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Expression Screening (expression + screening)
Selected AbstractsExpression screening of integral membrane proteins from Helicobacter pylori 26695PROTEIN SCIENCE, Issue 12 2007Georgios Psakis Abstract The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins ,40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions. [source] Molecular characterization of the response to chemotherapy in conventional osteosarcomas: Predictive value of HSD17B10 and IFITM2INTERNATIONAL JOURNAL OF CANCER, Issue 4 2009Sébastien Salas Abstract The therapy regimen of high-grade osteosarcoma includes chemotherapy followed by surgical resection and postoperative chemotherapy. The degree of necrosis following definitive surgery remains the only reliable prognostic factor and is used to guide the choice of postoperative chemotherapy. The aim of this study was to find molecular markers able to classify patients with an osteosarcoma as good or poor responders to chemotherapy before beginning treatment. Gene expression screening of 20 nonmetastatic high-grade osteosarcoma patients was performed using cDNA microarray. Expression of selected relevant genes was validated using QRT-PCR. Immunohistochemistry on tissue microarrays sections of 73 biopsies was performed to investigate protein expression. Fluorescent in situ hybridization was performed for RPL8 gene. We have found that HSD17B10 gene expression was up-regulated in poor responders and that immunohistochemistry expression of HSD17B10 on biopsy before treatment was correlatedto response to chemotherapy. Other results include correlationof IFITM2, IFITM3, and RPL8 gene expression to chemotherapy response. A statistical correlation was found between polysomy 8 or gain of RPL8 and good response to chemotherapy. These data suggest that HSD17B10, RPL8, IFITM2, and IFITM3 genes are involved in the response to the chemotherapy and that HSD17B10 may be a therapeutic target. RPL8 and IFITM2 may be useful in the assessment at diagnosis and for stratifying patients taking part in randomized trials. © 2009 UICC [source] Genes Associated With Alcohol Abuse and Tobacco Smoking in the Human Nucleus Accumbens and Ventral Tegmental AreaALCOHOLISM, Issue 7 2010Traute Flatscher-Bader Background:, The incidence of alcohol and tobacco co-abuse is as high as 80%. The molecular mechanism underlying this comorbidity is virtually unknown, but interactions between these drugs have important implications for the development of and recovery from drug dependence. Methods:, We investigated the effects of chronic tobacco and alcohol abuse and the interaction of the 2 behaviors on global gene expression in the human nucleus accumbens using cDNA microarrays and 20 alcoholic and control cases, with and without smoking comorbidity. Changes in gene expression were established by factorial ANOVA. Unsupervised hierarchical clustering was utilized to probe the strength of the data sets. Applying real-time PCR differential expression of candidate genes was confirmed in the nucleus accumbens and explored further in a second core region of the mesolimbic system, the ventral tegmental area. Results:, Subjecting the data sets derived from microarray gene expression screening to unsupervised hierarchical clustering tied the cases into distinct groups. When considering all alcohol-responsive genes, alcoholics were separated from nonalcoholics with the exception of 1 control case. All smokers were distinguished from nonsmokers based on similarity in expression of smoking-sensitive genes. In the nucleus accumbens, alcohol-responsive genes were associated with transcription, lipid metabolism, and signaling. Smoking-sensitive genes were predominantly assigned to functional groups concerned with RNA processing and the endoplasmic reticulum. Both drugs influenced the expression of genes involved in matrix remodeling, proliferation, and cell morphogenesis. Additionally, a gene set encoding proteins involved in the canonical pathway "regulation of the actin cytoskeleton" was induced in response to alcohol and tobacco co-abuse and included. Alcohol abuse elevated the expression of candidate genes in this pathway in the nucleus accumbens and ventral tegmental area, while smoking comorbidity blunted this induction in the ventral tegmental area. Conclusions:, The region-specific modulation of alcohol-sensitive gene expression by smoking may have important consequences for alcohol-induced aberrations within the mesolimbic dopaminergic system. [source] Expression screening of integral membrane proteins from Helicobacter pylori 26695PROTEIN SCIENCE, Issue 12 2007Georgios Psakis Abstract The efficiency of Helicobacter pylori as a mucosal pathogen is caused by unique soluble and integral membrane proteins, which allow its survival at acidic pH and successful colonization of the gastric environment. With about one-fourth of the H. pylori's proteome comprising integral membrane proteins, the need for solution of their three-dimensional (3D) structures becomes persistent as it can potentially drive the generation of more effective drugs. This study presents a medium-throughput approach for cloning and expression screening of integral membrane proteins from H. pylori (26695) using Escherichia coli as the expression host. One-hundred sixteen H. pylori targets were cloned into two different vector systems and heterologously expressed in E. coli. Eighty-four percent of these proteins displayed medium to high expression. No clear-cut correlation was found between expression levels and number of putative transmembrane spans, predicted functionality, and molecular mass. Nonetheless, expression of transporters and hypothetical proteins ,40 kDa with two to four transmembrane spans displayed generally high expression levels. To statistically strengthen the quality of the data from the medium-throughput approach, a comparison with data derived from robotic-based methodologies was conducted. Optimization of expression and solubilization conditions for selected targets was also performed. Seventeen targets have been purified and subjected to crystallization so far. Eighteen percent of these targets (2/17) produced crystals under specific sets of crystallization conditions. [source] Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINEACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2006Pedro M. Alzari The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation. [source] | |||||||||||||