Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.
Hu, Y., Rolfs, A., Bhullar, B., Murthy, T.V., Zhu, C., Berger, M.F., Camargo, A.A., Kelley, F., McCarron, S., Jepson, D., Richardson, A., Raphael, J., Moreira, D., Taycher, E., Zuo, D., Mohr, S., Kane, M.F., Williamson, J., Simpson, A., Bulyk, M.L., Harlow, E., Marsischky, G., Kolodner, R.D. and LaBaer, J.
Notes: The authors generated a collection of >5,000 clones containing predicted protein-coding sequences from Saccharomyces cerevisiae. Each clone was sequenced to verify the open reading frame (ORF) sequence. In addition, the ORFs from 257 clones that encode known or predicted transcription factors were transferred to a bacterial expression vector and expressed as GST-fusion proteins. GST-fusion proteins were purified using MagneGST™ Glutathione Particles in a 96-well format with a Biomek® FX automation workstation for all steps (cell lysis to elution of bound protein). Purified proteins were then applied to a protein-binding microarray to identify DNA sequences that were bound by each protein. (3783)
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