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Research

Pooled ORF Expression Technology (POET)

Using Proteomics to Screen Pools of Open Reading Frames for Protein Expression^*,S

William K. Gillette, Dominic Esposito, Peter H. Frank, Ming Zhou, Li-Rong Yu, Catherine Jozwik^¶, Xiuying Zhang^¶, Brighid McGowan^¶, David M. Jacobowitz^¶,||, Harvey B. Pollard^¶, Tong Hao^**, David E. Hill^**, Marc Vidal^**, Thomas P. Conrads, Timothy D. Veenstra and James L. Hartley^,

From the Protein Expression Laboratory and Laboratory of Proteomics and Analytical Technologies, Research Technology Program, SAIC-Frederick, Inc./NCI, National Institutes of Health, Frederick, Maryland 21702, ^¶ Department of Anatomy, Physiology, and Genetics, Uniformed Services University, Bethesda, Maryland 20814, ^** Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, and ^|| Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20892

We have developed a pooled ORF expression technology, POET, that uses recombinational cloning and proteomic methods (two-dimensional gel electrophoresis and mass spectrometry) to identify ORFs that when expressed are likely to yield high levels of soluble, purified protein. Because the method works on pools of ORFs, the procedures needed to subclone, express, purify, and assay protein expression for hundreds of clones are greatly simplified. Small scale expression and purification of 12 positive clones identified by POET from a pool of 688 Caenorhabditis elegans ORFs expressed in Escherichia coli yielded on average 6 times as much protein as 12 negative clones. Larger scale expression and purification of six of the positive clones yielded 47–374 mg of purified protein/liter. Using POET, pools of ORFs can be constructed, and the pools of the resulting proteins can be analyzed and manipulated to rapidly acquire information about the attributes of hundreds of proteins simultaneously.

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