Methods for expression of recombinant proteins using a Pichia pastoris cell-free system

Cell‐free protein synthesis is a powerful tool for engineering biology and has been utilized in many diverse applications, from biosensing and protein prototyping to biomanufacturing and the design of metabolic pathways. By exploiting host cellular machinery decoupled from cellular growth, proteins can be produced in vitro both on demand and rapidly. Eukaryotic cell‐free platforms are often neglected due to perceived complexity and low yields relative to their prokaryotic counterparts, despite providing a number of advantageous properties. The yeast Pichia pastoris (also known as Komagataella phaffii) is a particularly attractive eukaryotic host from which to generate cell‐free extracts, due to its ability to grow to high cell densities with high volumetric productivity, genetic tractability for strain engineering, and ability to perform post‐translational modifications. Here, we describe methods for conducting cell‐free protein synthesis using P. pastoris as the host, from preparing the cell lysates to protocols for both coupled and linked transcription‐translation reactions. By providing these methodologies, we hope to encourage the adoption of the platform by new and experienced users alike

Desinfestação de explantes radiculares de bacurizeiro.

Este trabalho teve como objetivo avaliar a desinfestação de explantes radiculares de bacurizeiro para o seu estabelecimento in vitro

Overview of NASA FINESSE (Field Investigations to Enable Solar System Science and Exploration) Science and Exploration Results

NASA's FINESSE (Field Investigations to Enable Solar System Science and Exploration) project is focused on a science and exploration field-based research program to generate strategic knowledge in preparation for human and robotic exploration of other planetary bodies including our moon, Mars' moons Phobos and Deimos, and near-Earth asteroids. Scientific study focuses on planetary volcanism (e.g., the formation of volcanoes, evolution of magma chambers and the formation of multiple lava flow types, as well as the evolution and entrapment of volatile chemicals) and impact cratering (impact rock modification, cratering mechanics, and the chronologic record). FINESSE conducts multiple terrestrial field campaigns (Craters of the Moon National Monument and Preserve in Idaho for volcanics, and West Clearwater Impact Structure in Canada for impact studies) to study such features as analogs relevant to our moon, Phobos, Deimos, and asteroids. Here we present the science and exploration results from two deployments to Idaho (2014, 2015) and our first deployment to Canada (2014). FINESSE was selected as a research team by NASA's Solar System Exploration Research Virtual Institute (SSERVI). SSERVI is a joint effort by NASA's Science Mission Directorate (SMD) and Human Exploration and Operations Mission Directorate (HEOMD)

Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform

Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT® SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354–359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two 15N-, 13C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline

Direct 2D spatial coherence determination using the Fourier analysis method Multi parameter characterization of the P04 beamline at PETRA III

We present a systematic 2D spatial-coherence analysis of the soft-X-ray beamline P04 at PETRA III for various beamline configurations. The influence of two different beam-defining apertures on the spatial coherence properties of the beam is discussed and optimal conditions for coherence-based experiments are found. A significant degradation of the spatial coherence in the vertical direction has been measured and sources of this degradation are identified and discussed. The Fourier-analysis method, which gives fast and simple access to the 2D spatial coherence function of the X-ray beam, is used for the experiment. Here, we exploit the charge scattering of a disordered nanodot sample allowing the use of arbitrary X-ray photon energies with this method

Megahertz-rate ultrafast X-ray scattering and holographic imaging at the European XFEL

The advent of X-ray free-electron lasers (XFELs) has revolutionized fundamental science, from atomic to condensed matter physics, from chemistry to biology, giving researchers access to X-rays with unprecedented brightness, coherence and pulse duration. All XFEL facilities built until recently provided X-ray pulses at a relatively low repetition rate, with limited data statistics. Here, results from the first megahertz-repetition-rate X-ray scattering experiments at the Spectroscopy and Coherent Scattering (SCS) instrument of the European XFEL are presented. The experimental capabilities that the SCS instrument offers, resulting from the operation at megahertz repetition rates and the availability of the novel DSSC 2D imaging detector, are illustrated. Time-resolved magnetic X-ray scattering and holographic imaging experiments in solid state samples were chosen as representative, providing an ideal test-bed for operation at megahertz rates. Our results are relevant and applicable to any other non-destructive XFEL experiments in the soft X-ray range

Department of Animal Sciences research and reviews: beef and sheep

Relationship of a PCR-SSCP at the Bovine calpastatin locus with calpastatin activity and meat tenderness / H. Y. Chung, M. E. Davis, H. C. Hines, and D. M. Wulf -- Effects of calpain proteolysis and calpain genotypes on meat tenderness of angus bulls / H. Y. Chung, M. E. Davis, H. C. Hines, and D. M. Wulf -- Identification of genetic markers for growth and carcass traits in beef cattle / W. Ge, M. E. Davis, H. C. Hines, and K. M. Irvin -- Relationships of polymorphisms in the bovine leptin gene with differences in beef carcass traits / K. Tessanne, H. C. Hines, and M. E. Davis -- Effects of rate of gain during periods of restricted intake on performance and carcass characteristics in steers fed to achieve step-wise increases in rate of gain / J. E. Rossi and S. C. Loerch -- Effects of feeding regimen and days fed on performance and carcass characteristics of feedlot steers / J. E. Rossi, S. C. Loerch, S. J. Moeller, and J. P. Schoonmaker -- Effect of an accelerated finishing program on performance and carcass characteristics of early weaned bulls and steers / J. P. Schoonmaker, S. C. Loerch, F. L. Fluharty, T. B. Turner, S. J. Moeller and J. E. Rossi -- Yeast-mineral mixes and beef-cattle performance in fescue-based grazing systems: preliminary report / S. Boyles, W. Shriver, and D. Kobs -- Forage and animal evaluation of heifers at Indian Lake Hydrologic Unit CRP stocker grazing demonstration / S. L. Boyles, B. W. Stoll, and T. L. Dobbels -- Beef quality is every cattleman's business: education program / J. Yates and S. Boyles -- Effects of pelleted alfalfa and whole-shelled corn combinations on lamb growth and carcass characteristics / F. L. Fluharty -- Effects of feeding pelleted, ensiled, or a combination of pelleted and ensiled alfalfa on lamb growth and carcass characteristics / F. L. Fluharty, G. D. Lowe, and D. D. Clevenger -- Effects of corn silage vs. alfalfa haylage on lamb growth and carcass characteristics in forage-based finishing systems / F. L. Fluharty, G. D. Lowe, and D. D. Clevenger -- Effects of feed-delivery system and corn processing on lamb growth and carcass characteristics / F. L. Fluharty, G. D. Lowe, and D. D. Clevenger -- Effects of pen floor type and bedding on lamb growth and carcass characteristics / F. L. Fluharty, G. D. Lowe, and D. D. Clevenger -- A PCR-SSCP polymorphism detected in the 5' flanking region of the ovine IGF-I gene / A. Yilmaz, M. E. Davis, and H. C. Hine