Coexpression,copurification of Sacchromyces cerevisiae monopolin complex and analysis of Csm1/Lrs4 complex stoichiometry
Date Issued
2004
Date
2004
Author(s)
Hsu, Kuo-Sheng
DOI
zh-TW
Abstract
In budding yeast, Saccharomyces cerevisiae, Mam1, Csm1 and Lrs4 form monopolin complex around sister centromeres during meiosis I for sister chromatids cosegregation. Sister kinetochores can be clamped togethor by monopolin complex to attach the same oriented poles during metaphase and the sister chromatids will be pulled to the same cell after the first cell division of meiosis. For studying the protein complex, we used the high-throughput screening of soluble recombinant proteins method to obtain the clones which could overexpress the target fusion protein. By transforming two hetero-locus plasmids to E. coli., BL21-CodonPlus(DE3), Csm1/Lrs4 or Mam1/Csm1/Lrs4 complex was coexpressed in the double antibiotic selected strains. Using Ni-NTA or amylase affinity column, we have purified His6-Csm1, His6-Csm1/Lrs4 and MBP-Mam1/His6-Csm1/Lrs4 protein complex indepedently. Futhermore, we also determined the stoichiometry of Csm1/Lrs4 complex by combining the gel filtration and the sucrose gradient velocity sedimentation assay. In conclusion, we confirmed Mam1, Csm1 and Lrs4 can form monopolin complex in vitro and Csm1 can form homotrimer alone and heterohexamer with Lrs4 via the interaction of their coiled coil domains.
Subjects
酵母菌monopolin蛋白複合體
Csm1/Lrs4 蛋白複合體
monopolin complex
Csm1/Lrs4 complex
Type
other
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