One-step purification of assembly-competent tubulin from diverse eukaryotic sources.
We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research.
PubMed ID: 22993214
PMC ID: PMC3496613
Article link: Mol Biol Cell.
Genes referenced: alb ckap5 dnajb1 herpud1 hsp70 hsp90aa1.1 hspa1l
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|FIGURE 1:. The purification matrix. (A) The domain structure of S. cerevisiae Stu2 (YLR045C) protein. TOG1 spans residues 8–280, and TOG2 spans residues 326–550. (B) GST-TOG fusion constructs used in this study. (C) Expression and purification of the GST-TOG1/2 fusion protein after overexpression in E. coli. Lanes: Total cell extract before induction (UI) and after induction (I) and the glutathione elute (E).|
|FIGURE 2:. Purification of S. frugiperda (SF+) tubulin. (A) Samples throughout the purification were analyzed by SDS–PAGE and stained with Coomassie blue. The lanes are as follows: crude extract (Crude), high-speed extract (Cleared), TOG column flow through (Flow), eluate, desalted eluate, concentrated eluate (conc.). (B) The crude extract, cleared extract, and TOG column flowthrough were transferred to nitrocellulose and probed with DM1 α-tubulin antibody. Tubulin in the extract was >95% depleted by a single pass over the TOG column. (C) Polymerization and sedimentation of SF+ tubulin. Tubulin was polymerized in the presence of GTP and glycerol and the monomer and polymer pools separated by sedimentation over glycerol cushions. Left lanes, total assembly reaction (Total), supernatant (Sup), and pellet. Right lanes, supernatant (Sup), pellet, and combined supernatant and pellet. (D) SF+ tubulin was polymerized in GTP and glycerol, stabilized with Taxol, deposited on formvar-coated grids, and negatively stained. Images are at 39,000× and 6600× magnification.|
|FIGURE 3:. Purification of tubulins from various species and tissue types. Samples collected throughout each purification were analyzed by SDS–PAGE and stained with Coomassie blue. (A) H. sapiens HEK293 cells. (B) S. frugiperda SF+ cells. (C) C. elegans whole N2 worms. (D) S. cerevisiae cells. (E) X. laevis eggs. (F) C. reinhardtii flagella.|
|FIGURE 4:. Efficiency of tubulin depletion and elution. (A) Tubulin was detected in samples taken from the crude extract, cleared crude extract, and the TOG column flowthrough by Western blotting using an α-tubulin antibody: depletion of C. elegans extracts (98%), depletion of X. laevis extracts (94%). (B) A 100-μl amount of TOG1/2 resin was incubated either with bovine serum albumin or buffer, followed by 1 mg of porcine tubulin. The resin was then washed with buffers used in a standard SF+ tubulin purification and then boiled in SDS–PAGE sample buffer. These two samples along with known amounts of porcine tubulin were run on SDS–PAGE and analyzed on Western blots using an α-tubulin antibody (DM1α).|
|FIGURE 5:. Polymerization competence of tubulins. Electron micrographs of microtubules polymerized from C. elegans (A) and X. laevis (B) tubulin. Tubulins were polymerized in GTP and glycerol, stabilized with Taxol, and then deposited on formvar-coated grids and negatively stained. (C) Human tubulin was polymerized in the presence of GTP and glycerol and the monomer and polymer pools separated by sedimentation over glycerol cushions. Left four lanes, serial dilutions of the total assembly reaction; middle lane, supernatant (Sup); right lane, pellet.|
|FIGURE 6:. Identification and removal of proteins copurifying with tubulin. (A) A Coomassie blue–stained gel with tubulins purified from HEK293, SF+, frog egg extracts, and whole worms. Mass spectrometry was used to identify the labeled bands: (1) Hsp90 (83 kDa), (2) Hsp70 (71 kDa), (3) Sti1 (62 kDa), (4) Aldolase (39 kDa), (5) Hsp40 (38 kDa). (a) Hsp90 (80 kDa), (b) Hsp70 (70 kDa), (c) Sti1-like (45 kDa), (d) Sti1 (37 kDa), (e) Hsp40 (36 kDa), (f) ZK856.7 (13 kDa). (B, C) Purification of C. elegans tubulin without (B) and with (C) an ATP wash step. Samples taken throughout the purification were run on SDS–PAGE and stained with Coomassie blue.|