title: Pooled ORF expression technology (POET): using proteomics to screen pools of open reading frames for protein expression.
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.
authors.affiliation: Protein Expression Laboratory, Research Technology Program, SAIC-Frederick, Inc./NCI, National Institutes of Health, Frederick, Maryland 21702, USA.
authors.forename: William K
authors.initials: WK
authors.lastname: Gillette
authors.forename: Dominic
authors.initials: D
authors.lastname: Esposito
authors.forename: Peter H
authors.initials: PH
authors.lastname: Frank
authors.forename: Ming
authors.initials: M
authors.lastname: Zhou
authors.forename: Li-Rong
authors.initials: LR
authors.lastname: Yu
authors.forename: Catherine
authors.initials: C
authors.lastname: Jozwik
authors.forename: Xiuying
authors.initials: X
authors.lastname: Zhang
authors.forename: Brighid
authors.initials: B
authors.lastname: McGowan
authors.forename: David M
authors.initials: DM
authors.lastname: Jacobowitz
authors.forename: Harvey B
authors.initials: HB
authors.lastname: Pollard
authors.forename: Tong
authors.initials: T
authors.lastname: Hao
authors.forename: David E
authors.initials: DE
authors.lastname: Hill
authors.forename: Marc
authors.initials: M
authors.lastname: Vidal
authors.forename: Thomas P
authors.initials: TP
authors.lastname: Conrads
authors.forename: Timothy D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: James L
authors.initials: JL
authors.lastname: Hartley
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics* / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli; D020869:Gene Expression Profiling; D016366:Open Reading Frames / Q000235:genetics*; D020543:Proteome / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins
chemical_list: D029742:Caenorhabditis elegans Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins