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1.
Langmuir ; 32(32): 8141-53, 2016 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-27434827

RESUMO

The selective engineering of conjugated polyelectrolyte (CPE)-phospholipid interfaces is poised to play a key role in the design of advanced biomedical and biotechnological devices. Herein, we report a strategic study to investigate the relationship between the charge of the CPE side group and their association with zwitterionic phospholipid bilayers. The interaction of dipalmitoylphosphatidylcholine (DPPC) phospholipid vesicles with a series of poly(thiophene)s bearing zwitterionic, cationic, or anionic terminal groups (P3Zwit, P3TMAHT and P3Anionic, respectively) has been probed. Although all CPEs showed an affinity for the zwitterionic vesicles, the calculated partition coefficients determined using photoluminescence spectroscopy suggested preferential incorporation within the lipid bilayer in the order P3Zwit > P3Anionic ≫ P3TMAHT. The polarity probe Prodan was used to further qualify the position of the CPE inside the vesicle bilayers via Förster resonance energy transfer (FRET) studies. The varying proximity of the CPEs to Prodan was reflected in the Stern-Volmer quenching constants and decreased in the order P3Anionic > P3TMAHT ≫ P3Zwit. Dynamic light scattering measurements showed an increase in the hydrodynamic diameter of the DPPC vesicles upon addition of each poly(thiophene), but to the greatest extent for P3Anionic. Small-angle neutron scattering studies also revealed that P3Anionic specifically increased the thickness of the headgroup region of the phospholipid bilayer. Epifluorescence and atomic force microscopy imaging showed that P3TMAHT formed amorphous agglomerates on the vesicle surface, P3Zwit was buried throughout the bilayer, and P3Anionic formed a shell of protruding chains around the surface, which promoted vesicle fusion. The global data indicate three distinctive modes of interaction for the poly(thiophene)s within DPPC vesicles, whereby the nature of the association is ultimately controlled by the pendant charge group on each CPE chain. Our results suggest that charge-mediated self-assembly may provide a simple and effective route to design luminescent CPE probes capable of specific localization within phospholipid membranes.


Assuntos
Membrana Celular/química , Bicamadas Lipídicas/química , Modelos Químicos , Fosfolipídeos/química , Polímeros/química , Tiofenos/química , Transferência Ressonante de Energia de Fluorescência
2.
Phys Chem Chem Phys ; 18(18): 12423-7, 2016 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-27098999

RESUMO

The anionic conjugated polyelectrolyte, poly[3-(6-sulfothioatehexyl)thiophene] (P3Anionic), functions as a highly sensitive probe of membrane order, uniquely capable of sequentially detecting the three key phase transitions occurring within model phospholipid bilayers. The observed sensitivity is the result of charge-mediated, selective localisation of P3Anionic within the head-groups of the phospholipid bilayer.


Assuntos
Membrana Celular/química , Transição de Fase , Polieletrólitos/química , Polímeros/química , Ácidos Sulfônicos/química , Tiofenos/química , Cor , Bicamadas Lipídicas/química , Modelos Moleculares , Conformação Molecular , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
3.
J Phys Chem B ; 119(7): 3231-41, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25590689

RESUMO

We report on the structural and colorimetric effects of interaction of aqueous ∼0.06-1% poly[9,9-bis(2-ethylhexyl)fluorene]-b-poly[3-(6-trimethylammoniumhexyl)thiophene] bromide (PF2/6-P3TMAHT) with double-stranded DNA to form PF2/6-P3TMAHT(DNA)x where x is the molar ratio of DNA base pairs to P3TMAHT repeat units; x = 0.5 equals the nominal charge neutralization. PF2/6-P3TMAHT forms 20-40 nm sized particles with PF2/6 core and hydrated P3TMAHT exterior. The polymer particles form loose one-dimensional chains giving micrometer long branched chains (0.19 ≤ x ≤ 0.76) and subsequently randomly shaped aggregates (x = 1.89) upon DNA addition. Compaction of the P3TMAHT block and the 20-30 nm sized core is observed for x = 0.38-0.76 and attributed to the DNA merged within P3TMAHT domain with this structure disassembling with DNA excess. Structural transformations are followed by chromic changes seen as color changes from deep red (x < 0.076) to yellow (x = 0.19), nearly colorless (x = 0.38-0.76), and back to orange (x = 1.89). Both absorption and photoluminescence spectra display the distinct fluorene and thiophene bands and subsequent blue and red shifts when passing x = 0.5. Thiophene photoluminescence (PL) is significantly quenched by DNA with increasing x, and the changing P3TMAHT/PF2/6 band ratio allows quantitative DNA detection. Sixteen-fold dilution does not change aggregate structure, but PL is blue-shifted, indicating weakened intermolecular interactions.


