The economics of inclusion body processing

Many recombinant proteins are often over-expressed in host cells, such as Escherichia coli, and are found as insoluble and inactive protein aggregates known as inclusion bodies (IBs). Recently, a novel process for IB extraction and solubilisation, based on chemical extraction, has been reported. While this method has the potential to radically intensify traditional IB processing, the process economics of the new technique have yet to be reported. This study focuses on the evaluation of process economics for several IB processing schemes based on chemical extraction and/or traditional techniques. Simulations and economic analysis were conducted at various processing conditions using granulocyte macrophage-colony stimulating factor, expressed as IBs in E. coli, as a model protein. In most cases, IB processing schemes based on chemical extraction having a shorter downstream cascade demonstrated a competitive economic edge over the conventional route, validating the new process as an economically more viable alternative for IB processing.

Relation between cell disruption conditions, cell debris particle size, and inclusion body release

The efficiency of physical separation of inclusion bodies from cell debris is related to cell debris size and inclusion body release and both factors should be taken into account when designing a process. In this work, cell disruption by enzymatic treatment with lysozyme and cellulase, by homogenization, and by homogenization with ammonia pretreatment is discussed. These disruption methods are compared on the basis of inclusion body release, operating costs, and cell debris particle size. The latter was measured with cumulative sedimentation analysis in combination with membrane-associated protein quantification by SDS-PAGE and a spectrophotometric pepticloglycan quantification method. Comparison of the results obtained with these two cell debris quantification methods shows that enzymatic treatment yields cell debris particles with varying chemical composition, while this is not the case with the other disruption methods that were investigated. Furthermore, the experiments show that ammonia pretreatment with homogenization increases inclusion body release compared to homogenization without pretreatment and that this pretreatment may be used to control the cell debris size to some extent. The enzymatic disruption process gives a higher product release than homogenization with or without ammonia pretreatment at lower operating costs, but it also yields a much smaller cell debris size than the other disruption process. This is unfavorable for centrifugal inclusion body purification in this case, where cell debris is the component going to the sediment and the inclusion body is the floating component. Nevertheless, calculations show that centrifugal separation of inclusion bodies from the enzymatically treated cells gives a high inclusion body yield and purity. (C) 2004 Wiley Periodicals, Inc.

La texture et la granulométrie de la moulée influencent les concentrations d’acide propionique et butyrique de même que la prévalence d’Escherichia coli dans le tractus gastro-intestinal du porc d’engraissement

Certaines stratégies alimentaires sont actuellement considérées pour remplacer l’usage des antimicrobiens dans les fermes porcines. Les objectifs de cette étude étaient d'évaluer l'effet de la granulométrie et de la texture des aliments sur les concentrations d'acides gras volatils intestinaux, la composition des populations pathogènes et commensales d’E. coli et sur les performances de croissance des porcs. Des porcs d'engraissement (n= 840) ont reçu l'une des six diètes suivantes: moulée texturée 500, 750 et 1250 µm et moulée cubée 500, 750 et 1250 µm. Le gain de poids a été mesuré à chaque changement de formulation de moulée. À l'abattoir, les contenus du caecum et du côlon de 165 porcs ont été échantillonnés pour le dénombrement des E. coli par PCR quantitatif (qPCR) et pour la quantification des AGV. Le gène yccT a été utilisé pour dénombrer les E. coli totaux. Une diminution du taux de conversion alimentaire a été associée avec la moulée cubée et/ou la moulée de 500 µm. Les concentrations d’acide propionique et butyrique, et ce tant au niveau du caecum que du côlon, étaient plus élevées chez les porcs recevant de la moulée texturée que chez ceux recevant de la moulée cubée. Du point de vue de la granulométrie, les concentrations caecales et du côlon d’acide butyrique étaient plus élevées chez les porcs alimentés avec de la moulée de 1250 µm que chez ceux recevant de la moulée de 500 µm. D'autre part, les niveaux intestinaux d’E. coli totaux étaient plus élevés pour les porcs nourris avec de la moulée cubée que pour ceux ayant reçu de la moulée texturée. Les résultats ont montré que la moulée texturée est associée à des performances de croissance plus faibles mais à des changements intestinaux favorables.

