Our goal is to develop high throughput (HTP) gene cloning, gene expression and protein purification protocols that will ultimately produce active, recombinant versions of virtually any gene or set of genes regardless of source. A problem with single open-reading frame (ORF), robotic-based expression systems is that they intrinsically select for cytoplasmic, homomeric, unmodified, cofactor-less proteins, with an overall success rate in producing active proteins of probably less than 10%. Yet, analyses of complete microbial genomes suggest that more than half of the ORFs may be part of multisubunit proteins, about 30% likely encode membrane-bound proteins, and perhaps 10% encode proteins that contain complex cofactors which the host is unlikely to 'insert'. Our objectives are to develop a HTP-multifaceted expression and protein purification system that will also accommodate genes encoding proteins that are multisubunit, membrane-bound and/or contain complex cofactors, in addition to homomeric, cofactor-less proteins.
 
To develop this technology, the genome of the prokaryote, Pyrococcus furiosus, is being used as a model system. The P. furiosus genome contains approximately 2,200 ORFs with about half encoding (conserved) hypothetical proteins. About 700 of the ORFs are predicted to be organized in operons, suggesting that they encode either multisubunit complexes or include accessory proteins for assembly of the active enzyme. Using the HTP-system with multiple expression systems in various hosts, all 2,200 ORFs will be expressed individually and, where genome analyses indicate, as multiple ORFs. This will ultimately yield a complete expression library of P. furiosus, representing all proteins, from the simplest, cytoplasmic protein to the most complex membrane protein assembly.
 
Using the results from the P. furiosus system, protocols will then be developed whereby the successful expression of any given gene can be predicted based on sequence and, where available, genomic analyses. Predictions will be tested using known and unknown ORFs represented in other prokaryotic genomes and in eukaryotic cDNA libraries. In the longer term, methodologies will be incorporated into the HTP-system to accommodate various protein modifications commonly employed in eukaryotic systems.

Currently, cloning of the P. furiosus genome is about 86% complete. We are currently developing robotic protocols on the 96-well scale for cell fractionation and ELISA analysis to determine the optimal conditions for heterologous protein expression. We are also beginning development of systems for expression of membrane proteins, protein complexes, and cofactors.

For more information see our webpage: http://adams.bmb.uga.edu/

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