Pooled ORF Expression Technology (POET)

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Pooled ORF expression technology (POET): using proteomics to screen pools of open reading frames for protein expression.

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 expressio...

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Identification of highly expressed, soluble proteins using an improved, high-throughput pooled ORF expression technology.

This article describes an improved pooled open reading frame (ORF) expression technology (POET) that uses recombinational cloning and solution-based tandem mass spectrometry (MS/MS) to identify ORFs that yield high levels of soluble, purified protein when expressed in Escherichia coli. Using this method, three identical pools of 512 human ORFs were subcloned, purified, and transfected into thre...

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independent replicate experiments. Deletion of these genes affects the fitness of mutant strains in iron deficiency. The requirement for growth of each gene was quantified as a log 2 ratio of growth of the corresponding mutant strain in YPD with BPS compared to YPD alone. The log 2 fitness represents the average of the significant values in these experiments. A negative value indicates sensitiv...

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ژورنال

عنوان ژورنال: Molecular & Cellular Proteomics

سال: 2005

ISSN: 1535-9476

DOI: 10.1074/mcp.m500128-mcp200