Pooled ORF Expression Technology (POET)

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. 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. Projects aiming to convert the thousands of genes made accessible by genomic sequences into their corresponding proteins have met with limited success (1Service R.F. Tapping DNA for structures produces a trickle.Science. 2002; 298: 948-950Google Scholar) despite the expenditure of significant resources (2Lattman E. The state of the Protein Structure Initiative.Proteins. 2004; 54: 611-615Google Scholar, 3Frazier M.E. Johnson G.M. Thomassen D.G. Oliver C.E. Patrinos A. Realizing the potential of the genome revolution: the genomes to life program.Science. 2003; 300: 290-293Google Scholar). Expression of recombinant proteins in Escherichia coli, the primary host organism for high throughput applications, has been especially unsuccessful for metazoan proteins. For example, one effort directed at producing Caenorhabditis elegans proteins successfully purified only about 2% of those attempted (4Luan C.H. Qiu S. Finley J.B. Carson M. Gray R.J. Huang W. Johnson D. Tsao J. Reboul J. Vaglio P. Hill D.E. Vidal M. Delucas L.J. Luo M. High-throughput expression of C. elegans proteins.Genome Res. 2004; 14: 2102-2110Google Scholar). In the standard approach to high throughput protein expression and purification used in these programs, genes are cloned individually into an expression vector, introduced into an expression host, and expressed in separate cultures. Each culture is subsequently tested for expression and solubility of the corresponding protein at which point new cultures of positive clones are grown and induced so that protein purification can be attempted. Even with intensive use of robotics, the logistics and costs of this strategy are considerable when thousands of genes are put into such a pipeline. Here we describe a method called pooled ORF expression technology (POET) 1The abbreviations used are: POET, pooled ORF expression technology; attL, site-specific recombination sites for Gateway cloning; LR Clonase, site-specific recombination enzyme mixture for Gateway cloning; 2