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Lac Repressor (lac + repressor)
Selected AbstractsEngineering LacI for Self-Assembly of Inorganic Nanoparticles on DNA Scaffold through the Understanding of LacI Binding to Solid SurfacesADVANCED FUNCTIONAL MATERIALS, Issue 8 2009Haibin Chen Abstract The potential of utilizing the DNA binding protein lac repressor (LacI) to organize inorganic nanoparticles (NPs) is explored in this study. A peptide cognitive of both SiO2 and TiO2 simultaneously (STB1, -CHKKPSKSC-) is genetically engineered into the C-terminus of LacI to give LacI-STB1, and the inserted STB1 peptides in the context of LacI-STB1 molecules are shown to actively interact with both SiO2 and TiO2. Wild-type LacI is found to interact with the two surfaces at its flexible N-terminal DNA binding domain, and LacI-STB1 exhibits much stronger binding affinity to both surfaces by harnessing a second binding region (STB1 peptide) fused at its C-terminus. The quantitative analysis of binding kinetics reveals that, compared to wild-type LacI with one binding region (N-terminus), two remote binding regions (N-terminus and C-terminus) in LacI-STB1 do not lead to faster adsorption rates to the two surfaces, but remarkably slow down the desorption rates. Finally, using LacI-STB1 as a linker, the successful assembly of a sandwich nanostructure of DNA/LacI-STB1/TiO2 NPs is demonstrated using surface plasmon resonance (SPR) measurements and TEM. The demonstrated LacI-STB1-mediated assembly of TiO2 NPs on DNA scaffold may provide a generic platform for controlled spatial arrangement of various nanoparticles of engineering interest. [source] Synthesis of new S -glycodendrimer toward activation of lac operon transcription for protein biosynthesisJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 1 2009Akinori Takasu To enable gene transcription or lac operon transcription, isopropyl ,- D -thiogalactoside (IPTG) and allolactose can bind to the lac repressor. New S -glycodendrimers for activation of the lac operon were synthesized by S -glycosidation and DCC-HOBt coupling with a poly(amidoamine) dendrimer. Expression of artificial protein was performed for Escherichia coli using these glycodendrimers as the inducers. Cells encoded with green fluorescent protein (GFP) were induced with the glycoconjugates. After expression at 37 °C for 4 h, fluorescence emissions were actually observed through visual observation, which indicated that S -glycodendrimer acted as an inducer for protein biosynthesis. Quantitative analysis using fluorescence spectrometer was carried out to evaluate the activity. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] [source] A hierarchical Bayesian model for predicting the functional consequences of amino-acid polymorphismsJOURNAL OF THE ROYAL STATISTICAL SOCIETY: SERIES C (APPLIED STATISTICS), Issue 1 2005Claudio J. Verzilli Summary., Genetic polymorphisms in deoxyribonucleic acid coding regions may have a phenotypic effect on the carrier, e.g. by influencing susceptibility to disease. Detection of deleterious mutations via association studies is hampered by the large number of candidate sites; therefore methods are needed to narrow down the search to the most promising sites. For this, a possible approach is to use structural and sequence-based information of the encoded protein to predict whether a mutation at a particular site is likely to disrupt the functionality of the protein itself. We propose a hierarchical Bayesian multivariate adaptive regression spline (BMARS) model for supervised learning in this context and assess its predictive performance by using data from mutagenesis experiments on lac repressor and lysozyme proteins. In these experiments, about 12 amino-acid substitutions were performed at each native amino-acid position and the effect on protein functionality was assessed. The training data thus consist of repeated observations at each position, which the hierarchical framework is needed to account for. The model is trained on the lac repressor data and tested on the lysozyme mutations and vice versa. In particular, we show that the hierarchical BMARS model, by allowing for the clustered nature of the data, yields lower out-of-sample misclassification rates compared with both a BMARS and a frequen-tist MARS model, a support vector machine classifier and an optimally pruned classification tree. [source] Conditional gene silencing utilizing the lac repressor reveals a role of SHP-2 in cagA -positive Helicobacter pylori pathogenicityCANCER SCIENCE, Issue 5 2004Megumi Higuchi RNA interference (RNAi) is a newly described biological phenomenon mediated by small interfering RNA (siRNA) that targets mRNA for degradation by cellular enzymes and has become a powerful method for studying gene functions in mammalian systems. The development of systems for inducing siRNA expression should enable examination of acute loss-of-function phenotypes in a cell of interest without the need to consider lethality or epigenetic adaptation of cells. We describe in this report an inducible siRNA expression system made by combined utilization of the RNA polymerase III-dependent promoter H1 and the bacterial lac repressor. Using this system, we established AGS gastric epithelial cells in which expression of SHP-2, a cellular tyrosine phosphatase known to specifically bind the Helicobacter pylori virulence factor CagA, is conditionally and reversibly silenced by the lactose analog isopropyl-1-thio-,-D-galactopyranoside (IPTG). Upon expression in AGS cells, CagA provoked a morphological transformation, termed the hummingbird phenotype, which is associated with CagA virulence. This morphogenetic activity of CagA was totally abolished when SHP-2 expression was silenced by inducible siRNA expression in AGS cells. Our results indicate that SHP-2 is a critical downstream effector of H. pylori CagA. The conditional gene silencing system described here should become a powerful tool for investigating the roles of cancer-related genes through a reversed genetic approach. [source] | ||||||||||