Expression and Purification of EPHA2 Tyrosine Kinase Domain for Crystallographic and NMR Studies

The receptor tyrosine kinase EPHA2 is overexpressed in several cancers (breast, head and neck, non‐small‐cell lung cancer). Small‐molecule‐based inhibition of the EPHA2 kinase domain (KD) is seen as an important strategy for therapeutic intervention. However, obtaining structural information by crystallography or NMR spectroscopy for drug discovery is severely hampered by the lack of pure, homogeneous protein. Here, different fragments of the EPHA2 KD were expressed and purified from both bacterial (Escherichia coli, BL21(DE3) cells) and insect cells (Spodoptera frugiperda, Sf9 cells).¹H,¹⁵N HSQC was used to determine the proper folding and homogeneity of all the constructs. Protein from E. coli was well‐folded but unstable, and it did not crystallize. However, a construct (D596–G900) produced in Sf9 cells yielded homogenous, well‐folded protein that crystallized readily, thereby resulting in eleven new EPHA2–ligand crystal structures. We have also established a strategy for selective and uniform ¹⁵N‐amino acid labeling of EPHA2 KD in Sf9 cells for investigating dynamics and EPHA2–drug interactions by NMR.     

The Effect of Water Vapor on the Particle Structure and Size of Silica Nanoparticles During Sintering

In this article, the influence of the water vapor concentration on structural changes of SiO₂ aerosol nanoparticle agglomerates during tempering was studied. The presence of water vapor in the carrier gas was shown to strongly accelerate the kinetics of sintering. While dry sintering at temperatures between 1100°C and 1500°C generated aggregates only, the addition of water to the process yields individual, completely coalesced nanoparticles at a temperature of 1300°C. Furthermore, depending on the water vapor concentration and temperature of the process, evaporation and condensation processes could be observed, leading to bimodal size distributions. The results prove the significant role of the water concentration in high temperature synthesis of silica and may be used to improve the control over morphology and specific surface area in these processes.     

Synthesis of Functionalized Indolizines by Lewis Acid‐Mediated Cyclocondensation of 3‐(Pyridin‐2‐yl)‐propiolates with Enones

Functionalized indolizines were prepared by Lewis acid‐catalyzed cyclizations of 3‐(pyridin‐2‐yl)‐propiolates with enones. This new type of reaction provides a convenient and regioselective approach to ester‐substituted indolizine derivatives which are not readily available by other methods. Based on density function theory (DFT) calculations, a mechanism of the reaction has been suggested.     

Single-photon emission from single InGaAs/GaAs quantum dots grown by droplet epitaxy at high substrate temperature

ABSTRACT: The authors report single-photon emission from InGaAs quantum dots grown by droplet epitaxy on (100) GaAs substrates using a solid-source molecular beam epitaxy system at elevated substrate temperatures above 400 [DEGREE SIGN]C without post-growth annealing. High-resolution micro-photoluminescence spectroscopy exhibits sharp excitonic emissions with lifetimes ranging from 0.7 to 1.1 ns. The coherence properties of the emitted photons are investigated by measuring the first-order field correlation function.     

Recombinant yeast technology at the cutting edge: robust tools for both designed catalysts and new biologicals

Health and safety concerns, enhanced quality criteria, and environmental sustainability, have prompted investigations into production using recombinant yeasts as a feasible alternative for isolation of proteins from natural animal or plant sources, as well as for processes utilising either mammalian cell cultures or bacterial systems. An overview of recent research papers and review articles provides readers with a comprehensive insight into the field of next-generation yeast expression systems. Major breakthroughs in recombinant yeast technology linked to Pichia pastoris are (i) the public availability of tools to generate proteins with tailored and highly homogenous N-glycan structures, similar to the forms assembled in humans, (ii) the recent accomplishment of the annotation of its genome sequence, and finally, (iii) the presence of the first few (non-glycosylated) therapeutic proteins in Pichia on the market. The P. pastoris expression platform is now well developed, as proven by multiple products used in human and veterinary medicine and in industry (e.g., enzymes for chemical synthesis and for the modification/synthesis of pharmaceuticals, drug target proteins used for structural analysis or for high throughput screening, proteins for diagnostics, proteinous biomaterials, vaccines, and therapeutic proteins). Nevertheless, the complexity of protein analysis (monitoring) continues to restrict process development for recombinant products. Drawing on combined expertise in molecular biology and process technology, the Institute of Biotechnology (IBT) at the Zurich University of Applied Science (ZHAW) and its international partners have developed solutions which (i) fully eliminate (or partially reduce) the use of methanol, which is undesirable in high-cell-density and high-productivity processes, (ii) match both strain construction and process design with the target protein characteristics to the benefit of the cells' physiological shape, and (iii) allow multi-gene expressions to be balanced to achieve custom tailored and reproducible protein quality at the level of (engineered) posttranslational modifications. In addition to enabling superior product quality specifications to be achieved with reduced development time, these innovations have helped the industries involved to minimise financial risks and the risk of failure, as well as create an opportunity for (new) drugs with improved functionality at low cost.     

Effiziente Herstellung von Kohlenstofffasern

Efficient production of carbon fibres using new technologies Innovative process technologies for carbonization of precursor fibres, which can substitute conventional oven technologies and raise the efficiency of carbon fibre production are presented. The process technologies are based on the substitution of convective heat transfer by microwave plasma or direct fiber heating. In microwave-assisted plasma treatment, the heating of the fibres is carried out both by microwave absorption in the fibre volume and energy input into the fibre surface by means of the plasma species. During electrical carbonization, the fibres are heated by means of an electrical current flow. The processes can be used separately, one after the other (also in combination with conventional furnace technology) or as hybrid processes.     

High Yield,Reproducible and Quasi‐Automated Bilayer Formation in a Microfluidic Format

A microfluidic platform is reported for various experimentation schemes on cell membrane models and membrane proteins using a combination of electrical and optical measurements, including confocal microscopy. Bilayer lipid membranes (BLMs) are prepared in the device upon spontaneous and instantaneous thinning of the lipid solution in a 100‐μm dry‐etched aperture in a 12.5‐μm thick Teflon foil. Using this quasi‐automated approach, a remarkable 100% membrane formation yield is reached (including reflushing in 4% of the cases), and BLMs are stable for up to 36 h. Furthermore, the potential of this platform is demonstrated for (i) the in‐depth characterization of BLMs comprising both synthetic and natural lipids (1,2‐diphytanoyl‐sn‐glycero‐3‐phosphocholine (DPhPC) and L ‐α‐phosphatidylcholine (L ‐α‐PC)/cholesterol, respectively) in terms of seal resistance, capacitance, surface area, specific capacitance, and membrane hydrophobic thickness; (ii) confocal microscopy imaging of phase separation in sphingomyelin/L‐α‐PC/cholesterol ternary membranes; (iii) electrical measurements of individual nanopores (α‐hemolysin, gramicidin); and (iv) indirect assessment of the alteration of membrane properties upon exposure to chemical stimuli using the natural nanopore gramicidin as a sensor.