Analysis of tape tether survival in LEO against orbital debris

The low earth orbit (LEO) environment contains a large number of artificial debris, of which a significant portion is due to dead satellites and fragments of satellites resulted from explosions and in-orbit collisions. Deorbiting defunct satellites at the end of their life can be achieved by a successful operation of an Electrodynamic Tether (EDT) system. The effectiveness of an EDT greatly depends on the survivability of the tether, which can become debris itself if cut by debris particles; a tether can be completely cut by debris having some minimal diameter. The objective of this paper is to develop an accurate model using power laws for debris-size ranges, in both ORDEM2000 and MASTER2009 debris flux models, to calculate tape tether survivability. The analytical model, which depends on tape dimensions (width, thickness) and orbital parameters (inclinations, altitudes) is then verified with fully numerical results to compare for different orbit inclinations, altitudes and tape width for both ORDEM2000 and MASTER2009 flux data.

Bare-tape scaling laws for de-orbit missions in a space debris environment

El entorno espacial actual hay un gran numero de micro-meteoritos y basura espacial generada por el hombre, lo cual plantea un riesgo para la seguridad de las operaciones en el espacio. La situación se agrava continuamente a causa de las colisiones de basura espacial en órbita, y los nuevos lanzamientos de satélites. Una parte significativa de esta basura son satélites muertos, y fragmentos de satélites resultantes de explosiones y colisiones de objetos en órbita. La mitigación de este problema se ha convertido en un tema de preocupación prioritario para todas las instituciones que participan en operaciones espaciales. Entre las soluciones existentes, las amarras electrodinámicas (EDT) proporcionan un eficiente dispositivo para el rápido de-orbitado de los satélites en órbita terrestre baja (LEO), al final de su vida útil. El campo de investigación de las amarras electrodinámicas (EDT) ha sido muy fructífero desde los años 70. Gracias a estudios teóricos, y a misiones para la demostración del funcionamiento de las amarras en órbita, esta tecnología se ha desarrollado muy rápidamente en las últimas décadas. Durante este período de investigación, se han identificado y superado múltiples problemas técnicos de diversa índole. Gran parte del funcionamiento básico del sistema EDT depende de su capacidad de supervivencia ante los micro-meteoritos y la basura espacial. Una amarra puede ser cortada completamente por una partícula cuando ésta tiene un diámetro mínimo. En caso de corte debido al impacto de partículas, una amarra en sí misma, podría ser un riesgo para otros satélites en funcionamiento. Por desgracia, tras varias demostraciones en órbita, no se ha podido concluir que este problema sea importante para el funcionamiento del sistema. En esta tesis, se presenta un análisis teórico de la capacidad de supervivencia de las amarras en el espacio. Este estudio demuestra las ventajas de las amarras de sección rectangular (cinta), en cuanto a la probabilidad de supervivencia durante la misión, frente a las amarras convencionales (cables de sección circular). Debido a su particular geometría (longitud mucho mayor que la sección transversal), una amarra puede tener un riesgo relativamente alto de ser cortado por un único impacto con una partícula de pequeñas dimensiones. Un cálculo analítico de la tasa de impactos fatales para una amarra cilindrica y de tipo cinta de igual longitud y masa, considerando el flujo de partículas de basura espacial del modelo ORDEM2000 de la NASA, muestra mayor probabilidad de supervivencia para las cintas. Dicho análisis ha sido comparado con un cálculo numérico empleando los modelos de flujo el ORDEM2000 y el MASTER2005 de ESA. Además se muestra que, para igual tiempo en órbita, una cinta tiene una probabilidad de supervivencia un orden y medio de magnitud mayor que una amarra cilindrica con igual masa y longitud. Por otra parte, de-orbitar una cinta desde una cierta altitud, es mucho más rápido, debido a su mayor perímetro que le permite capturar más corriente. Este es un factor adicional que incrementa la probabilidad de supervivencia de la cinta, al estar menos tiempo expuesta a los posibles impactos de basura espacial. Por este motivo, se puede afirmar finalmente y en sentido práctico, que la capacidad de supervivencia de la cinta es bastante alta, en comparación con la de la amarra cilindrica. El segundo objetivo de este trabajo, consiste en la elaboración de un modelo analítico, mejorando la aproximación del flujo de ORDEM2000 y MASTER2009, que permite calcular con precisión, la tasa de impacto fatal al año para una cinta en un rango de altitudes e inclinaciones, en lugar de unas condiciones particulares. Se obtiene el numero de corte por un cierto tiempo en función de la geometría de la cinta y propiedades de la órbita. Para las mismas condiciones, el modelo analítico, se compara con los resultados obtenidos del análisis numérico. Este modelo escalable ha sido esencial para la optimización del diseño de la amarra para las misiones de de-orbitado de los satélites, variando la masa del satélite y la altitud inicial de la órbita. El modelo de supervivencia se ha utilizado para construir una función objetivo con el fin de optimizar el diseño de amarras. La función objectivo es el producto del cociente entre la masa de la amarra y la del satélite y el numero de corte por un cierto tiempo. Combinando el modelo de supervivencia con una ecuación