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 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.
Gillette
William K
WK
Protein Expression Laboratory, Research Technology Program, SAIC-Frederick, Inc./NCI, National Institutes of Health, Frederick, Maryland 21702, USA.
Esposito
Dominic
D
Frank
Peter H
PH
Zhou
Ming
M
Yu
Li-Rong
LR
Jozwik
Catherine
C
Zhang
Xiuying
X
McGowan
Brighid
B
Jacobowitz
David M
DM
Pollard
Harvey B
HB
Hao
Tong
T
Hill
David E
DE
Vidal
Marc
M
Conrads
Thomas P
TP
Veenstra
Timothy D
TD
Hartley
James L
JL
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
D029742:Caenorhabditis elegans Proteins; D020543:Proteome; D011993:Recombinant Fusion Proteins