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Insect Expression System > | MATERIALS AND METHODS Cloning and purification of yeast SWR1 complexes Genes for a fourteen subunit yeast SWR1 complex compris- ing Swr1 (residues 1–1514 with an N-terminal 8-histidine and C-terminal twin Strep-tag® ), actin, Arp4, Arp6, Bdf1, Yaf9, Swc2, Swc3, Swc4, Swc5, Swc6, Swc7, Rvb1 and Rvb2 were synthesized with codon bias for expression in insect cells (Genscript). | Get A Quote |
We have prepared recombinant fourteen subunit yeast SWR1 complex from insect cells using a modified MultiBac system. The 1.07 MDa recombinant protein complex has histone-exchange activity. Full exchange activity is realized with a single SWR1 complex bound to a nucleosome. We also prepared mutant complexes that lack a variety of subunits or combinations of subunits and these start to reveal roles for some of these subunits as well as indicating interactions between them in the full complex. Complexes containing a series of N-terminally and C-terminally truncated Swr1 subunits reveal further details about interactions between subunits as well as their binding sites on the Swr1 subunit. Finally, we present elec... More