dinámica de la amarra donde aparece la fuerza de Lorentz, se elimina el tiempo y se escribe la función objetivo como función de la geometría de la cinta y las propietades de la órbita. Este modelo de optimización, condujo al desarrollo de un software, que esta en proceso de registro por parte de la UPM. La etapa final de este estudio, consiste en la estimación del número de impactos fatales, en una cinta, utilizando por primera vez una ecuación de límite balístico experimental. Esta ecuación ha sido desarollada para cintas, y permite representar los efectos tanto de la velocidad de impacto como el ángulo de impacto. Los resultados obtenidos demuestran que la cinta es altamente resistente a los impactos de basura espacial, y para una cinta con una sección transversal definida, el número de impactos críticos debidos a partículas no rastreables es significativamente menor. ABSTRACT The current space environment, consisting of man-made debris and tiny meteoroids, poses a risk to safe operations in space, and the situation is continuously deteriorating due to in-orbit debris collisions and to new satellite launches. Among these debris a significant portion is due to dead satellites and fragments of satellites resulted from explosions and in-orbit collisions. Mitigation of space debris has become an issue of first concern for all the institutions involved in space operations. Bare electrodynamic tethers (EDT) can provide an efficient mechanism for rapid de-orbiting of defunct satellites from low Earth orbit (LEO) at end of life. The research on EDT has been a fruitful field since the 70’s. Thanks to both theoretical studies and in orbit demonstration missions, this technology has been developed very fast in the following decades. During this period, several technical issues were identified and overcome. The core functionality of EDT system greatly depends on their survivability to the micrometeoroids and orbital debris, and a tether can become itself a kind of debris for other operating satellites in case of cutoff due to particle impact; however, this very issue is still inconclusive and conflicting after having a number of space demonstrations. A tether can be completely cut by debris having some minimal diameter. This thesis presents a theoretical analysis of the survivability of tethers in space. The study demonstrates the advantages of tape tethers over conventional round wires particularly on the survivability during the mission. Because of its particular geometry (length very much larger than cross-sectional dimensions), a tether may have a relatively high risk of being severed by the single impact of small debris. As a first approach to the problem, survival probability has been compared for a round and a tape tether of equal mass and length. The rates of fatal impact of orbital debris on round and tape tether, evaluated with an analytical approximation to debris flux modeled by NASA’s ORDEM2000, shows much higher survival probability for tapes. A comparative numerical analysis using debris flux model ORDEM2000 and ESA’s MASTER2005 shows good agreement with the analytical result. It also shows that, for a given time in orbit, a tape has a probability of survival of about one and a half orders of magnitude higher than a round tether of equal mass and length. Because de-orbiting from a given altitude is much faster for the tape due to its larger perimeter, its probability of survival in a practical sense is quite high. As the next step, an analytical model derived in this work allows to calculate accurately the fatal impact rate per year for a tape tether. The model uses power laws for debris-size ranges, in both ORDEM2000 and MASTER2009 debris flux models, to calculate tape tether survivability at different LEO altitudes. The analytical model, which depends on tape dimensions (width, thickness) and orbital parameters (inclinations, altitudes) is then compared with fully numerical results for different orbit inclinations, altitudes and tape width for both ORDEM2000 and MASTER2009 flux data. This scalable model not only estimates the fatal impact count but has proved essential in optimizing tether design for satellite de-orbit missions varying satellite mass and initial orbital altitude and inclination. Within the frame of this dissertation, a simple analysis has been finally presented, showing the scalable property of tape tether, thanks to the survivability model developed, that allows analyze and compare de-orbit performance for a large range of satellite mass and orbit properties. The work explicitly shows the product of tether-to-satellite mass-ratio and fatal impact count as a function of tether geometry and orbital parameters. Combining the tether dynamic equation involving Lorentz drag with space debris impact survivability model, eliminates time from the expression. Hence the product, is independent of tether de-orbit history and just depends on mission constraints and tether length, width and thickness. This optimization model finally led to the development of a friendly software tool named BETsMA, currently in process of registration by UPM. For the final step, an estimation of fatal impact rate on a tape tether has been done, using for the first time an experimental ballistic limit equation that was derived for tapes and accounts for the effects of both the impact velocity and impact angle. It is shown that tape tethers are highly resistant to space debris impacts and considering a tape tether with a defined cross section, the number of critical events due to impact with non-trackable debris is always significantly low.

