212750; 171 mM NaCl, pH 7.0) medium and incubated overnight at 37°C. The bacteria were pelleted at 5000 relative centrifugal force (rcf) in sterilized centrifuge bottles, resuspended in 10 ml S-Basal (0.02 M K2
, 0.043 M KH2
, 0.1 M NaCl, 4 mg/l cholesterol), and transferred to 50-ml Falcon tubes (Fisher Scientific, Pittsburgh, PA). The resuspended pellet was stored at −80°C.
Nine large (8 cm) OP50/NGM plates were seeded with 15 adult hermaphrodites (N2) each. The worms were allowed to grow until starved and then collected into Falcon tubes from plates using S complete medium supplemented with penicillin, streptomycin, and nystatin. The worms were washed twice with the same medium. The suspension was then used to inoculate 2× 1 l of liquid culture consisting of S complete medium supplemented with nystatin. The worms were grown at 22°C with vigorous shaking to ensure sufficient oxygen supply (~200 rpm) for 2–3 d. More bacteria were added if growth slowed during this time. The worms were harvested by letting them settle 4 h at 4°C. Media was decanted, and worms were transferred to a 50-ml Falcon tube and then washed twice with cold M9 medium. As much media as possible was removed before storing the worms at −80°C. The worm pellet was resuspended in an equal volume of 1× BRB80 supplemented with protease inhibitor cocktail (Roche, Indianapolis, IN). The slurry was frozen dropwise in liquid nitrogen and ground to a powder with a mortar and pestle under liquid nitrogen and then allowed to thaw on ice. The tubulin purification was performed as per the SF+ protocol, except that the Tween/glycerol wash step was omitted.
Purification of S. cerevisiae tubulin
For purification of budding yeast tubulin, we used strain BY4741 (Brachmann et al., 1998
; Mat a, his3Δ1, leu2Δ0, met15Δ0, ura3Δ0). Nine liters of culture was grown at 30ºC with shaking at 180 rpm until OD600
of 1.5. Cells were pelleted and washed in doubly distilled H2
O, and pellets were ground in liquid nitrogen as previously described (Sorger et al., 1988
). Lysate powder was stored at −80ºC and thawed in lysis buffer before use (3 ml of lysis buffer was added to 1 g of lysate powder). Lysis buffer consisted of BRB80, 10% glycerol, and 0.2% Triton X-100 supplemented with 5 μg/ml DNase I, 1 mM DTT, 0.1 mM ATP, 1 mM GTP, 0.2 mM phenylmethylsulfonyl fluoride, 5 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml aprotinin. The lysate was stirred at 4ºC for 10 min and sonicated using a Branson digital sonifier model 450D with 1/8-inch tapered microtip (Branson, Danbury, CT) set at 50% amplitude in 5× 10-s intervals with pauses of 30 s between pulses. The lysate was then centrifuged at 235,000 × g
, 4ºC for 30 min. Fresh PMSF was added to the supernatant to 0.2 μg/ml.
The supernatant was passed through a TOG1 column preequilibrated with lysis buffer at 0.5 ml/min. The column was washed with BRB80 and eluted with BRB80 with 500 mM KCl. Peak fractions were determined by Bradford assay and pooled. Pooled eluates were desalted into BRB80 and concentrated using an Amicon Ultra-4 10K centrifugal filter device (Millipore). Glycerol was added to 10% before the tubulin was aliquoted, snap frozen in liquid nitrogen, and stored at −80ºC.
Purification of X. laevis egg tubulin
CSF-extract was prepared according to Murray (1991
). The extract was then diluted with an equal volume of BRB80 and centrifuged at 80,000 rpm in an S100AT6-0123 rotor (Sorvall-Hitachi) for 10 min at 4°C. The tubulin purification was performed as per the SF+ protocol, except that the Tween/glycerol wash step was omitted.
Purification of C. reinhardtii flagellar tubulin
The C. reinhardtii
cells (CC-2228 oda1 mt+) were grown in liquid Tris-acetate-phosphate medium (20 mM Tris HCl, 7 mM NH4
Cl, 0.40 mM MgSO4
, 0.34 mM CaCl2
, 2.5 mM Na3
, and 1000× diluted Hutner's trace elements (Gorman and Levine, 1965
), titrated to pH 7.0 with glacial acetic acid) with continuous aeration and 24 h of light at room temperature. Cultures (60 l) were grown to ~5 × 106
cells/ml. Cells were harvested and the axonemes were isolated by standard methods (Witman, 1986
). Briefly, cells were harvested by centrifugation (800 × g
for 7 min). They were deflagellated by 1.5 min of exposure to 4.2 mM dibucane-HCl. The deflagellation was quenched by 2× dilution and the addition of 0.5 mM EGTA. The flagella were separated from the cell bodies by centrifugation (1100 × g
for 7 min and 1100 rcf for 20 min on a 30% sucrose cushion). The collected flagella were concentrated by resuspending the pellet after centrifugation (28,000 rcf) in 10 ml of HMDE (30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 mM MgSO4
, 1 mM DTT, and 1 mM EGTA titrated to pH 7.4 with KOH) with 0.4 μM Pefabloc (Sigma-Aldrich, St. Louis, MO). The flagella were demembranated by the addition of 0.2% IGEPAL CA-630 (Sigma-Aldrich), and then they were washed in HMDE. The axonemes were stripped of many microtubule-associated proteins with 20 min of incubation in HMDE plus 0.6 M KCl. After centrifugation (45,000 × g
), the pellet was resuspended in HMDE plus 50 mM CaCl2
. It was sonicated for 10× 1-min-on/1-min-off cycles in an ice-cold sonicating bath to induce the axonemal microtubules to depolymerize. It was then centrifuged at 45,000 × g
for 10 min. The supernatant was diluted fivefold in HMDE to reduce the CaCl2
The tubulin purification was performed as per the SF+ protocol, except that 1) the Tween/glycerol wash step was omitted, 2) GTP was not included in the purification, and 3) the tubulin was eluted in 1× BRB80 containing 0.5 M KCl. The Chlamydomonas tubulin was diluted to 10 μM in BRB80, flash frozen, and stored at −80°C.
