Functional genomics at the interface of protein expression and biophysical analysis

Sammanfattning: Protein characterization is central to any functional genomics effort, hence protein expression and purification play fundamental roles in these initiatives. In this study strategies for protein expression in E. coli were analysed, and several parameters were investigated respect to high-level expression of functional proteins. Produced proteins were characterized using biophysical methods to determine their functional properties, including protein-ligand interaction, enzyme kinetics and thermostability analysis. Rare codons often prevent high-level expression of foreign genes in E. coli. Two rare-codon genes (with a content of about 30%) were used as a model system. The genes studied were a histone-like protein from Sulfolobus shibatae (Ssh10), and a glutaredoxin-like protein from Methanobacterium thermoautotrophicum (mtGrx). High-level expression and rapid purification of these rare-codon genes were achieved by the GST gene fusion system. Factors influencing expression patterns in E. coli of these two genes were studied using expression plasmids encoding wild-type genes, codon-optimized synthetic and GST-fusion genes. These constructs were expressed in BL21 (DE3), its LysS derivative (carrying lysozyme for T7 RNA polymerase), and modified strains carrying copies for rare codon tRNAs, and deletions in the RNAseE gene. The results underscore the importance of mRNA levels and RNA stability, but not necessarily tRNA abundance for efficient heterologous protein production in E. coli. A gene fusion system is an alternative method for rare codon gene expression compared to the synthetic gene. Hydrophobicity and membrane attachment of 11 beta-hydroxysteroid dehydrogenase type 1 (11 betaHSD1) has prevented its purification to homogeneity in quantities sufficient for detailed structural and mechanistic studies. Our study shows that by co-overexpressing GroEL/ES and adding an 11 beta-HSD1 inhibitor during protein synthesis, the accumulation of solubility has been increased by more than one order of magnitude. Stable monodisperse enzyme forms from human and guinea pig were purified by optimizing various solubilization systems for the chromatographic steps, including different detergents or chaotropic salts, and by adding cofactors and specific inhibitors of the enzyme. Protein interactions of beta-catenin with Tcf4 were studied. This feature plays a central role in the wntsignaling pathway and has been proposed as a possible site of intervention for the development of anticancer drugs. By performing Ala-scanning mutagenesis of all Tcf4 residues in the Tcf-beta-catenin interface, the binding energetics of these mutants were studied using isothermal titration calorimetry (ITC). Asp 16 in Tcf4 is a hot spot for manipulation of Tcf4-beta-catenin interaction and suggested as a possible anti-cancer drug target. Ssh10b is a representative of an archaeal histone-like class of transcriptional regulators, whose activity is regulated by Lys-acetylation. The thermal and chemical unfolding of Ssh10b has been characterized using differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy. Both experimental calorimetric and spectroscopic data fit well to a two-state unfolding model, suggesting that the Ssh10b dimer unfolds as a single cooperative unit. The low specific heat capacity change of 11 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (deltaG (T)) and results in a maximal deltaG of 9.5 kcal/mol at 320K and a deltaG of 6.0 kcal/mol at the optimal growth temperature of Suffiblobus. The sexual development of several plant species proceeds through selective elimination and maturation of floral organs in an initially bisexual floral meristem. The Tasselseed2 (TS2) gene is a key component in the cell-death pathway in maize. TS2 was produced as fusion protein to maltosebinding protein in E. coli. Substrate screening demonstrates that TS2 converts steroid hormones with specificities at positions 3 and 17 of the steroid backbone, besides several dicarbonyl compounds, thus establishing TS2 as a plant 3beta/17beta-hydroxysteroid dehydrogenase and carbonyl reductase. Our results suggest that TS2 converts specific hormones and acts as a pre-receptor control mechanism, in a similar manner as mammalian hydroxysteroid dehydrogenases.