Heavy minerals, grain size distribution, and ice-rafted debris in the Greenland Sea during 44 to 30 Ma

The widely accepted age estimate for the onset of glaciation in the Northern Hemisphere ranges between 2 and 15 million years ago (Ma). However, recent studies indicate the date for glacial onset may be significantly older. We report the presence of ice-rafted debris (IRD) in ~44 to 30 Ma sediments from the Greenland Sea, evidence for glaciation in the North Atlantic during the Middle Eocene to Early Oligocene. Detailed sedimentological evidence indicates that glaciers extended to sea level in the region, allowing icebergs to be produced. IRD may have been sourced from tidewater glaciers, small ice caps, and/or a continental ice sheet.

(Table 1) Grain size, sedimentation rates and accumulation rates of ODP Hole 152-919A coarse-sand ice-rafted debris

At mid- to high-latitude marine sites, ice-rafted debris (IRD) is commonly recognized as anomalously coarse-grained terrigenous material contained within a fine-grained hemipelagic or pelagic matrix (e.g., Conolly and Ewing, 1970; Ruddiman, 1977, doi:10.1130/0016-7606(1977)88<1813:LQDOIS>2.0.CO;2; Krissek, 1989, doi:10.2973/odp.proc.sr.104.114.1989; Jansen et al., 1990; Bond et al., doi:10.1038/360245a0, 1992; Krissek, 1995, doi:10.2973/odp.proc.sr.145.118.1995). The presence of such ice-rafted material is a valuable indicator of the presence of glacial ice at sea level on an adjacent continent, whereas the composition of the IRD can often be used to identify the location of the source area (e.g., Goldschmidt, 1995, doi:10.1016/0025-3227(95)00098-J). Because the amount of core recovered during Leg 163 was very limited, this shore-based, postcruise study focuses on materials recovered at a nearby site during Leg 152. In particular, this study examines sediments recovered at Site 919; these sediments were described as containing a significant ice-rafted component in the Leg 152 Initial Reports volume (Larsen, Saunders, Clift, et al., 1994, doi:10.2973/odp.proc.ir.152.1994). In this study, the sedimentary section from Site 919 has been examined with the goal of providing a detailed history of glaciations on Greenland and other landmasses adjacent to the Norwegian-Greenland Sea; this history ultimately will be calibrated using an oxygen isotope stratigraphy (Flower, 1998, doi:10.2973/odp.proc.sr.152.219.1998), although that calibration has not been completed at this time. Because ice-core studies of the Greenland Ice Sheet (GIS) have shown that the GIS changed dramatically, and in some cases extremely rapidly, during at least the last interglacial stage (GRIP Members, 1993, doi:10.1038/364203a0), a detailed IRD record from the Southeast Greenland margin should provide insight into the longer term behavior of this sensitive component of the Northern Hemisphere climate system.