Determination of protein
The level of tubulin in purified preparations was determined using the NanoDrop spectrophotomer and the estimated extinction coefficient for tubulin, 1.15 (mg/ml)−1 cm−1. Total protein in extracts was determined using either the Bradford assay or the bicinchoninic acid assay if buffers contained detergent.
Determination of tubulin depletion
Samples taken during the purification were resolved on SDS–PAGE and Western blotted using a pan-specific anti–α-tubulin monoclonal (DM1α) and a horseradish peroxidase–labeled secondary with enhanced chemiluminescence for detection. The level of tubulin was quantified using ImageJ software (Schneider et al., 2012
Determination of the elution efficiency
We loaded 1 mg of porcine brain tubulin onto 100 μl of TOG1/2 resin and then washed it and eluted the bound fraction under conditions similar to those described under purification of S. frugiperda tubulin. After the final high-salt elution, the resin was then boiled in SDS–PAGE sample buffer, and the amount of tubulin in this fraction was compared with known standards.
Negative-stain electron microscopy
Microtubules were polymerized in BRB80 containing 1 mM GTP for 30 min at 37ºC. Microtubules were stabilized with 10 μM Taxol and then adsorbed for 2 min onto 200-mesh, glow-discharged, carbon-coated copper grids with formvar support (Electron Microscopy Sciences, Hatfield, PA). The samples were washed with 1× BRB80, stained in 50 μl of 2% uranyl acetate for 30 s, blotted, and air-dried. Grids were imaged on a CM100 transmission electron microscope (Philips/FEI Corporation, Eindhoven, Holland) operating at 80 kV. Micrographs were acquired using AMTV600 software operating an Advantage HS-B camera (2000 × 2000 pixels; AMT, Danvers, MA).
Protein bands visualized by Coomassie staining were excised from the gel and their protein content digested with trypsin. The resulting peptide mixtures were extracted with 5% formic acid/50% acetonitrile and analyzed by liquid chromatography–tandem mass spectrometry on an Ultimate nanoLC system (Thermo Scientific Dionex, Amsterdam, Netherlands) interfaced online to a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) via a TriVersa robotic nanoflow ion source (Advion BioSciences, Ithaca NY) as described in Junqueira et al. (2008
). Acquired spectra were searched against the National Center for Biotechnology Information protein database without species restriction using MASCOT software, version 2.2.0 (Matrix Science, London, United Kingdom), with the following settings: precursor mass tolerance was ±5 ppm; fragment mass tolerance was ±0.5 Da; variable modifications were propionamide (C), oxidation (M), and acetylation (protein N-terminus); enzyme was trypsin; two missed cleavages were allowed. Hits were evaluated using Scaffold software, version 2_04_00 (Proteome Software, Portland, OR), with the following settings: minimal number of matching peptides, two; peptide probability, 95%; protein probability, 99%; protein false discovery rate was calculated as 0.1%.
S. cerevisiae tubulin DIC imaging and assay conditions
Porcine brain tubulin was purified as described (Gell et al., 2011
) and labeled with TAMRA (Invitrogen; Hyman et al., 1991
). Rhodamine-labeled GMPCPP microtubules (25% labeled tubulin) were grown in BRB80 and 1 mM GMPCPP (Jena Bioscience, Jena, Germany), incubated for 2 h at 37°C, pelleted in an Airfuge (Beckman), and resuspended in BRB80. The flow cell assembly and preparation was performed as described (Varga et al., 2006
), with the exception that anti-rhodamine antibody was used. The objective was heated to 29°C. Yeast tubulin at 3 μM in BRB80 containing 1 mM GTP and antifade solution (40 mM glucose, 40 μg/ml glucose-oxidase, 16 μg/ml catalase, 0.1 mg/ml casein, 0.1% Tween 20, 1% DTT) was added to the chamber. Images were collected every 1.03 s for 11 min using the LED-VE-DIC setup described earlier (Bormuth et al., 2007