Assuntos
Cátions/química , DNA/química , Fluorenos/química , Polímeros/química , Tiofenos/química , Água/química , Brometos/química , Cor , Microscopia Eletrônica de Transmissão , Estrutura Molecular , Espalhamento a Baixo Ângulo , Soluções , Análise Espectral , Difração de Raios X
4.
ACS Appl Mater Interfaces ; 6(14): 11758-65, 2014 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-24995578

RESUMO

Anionic, conjugated thiophene- and fluorene-based polyelectrolytes with alkylthiosulfate side chains undergo hydrolysis under formation of alkylthiol and dialkyldisulfide functions. The hydrolysis products can be deposited onto gold or silver surfaces by self-assembly from solutions of the anionic conjugated polyelectrolyte (CPE) precursors in polar solvents such as methanol. This procedure allows solution-based surface modifications of gold and silver electrodes using environmentally friendly solvents and enables the formation of conjugated polymer bilayers. The herein presented alkylthiosulfate-substituted CPEs are promising candidates for increasing the work function of gold and silver electrodes thus improving hole injection from such electrode assemblies into organic semiconductors.

5.
J Phys Chem B ; 117(46): 14576-87, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24143872

RESUMO

Aqueous mixtures of a dye-labeled non-ionic thermoresponsive copolymer and a conjugated cationic polyelectrolyte are shown to exhibit characteristic changes in fluorescence properties in response to temperature and to the presence of salts, enabling a double-stimuli responsiveness. In such mixtures at room temperature, i.e., well below the lower critical solution temperature (LCST), the emission of the dye is strongly quenched due to energy transfer to the polycation, pointing to supramolecular interactions between the two macromolecules. Increasing the concentration of salts weakens the interpolymer interactions, the extent of which is simultaneously monitored from the change in the relative emission intensity of the components. When the mixture is heated above its LCST, the transfer efficiency is significantly reduced, signaling a structural reorganization process, however, surprisingly only if the mixture contains salt ions. To elucidate the reasons behind such thermo- and ion-sensitive fluorescence characteristics, we investigate the effect of salts of alkali chlorides, in particular of NaCl, on the association behavior of these macromolecules before and after the polymer phase transition by a combination of UV-vis, fluorescence, and (1)H NMR spectroscopy with light scattering and small-angle neutron scattering measurements.

6.
Langmuir ; 28(33): 12348-56, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22839776

RESUMO

The absorption and photoluminescence spectra of the cationic conjugated polyelectrolyte poly[3-(6-trimethylammoniumhexyl)thiophene] (P3TMAHT) were observed to be dramatically altered in the presence of anionic surfactants due to self-assembly through ionic complex formation. Small-angle neutron scattering (SANS), UV/vis, and photoluminescence spectroscopy were used to probe the relationship between the supramolecular complex organization and the photophysical response of P3TMAHT in the presence of industrially important anionic surfactants. Subtle differences in the surfactant mole fraction and chemical structure (e.g., chain length, headgroup charge density, perfluorination) result in marked variations in the range and type of complexes formed, which can be directly correlated to a unique colorimetric and fluorimetric fingerprint. Our results show that P3TMAHT has potential as an optical sensor for anionic surfactants capable of selectively identifying distinct structural subgroups through dual mode detection.


Assuntos
Fenômenos Ópticos , Transição de Fase , Polímeros/química , Compostos de Amônio Quaternário/química , Tensoativos/química , Tiofenos/química , Ácidos Alcanossulfônicos/química , Fluorcarbonetos/química , Modelos Moleculares , Conformação Molecular
7.
Microb Cell Fact ; 9: 35, 2010 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-20487563

RESUMO

BACKGROUND: Bioprocess development of recombinant proteins is time consuming and laborious as many factors influence the accumulation of the product in the soluble and active form. Currently, in most cases the developmental line is characterised by a screening stage which is performed under batch conditions followed by the development of the fed-batch process. Performing the screening already under fed-batch conditions would limit the amount of work and guarantee that the selected favoured conditions also work in the production scale. RESULTS: Here, for the first time, high throughput multifactorial screening of a cloning library is combined with the fed-batch technique in 96-well plates, and a strategy is directly derived for scaling to bioreactor scale. At the example of a difficult to express protein, an RNase inhibitor, it is demonstrated that screening of various vector constructs and growth conditions can be performed in a coherent line by (i) applying a vector library with promoters and ribosome binding sites of different strength and various fusion partners together with (ii) an early stage use of the fed-batch technology. It is shown that the EnBase technology provides an easy solution for controlled cultivation conditions in the microwell scale. Additionally the high cell densities obtained provide material for various analyses from the small culture volumes. Crucial factors for a high yield of the target protein in the actual case were (i) the fusion partner, (ii) the use of of a mineral salt medium together with the fed-batch technique, and (iii) the preinduction growth rate. Finally, it is shown that the favorable conditions selected in the microwell plate and shake flask scales also work in the bioreactor. CONCLUSIONS: Cultivation media and culture conditions have a major impact on the success of a screening procedure. Therefore the application of controlled cultivation conditions is pivotal. The consequent use of fed-batch conditions from the first screening phase not only shortens the developmental line by granting that the selected conditions are relevant for the scale up, but in our case also standard batch cultures failed to select the right clone or conditions at all.


