Radiolarian assemblages of ODP Leg 117 sites

Significant numbers of radiolarians ranging in age from late middle Miocene to Recent were recovered from six sites drilled on the Oman margin and Owen Ridge. Sparse faunas were recovered from five additional sites on the Oman margin and one site on the Indus Fan. Detailed range charts and biozonations are presented for most sites. The radiolarian assemblages are peculiar in that numerous common tropical forms, some of which are biomarkers, are absent or very rare. In addition, some species not usually found in tropical assemblages are present. These forms, indicative of up welling conditions, fall into three categories: (1) endemic upwelling: species endemic to upwelling and not previously described from the Indian Ocean; (2) displaced temperate: temperate forms not usually found in tropical waters; and (3) enhanced tropical: tropical forms which are more abundant and/or robust in areas of upwelling. Comparison of the Oman margin/Owen Ridge fauna with that recovered from the Peru margin upwelling area (ODP Leg 112) suggests that the assemblage may be globally diagnostic of upwelling conditions. The onset of upwelling is marked by the appearance of siliceous biota at about 11.9 Ma, and there is some indication of a decrease in the strength of the upwelling signal at about 9.6 Ma. A strong pulse in, or strengthening of, the upwelling mechanism is indicated by a marked fauna change at 4.7 Ma. There is a weaker signal, implying a change in upwelling conditions, at about 1.5 Ma.

Composition of benthic taxa collected during POLARSTERN cruise ANT-XXI/5 (BENDEX) at Bouvet Island and Spiess Seamound

Bouvet (Bouvetøya) is a geologically young and very remote island just south of the Polar Front. Here we report samples taken during the RV "Polarstern" cruise ANTXXI/2 on 3 days in November 2003 and January 2004. This work was part of SCAR's EASIZ programme and intended, by providing data on the marine fauna of this "white gap" in the Atlantic sector of the Southern Ocean, to contribute to identifying the role of Bouvet in the faunal exchange between the Sub- and high Antarctic. While this goal demands extensive molecular analysis of the material sampled (future work), a checklist of the samples and data at hand widens the faunal and environmental inventory substantially. We suggest some preliminary conclusions on the relationship of Bouvet Island's fauna with that of other regions, such as Magellanic South America, the Antarctic Peninsula, and the high Antarctic Weddell Sea, which have been sampled previously. There seem to be different connections for individual higher taxa rather than a generally valid consistent picture.

[al-Kawākib al-durrīyah fī madḥ khayr al-barīyah, 16th century?].

Fine copy of al-Būṣīrī's poem in praise of the Prophet accompanied by elucidation in Persian and Turkish.

Gazophylacium Divinae dilectionis : petra pretiosior coeli,soli et sali. scilicet, Sanctus Petrus princeps apostolorum apposite, ac dilucide elucidatus, ac eximium exemplum Boni Pastoris Christi Domini ... cum indice tripartito /

Mode of access: Internet.

Midhat Paşa.

Mode of access: Internet.

Stellula catholicae orthodoxaeque fidei, ad Septemtrionem tenebricosis Iacobi Reneccii Pastoris Hamburgensis problematibus opposita.

Mode of access: Internet.

De versionibus Pastoris Hermae latinis

Diss.

Structural characterization of recombinant human CD81 produced in Pichia pastoris

Human CD81 (hCD81) protein has been recombinantly produced in the methylotrophic yeast Pichia pastoris. The purified protein, produced at a yield of 1.75 mg/L of culture, was shown to interact with Hepatitis C virus E2 glycoprotein. Immunofluorescent and flow cytometric staining of P. pastoris protoplasts with monoclonal antibodies specific for the second extracellular loop (EC2) of hCD81 confirmed the antigenicity of the recombinant molecule. Full-length hCD81 was solubilized with an array of detergents and subsequently characterized using circular dichroism (CD) and analytical ultracentrifugation. These biophysical techniques confirmed that the protein solution comprises a homogenous species possessing a highly-defined alpha-helical secondary structure. The predicted alpha-helical content of the protein from CD analysis (77.1%) fits remarkably well with what would be expected (75.2%) from knowledge of the protein sequence together with the data from the crystal structure of the second extracellular loop. This study represents the first biophysical characterization of a full-length recombinant tetraspanin, and opens the way for structure-activity analyses of this ubiquitous family of transmembrane proteins.

Developing a scalable model of recombinant protein yield from Pichia pastoris:the influence of culture conditions, biomass and induction regime

Background The optimisation and scale-up of process conditions leading to high yields of recombinant proteins is an enduring bottleneck in the post-genomic sciences. Typical experiments rely on varying selected parameters through repeated rounds of trial-and-error optimisation. To rationalise this, several groups have recently adopted the 'design of experiments' (DoE) approach frequently used in industry. Studies have focused on parameters such as medium composition, nutrient feed rates and induction of expression in shake flasks or bioreactors, as well as oxygen transfer rates in micro-well plates. In this study we wanted to generate a predictive model that described small-scale screens and to test its scalability to bioreactors. Results Here we demonstrate how the use of a DoE approach in a multi-well mini-bioreactor permitted the rapid establishment of high yielding production phase conditions that could be transferred to a 7 L bioreactor. Using green fluorescent protein secreted from Pichia pastoris, we derived a predictive model of protein yield as a function of the three most commonly-varied process parameters: temperature, pH and the percentage of dissolved oxygen in the culture medium. Importantly, when yield was normalised to culture volume and density, the model was scalable from mL to L working volumes. By increasing pre-induction biomass accumulation, model-predicted yields were further improved. Yield improvement was most significant, however, on varying the fed-batch induction regime to minimise methanol accumulation so that the productivity of the culture increased throughout the whole induction period. These findings suggest the importance of matching the rate of protein production with the host metabolism. Conclusion We demonstrate how a rational, stepwise approach to recombinant protein production screens can reduce process development time.

Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris

We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1. © 2006 Elsevier Inc. All rights reserved.

Which yeast species shall I choose? Saccharomyces cerevisiae versus Pichia pastoris (review)

Having decided on yeast as a production host, the choice of species is often the first question any researcher new to the field will ask. With over 500 known species of yeast to date, this could pose a significant challenge. However, in reality, only very few species of yeast have been employed as host organisms for the production of recombinant proteins. The two most widely used, Saccharomyces cerevisiae and Pichia pastoris, are compared and contrasted here.

Improving recombinant eukaryotic membrane protein yields in Pichia pastoris:the importance of codon optimization and clone selection

In the last 15 years, 80% of all recombinant proteins reported in the literature were produced in the bacterium, Escherichia coli, or the yeast, Pichia pastoris. Nonetheless, developing effective general strategies for producing recombinant eukaryotic membrane proteins in these organisms remains a particular challenge. Using a validated screening procedure together with accurate yield quantitation, we therefore wished to establish the critical steps contributing to high yields of recombinant eukaryotic membrane protein in P. pastoris. Whilst the use of fusion partners to generate chimeric constructs and directed mutagenesis have previously been shown to be effective in bacterial hosts, we conclude that this approach is not transferable to yeast. Rather, codon optimization and the preparation and selection of high-yielding P. pastoris clones are effective strategies for maximizing yields of human aquaporins.

Antifoam addition to shake flask cultures of recombinant Pichia pastoris increases yield

Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.