The completion of a growing number of genomes has driven the structural genomics field with the aim of determining many thousand structures within the next few years. This requires substantial optimization of every step in a crystallographic structure determination. In this aspect, one of the most important techniques now routinely used is the production of selenomethionine (SeMet) substituted recombinant proteins. In some ways SeMet has advanced the crystallography field as polymerase chain reaction (PCR) has advanced molecular biology. SeMet aids in positioning methionines in the polypeptide chain as well as for phase determination by multiwavelength anomalous dispersion (MAD) allowing more efficient structure determinations. SeMet is typically utilized for expression of recombinant proteins from Escherichia coli, but there is a growing need for expression in eukaroyotic cells as the number of eukaryotic genomes completed increases. The yeast Saccharomyces cerevisiae offers powerful genetic systems and high level production of endogenous and exogenous eukaryotic proteins with low cost and ease of growth. The routine production of completely substituted SeMet proteins for high resolution structural analysis is far from achieved in yeast. The goals of this high risk/high impact proposal are to bring together expertise in yeast genetics and selenium biology to develop yeast strains for the production of SeMet proteins for structural analysis. The factors identified to confer SeMet resistance will further provide insight into the biology of selenium toxicity. Additionally, the screens utilized to develop SeMet resistance will result in the identification of gene products that can also be targeted in Pichia pastoris or baculovirus infected cells to enhance SeMet tolerance and thus occupancy in the proteins produced from these systems. By increasing our repertoire of available choices as described in this proposal we can maximize the ability to screen protein production for high throughput structural approaches, utilize the many available cloned and tagged yeast proteins for enhancing our molecular understanding of protein function, further optimize other eukaryotic expression systems for SeMet incorporation and develop a better understanding of Se toxicity. ? ? ?
|Bockhorn, Jessica; Balar, Bharvi; He, Dongming et al. (2008) Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance. Proc Natl Acad Sci U S A 105:17682-7|
|Seitomer, Eden; Balar, Bharvi; He, Dongming et al. (2008) Analysis of Saccharomyces cerevisiae null allele strains identifies a larger role for DNA damage versus oxidative stress pathways in growth inhibition by selenium. Mol Nutr Food Res 52:1305-15|