Assuntos
Escherichia coli/citologia , Microbiologia Industrial/métodos , Análise em Microsséries/métodos , Técnicas Bacteriológicas , Reatores Biológicos , Células Clonais , Meios de Cultura , Projetos Piloto
8.
Chemistry ; 14(29): 8972-9, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18696525

RESUMO

We report a series of [2]borametallocenophanes of Ti, Zr, and Hf with various ligand systems. The ligands have been synthesized in high yields starting from 1,2-dibromo-1,2-bis(dimethylamino)diborane(4) upon reaction with Na[C5H5] and Li[C13H9], respectively. All compounds were fully characterized by multinuclear NMR spectroscopy and, for selected examples, by X-ray analysis.

9.
Appl Microbiol Biotechnol ; 76(6): 1413-22, 2007 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-17684739

RESUMO

The functional analysis of individual proteins or of multiprotein complexes - since the completion of several genome sequencing projects - is in focus of current scientific work. Many heterologous proteins contain disulfide-bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degP-promoter-based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies.


Assuntos
Escherichia coli/metabolismo , Periplasma/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Escherichia coli/genética , Expressão Gênica , Genes Reporter , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas Periplásmicas/genética , Proteínas Periplásmicas/metabolismo , Regiões Promotoras Genéticas/genética , Dobramento de Proteína , Proteínas Recombinantes/biossíntese , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Solubilidade
10.
Appl Microbiol Biotechnol ; 75(2): 397-406, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17221192

RESUMO

The expression of heterologous proteins in the cytoplasm of Escherichia coli is often accompanied by limitations resulting in uncontrollable fermentation processes, increased rates of cell lysis, and thus limited yields of target protein. To deal with these problems, reporter tools are required to improve the folding properties of recombinant protein. In this work, the well-known sigma(32)-dependent promoters ibpAB and fxsA were linked in a tandem promoter (ibpfxs), fused with the luciferase reporter gene lucA to allow enhanced monitoring of the formation of misfolded proteins and their aggregates in E. coli cells. Overexpression of MalE31, a folding-defective variant of the maltose-binding protein, and other partially insoluble heterologous proteins showed that the lucA reporter gene was activated in the presence of these misfolded proteins. Contrary to this, the absence of damaged proteins or overexpression of mostly soluble proteins led to a reduced level of luciferase induction. Through performing expression of aggregation-prone proteins, we were able to demonstrate that the ibpfxs::lucA reporter unit is 2.5-4.5 times stronger than the single reporter units ibp::lucA and fxs::lucA. Data of misfolding studies showed that this reporter system provides an adequate tool for in vivo folding studies in E. coli from microtiter up to fermentation scales.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Genes Reporter , Proteínas de Choque Térmico/metabolismo , Sistemas On-Line , Regiões Promotoras Genéticas , Dobramento de Proteína , Fator sigma/metabolismo , Reatores Biológicos , Biotecnologia/métodos , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Fermentação , Regulação Bacteriana da Expressão Gênica , Resposta ao Choque Térmico , Luciferases/genética , Luciferases/metabolismo , Solubilidade
11.
Protein Expr Purif ; 52(2): 478-84, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17188891

RESUMO

Quantitative and folding reporters are adequate tools to optimize recombinant protein expression in various host organisms, including Escherichia coli. To determine the yield of soluble active protease from the tobacco etch virus (TEV), we developed a single-molecule assay based on the fluorogenic substrate ANA-QS-MCA. This substrate consists of a 10 amino acid peptide (ENLYFQSGTK) containing the proteolytic cleavage sequence of the TEV protease. The peptide works as a linker N-terminally tagged with a fluorescent donor group (7-Methoxycoumarin-4-yl)acetyl (MCA) and C-terminally tagged with the acceptor group 5-Amino-2-nitrobenzoic acid (ANA). Fluorescence can be observed after specific cleavage of the substrate at the Gln-Ser bond by active TEV protease. Purified His-tagged TEV protease was used for in vitro analysis. Through determination of proteolytic activity in living E. coli cells and through application of Confocal Laser-Scanning-Microscopy we demonstrate that the peptide is well suited to in vivo expression analysis. This provides an effective tool to monitor the accumulation of active recombinant TEV protease in crude extracts and intact cells.


Assuntos
Endopeptidases/metabolismo , Potyvirus/enzimologia , Endopeptidases/química , Endopeptidases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes , Expressão Gênica , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Inorg Chem ; 45(14): 5275-7, 2006 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-16813387

RESUMO

In the present paper, we describe the first structural characterization of cymantrenyl(dihalo)borane and report on its use for the synthesis of novel cymantrenylboryl complexes.

13.
J Am Chem Soc ; 127(10): 3282-3, 2005 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-15755137

RESUMO

The first [2]borametallocenophanes of Zr and Hf are reported, which were obtained from 1,2-bis(dimethylamino)-1,2-dicyclopentadienyldiborane(4). The constitution of these complexes in solution and, for the Hf complex, in the crystalline state is discussed. The title complexes were studied as Ziegler-Natta-type catalyst precursors for the polymerization of ethylene. Both complexes produced PE of high molecular mass up to 1.6 x 106 g/mol, and very interestingly, the Hf complex proved to be as active as its Zr analogue.

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