Purification of assembly-competent tubulin from Saccharomyces cerevisiae

Article Article scientifique Article Purification of assembly-competent tubulin from Saccharomyces cerevisiae eng 1992 author Bellocq Christine 22920 author Andrey-Tornare Isabelle author Paunier Mathey Doret Anne-Marie 25478 author Maeder Bernard 27495 author Paturle Lawrence author Job Didier author Haiech Jacques author Edelstein Stuart 22770 343-349 1446683 0014-2956 We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels. The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure. In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to Sepharose-4B. Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure. Upon addition of porcine brain microtubule-associated proteins or DEAE-dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples. Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values. Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process. Animals Blotting Western Chromatography Gel Electrophoresis Polyacrylamide Gel Hydrogen-Ion Concentration Microtubule-Associated Proteins/chemistry/ultrastructure Microtubules/chemistry/ultrastructure Saccharomyces cerevisiae/chemistry Swine Tubulin/chemistry/isolation & purification Dewey 540 28 Section de chimie et biochimie 14 false European journal of biochemistry 210 1 79a73f4f-cf65-4b7e-8381-7f95f7825283 Article (Published version) Restricted C.Bellocq.pdf 915172 application/pdf restrictedAccess Faculté des sciences / Section de chimie et biochimie

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Purification of assembly-competent tubulin from Saccharomyces cerevisiae	European journal of biochemistry			1992	437	0

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Bellocq, Christine