Membranes for the Life Sciences, Volume 1
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The vpu gene optimized for expression in E. The same pair of restriction enzymes were also used to linearize the pET26 b vector by Novagen. The restriction digestion reactions were analyzed by agarose gel electrophoresis and the desired DNA bands were excised and purified using a QIAquick gel extraction kit Qiagen; cat The resulting bacterial colonies were screened using colony screen PCR using gene specific primers oTMs and and plasmid specific primers, oTMs and Finally the cultures were induced for expression with 0. The cells were lysed by double passage through a microfluidizer Microfluidics Microfluidizer.
The insoluble fraction, containing the inner and outer membranes among other insoluble materials like protein inclusion bodies, was washed once by repeated re-suspension 50 mL of ice-cold PBS with protease inhibitor cocktail and centrifugation. Aqueous and non-aqueous fractions from mL cultures were prepared as described above. The sample was then loaded onto the column, and passed twice.
Filter Holders and Membranes
The column was washed with ten bed volumes of wash buffers containing various combinations of salt and imidazole, for example, 20 mM HEPES, pH 7. For preparatory separations, a 1 mL sample of concentrated PelB-Vpu was loaded onto the SEC column and chromatography was performed at a flow rate of 0. The protein elution was detected by absorption at nm. The culture was grown and induced with 0.
A deconstructed version of human CD4 containing the transmembrane and the cytoplasmic domains with an N-terminal fusion of maltose binding protein was expressed in BL21 cells. The protein was extracted and solubilized in buffer containing 0. Following electrophoresis, gels were stained by Coomassie brilliant blue, subjected to silver staining, or processed for immuno-blotting.
For immuno-blotting, the acrylamide gel was first rinsed with water and equilibrated in a non-denaturing buffer 25 mM TRIS base and mM glycine. The nitrocellulose membrane Bio-Rad; cat — was also equilibrated in the non-denaturing buffer. The gel and the membrane were sandwiched between extra-thick blot filter papers VWR; cat — soaked in the non-denaturing buffer and proteins were electro- blotted for 15 min at 15 V using a Bio-Rad Transfer-blot SD Semi-dry Transfer Cell.
Following additional 3x 15 min washes, the nitrocellulose membrane was then soaked in Immunocruz western blotting luminol reagent Santa Cruz Biotechnology; cat sc , exposed to a chemiluminescence sensitive film GE Heathcare; cat for the optimum time of exposure and the film was developed with a Konica SRXA system. An external calibration spot prepared in a similar manner was placed near the PelB-Vpu protein spot. The final results represented the average of 10 separate spectra.
Nanostructured Polymer Membranes, Volume 1: Processing and Characterization
CD spectra for the protein were recorded from to nm using a 0. Output spectra were generated based on an accumulation of five scans. Ten consecutive measurements, each with an integration time of 20 s, were averaged. Hydrodynamic size of the particles was estimated with the instrument software using the following parameters: refractive index 1.
We thank Drs. Data curation: AD. Browse Subject Areas?
Click through the PLOS taxonomy to find articles in your field. Data Availability: All relevant data are within the paper. Introduction Structural analysis of membrane proteins has traditionally been bottlenecked by the lack of sufficiently large amounts of pure, properly folded and functional membrane proteins.
Download: PPT. Fig 1. Optimization of the vpu gene for expression in E. Fig 2.
Organization of the expression cassette within the plasmid pTM Expression in E. Fig 3. Vpu fractionates with the water insoluble fraction and can be subsequently solubilized by detergents. Vpu is targeted to the membranes and inserted as a type I membrane protein In mammalian cells, the Vpu transmembrane domain TMD serves as a signal-anchor to target the protein for insertion into the membrane, but when expressed in bacteria, the TMD does not support this insertion function, resulting in the accumulation and aggregation of the protein in inclusion bodies [ 38 ].
Fig 4. Vpu fractionates with the membrane fraction and inclusion bodies dependent on time post-induction with IPTG. Fig 5. Vpu is inserted into the bacterial inner membrane with its C-terminal domain within the cytoplasm.
Fig 6. Protein analysis of the talon metal affinity chromatography purification of PelB-Vpu. Fig 7. Fig 8.
Osmotically Driven Membrane Processes
Fig 9. Dynamic light scattering measurements show that PelB-Vpu is monodispersed. Fig Conclusions The requirement to produce substantial quantities of pure, folded, functional and monodispersed protein is a a major factor that hampers elucidation of membrane protein structural models. Table 1. Highly-expressed human gene set used in codon usage optimization. Detergent extraction Aqueous and non-aqueous fractions from mL cultures were prepared as described above. Determination of PelB-Vpu orientation in the E.
Co -immunoprecipitation with CD4 A deconstructed version of human CD4 containing the transmembrane and the cytoplasmic domains with an N-terminal fusion of maltose binding protein was expressed in BL21 cells. Acknowledgments We thank Drs. References 1. A novel gene of HIV-1, vpu, and its kilodalton product. Identification of a protein encoded by the vpu gene of HIV Human-immunodeficiency-virus-typeencoded V pu protein is phosphorylated by casein kinase II.
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Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. Journal of Bacteriology. Arvola M, Keinanen K. Characterization of the ligand-binding domains of glutamate receptor GluR -B and GluR-D subunits expressed in Escherichia coli as periplasmic proteins.
Journal of Biological Chemistry. High-level temperature-induced synthesis of an antibody VH-domain in Escherichia coli using the PelB secretion signal. Expression of single-chain antibody fragments scFv specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana. Plant Molecular Biology. Extracellular production of phospholipase D of Streptomyces antibioticus using recombinant Escherichia coli.
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View Article Google Scholar X-ray structure of a prokaryotic pentameric ligand-gated ion channel. A mucosally targeted subunit vaccine candidate eliciting HIV-1 transcytosis-blocking Abs. Transcytosis-blocking abs elicited by an oligomeric immunogen based on the membrane proximal region of HIV-1 gp41 target non-neutralizing epitopes. Curr HIV Res.