Purification of assembly-competent tubulin from Saccharomyces cerevisiae.

摘要: 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 overproduce both the alpha and beta subunits of tubulin, under control galactose promoter, approximately 10-times wild-type levels. first step purification is carried out with high-speed supernatant lysed loaded onto DEAE-Sephadex column; after this preparation 30% pure. In second step, fractions an immunoaffinity column prepared by coupling anti-(alpha-tubulin) monoclonal antibody YL 1/2 Sepharose-4B. Following elution 0.8 M KCl, present in peak 90% Upon addition porcine brain microtubule-associated proteins or DEAE-dextran, functionally active for assembly microtubules, as visualized electron microscopy on negatively stained samples. Virtually identical microtubule structures produced parallel experiments differences observed only at acidic pH values. Overall, relatively simple provides useful tool production functional suitable structural studies investigations process.