Pharmaceutical Sciences Faculty Publications

Pooled ORF Expression Technology (POET): Using Proteomics to Screen Pools of Open Reading Frames for Protein Expression

Document Type

Article

Publication Date

11-1-2005

Journal Title

Molecular & Cellular Proteomics

ISSN

1535-9476

Volume

4

Issue

11

First Page

1647

Last Page

1652

DOI

10.1074/mcp.M500128-MCP200

PubMed ID

16113400

Abstract

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.

Keywords

Caenorhabditis elegans, proteins, cloning, molecular, electrophoresis, gel, escherichia coli, gene expression profiling, proteome, proteomics, recombinant fusion proteins

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