Grain size distributions of Magneli phases and metallic titanium in chondritic porous interplanetary dust particles

A mineralogical survey of chondritic interplanetary dust particles (IDPs)showed that these micrometeorites differ significantly in form and texture from components of carbonaceous chondrites and contain some mineral assemblages which do not occur in any meteorite class1. Models of chondritic IDP mineral evolution generally ignore the typical (ultra-) fine grain size of consituent minerals which range between 0.002-0.1µm in size2. The chondritic porous (CP) subset of chondritic IDPs is probably debris from short period comets although evidence for a cometary origin is still circumstantial3. If CP IDPs represent dust from regions of the Solar System in which comet accretion occurred, it can be argued that pervasive mineralogical evolution of IDP dust has been arrested due to cryogenic storage in comet nuclei. Thus, preservation in CP IDPs of "unusual meteorite minerals", such as oxides of tin, bismuth and titanium4, should not be dismissed casually. These minerals may contain specific information about processes that occurred in regions of the solar nebula, and early Solar System, which spawned the IDP parent bodies such as comets and C, P and D asteroids6. It is not fully appreciated that the apparent disparity between the mineralogy of CP IDPs and carbonaceous chondrite matrix may also be caused by the choice of electron-beam techniques with different analytical resolution. For example, Mg-Si-Fe distributions of Cl matrix obtained by "defocussed beam" microprobe analyses are displaced towards lower Fe-values when using analytical electron microscope (AEM)data which resolve individual mineral grains of various layer silicates and magnetite in the same matrix6,7. In general, "unusual meteorite minerals" in chondritic IDPs, such as metallic titanium, Tin01-n(Magneli phases) and anatase8 add to the mineral data base of fine-grained Solar System materials and provide constraints on processes that occurred in the early Solar System.

Expression of the extracellular domain of the human heat-stable enterotoxin receptor in Escherichia coli and generation of neutralizing antibodies

The entire extracellular domain of the human heat-stable enterotoxin (ST) receptor as well as a truncated N-terminal domain were cloned as glutathione S-transferase fusion proteins and expressed in Escherichia coli. The recombinant fusion proteins were purified from both the cytosol and the inclusion body fractions by selective detergent extraction followed by glutathione-agarose affinity chromatography. The purified protein, corresponding to the entire extracellular domain, bound the stable toxin peptide with an affinity comparable to that of the native receptor characterized from the human colonic T84 cell line. No binding was observed with the N-terminal truncated fragment of the receptor under similar conditions, Polyclonal antibodies were raised to the entire extracellular domain fusion protein as well as the truncated extracellular domain fusion protein, and the antibodies were purified by affinity chromatography. Addition of the purified antibodies to T84 cells inhibited ST binding and abolished ST-mediated cGMP production, indicating that critical epitopes involved in ligand interaction are present in the N-terminal fragment of the receptor, Purified antibodies recognized a single protein of M(r) 160,000 Da on Western blotting with T84 membranes, corresponding to a size of the native glycosylated receptor in T84 cells. These studies are the first report of the expression, purification, and characterization of any member of the guanylyl cyclase family of receptors in E. coli and show that binding of the toxin to the extracellular domain of the receptor is possible in the absence of any posttranslational modifications such as glycosylation. The recombinant fusion proteins as well as the antibodies that we have generated could serve as useful tools in the identification of critical residues of the extracellular domain involved in ligand interaction.

Specific polyadenylation and purification of total messenger RNA from Escherichia coli

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages, We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese, The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA, Withdrawal of Mg2+ from the polyadenylation reaction rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes, Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species, Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis, Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.

Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.