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.

The effect of lignin and inorganic species in biomass on pyrolysis oil yields, quality and stability

This paper investigates four reference fuels and three low lignin Lolium Festuca grasses which were subjected to pyrolysis to produce pyrolysis oils. The oils were analysed to determine their quality and stability, enabling the identification of feedstock traits which affect oil stability. Two washed feedstocks were also subjected to pyrolysis to investigate whether washing can enhance pyrolysis oil quality. It was found that the mineral matter had the dominate effect on pyrolysis in compared to lignin content, in terms of pyrolysis yields for organics, char and gases. However the higher molecular weight compounds present in the pyrolysis oil are due to the lignin derived compounds as determined by results of GPC and liquid-GC/MS. The light organic fraction also increased in yield, but reduced in water content as metals increased at the expense of the lignin content. It was found that the fresh oil and aged oil had different compound intensities/concentrations, which is due to a large number of reactions occurring when the oil is aged day by day. These findings agree with previous reports which suggest that a large amount of re-polymerisation occurs as levoglucosan yields increase during the aging progress, while hydroxyacetaldehyde decrease. In summary the paper reports a window for producing a more stable pyrolysis oil by the use of energy crops, and also show that washing of biomass can improve oil quality and stability for high ash feedstocks, but less so for the energy crops.

The implementation of a design of experiments strategy to increase recombinant protein yields in yeast (review)

Biological processes are subject to the influence of numerous factors and their interactions, which may be non-linear in nature. In a recombinant protein production experiment, understanding the relative importance of these factors, and their influence on the yield and quality of the recombinant protein being produced, is an essential part of its optimisation. In many cases, implementing a design of experiments (DoE) approach has delivered this understanding. This chapter aims to provide the reader with useful pointers in applying a DoE strategy to improve the yields of recombinant yeast cultures.

The effect of antifoam addition on protein production yields

Pichia pastoris is a widely used host for recombinant protein production. The foaming associated with culturing it on a large scale is commonly prevented by the addition of chemical antifoaming agents or "antifoams." Unexpectedly, the addition of a range of antifoams to both shake flask and bioreactor cultures of P. pastoris has been shown to alter the total yield of the recombinant protein being produced. Possible explanations for this are that the presence of the antifoam increases the total amount of protein being produced and secreted per cell or that it increases the density of the culture. Antifoaming agents may therefore have specific effects on the growth and yield characteristics of recombinant cultures, in addition to their primary action as de-foamers.

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.

Identification of the HLA-DM/HLA-DR interface

Human leukocyte antigen (HLA)-DM is a critical participant in antigen presentation that catalyzes the dissociation of the Class II-associated Invariant chain-derived Peptide (CLIP) from the major histocompatibility complex (MHC) Class II molecules. There is competition amongst peptides for access to an MHC Class II groove and it has been hypothesised that DM functions as a 'peptide editor' that catalyzes the replacement of one peptide for another within the groove. It is established that the DM catalyst interacts directly with the MHC Class II but the precise location of the interface is unknown. Here, we combine previously described mutational data with molecular docking and energy minimisation simulations to identify a putative interaction site of >4000A2 which agrees with known point mutational data for both the DR and DM molecule. The docked structure is validated by comparison with experimental data and previously determined properties of protein-protein interfaces. A possible dissociation mechanism is suggested by the presence of an acidic cluster near the N terminus of the bound peptide.

Watermark-only security attack on DM-QIM watermarking:vulnerability to guided key guessing

This paper addresses the security of a specific class of common watermarking methods based on Dither modulation-quantisation index modulation (DM-QIM) and focusing on watermark-only attacks (WOA). The vulnerabilities of and probable attacks on lattice structure based watermark embedding methods have been presented in the literature. DM-QIM is one of the best known lattice structure based watermarking techniques. In this paper, the authors discuss a watermark-only attack scenario (the attacker has access to a single watermarked content only). In the literature it is an assumption that DM-QIM methods are secure to WOA. However, the authors show that the DM-QIM based embedding method is vulnerable against a guided key guessing attack by exploiting subtle statistical regularities in the feature space embeddings for time series and images. Using a distribution-free algorithm, this paper presents an analysis of the attack and numerical results for multiple examples of image and time series data.

Watermark-only security attack on DM-QIM watermarking:vulnerability to guided key guessing

This paper addresses the security of a specific class of common watermarking methods based on Dither modulation-quantisation index modulation (DM-QIM) and focusing on watermark-only attacks (WOA). The vulnerabilities of and probable attacks on lattice structure based watermark embedding methods have been presented in the literature. DM-QIM is one of the best known lattice structure based watermarking techniques. In this paper, the authors discuss a watermark-only attack scenario (the attacker has access to a single watermarked content only). In the literature it is an assumption that DM-QIM methods are secure to WOA. However, the authors show that the DM-QIM based embedding method is vulnerable against a guided key guessing attack by exploiting subtle statistical regularities in the feature space embeddings for time series and images. Using a distribution-free algorithm, this paper presents an analysis of the attack and numerical results for multiple examples of image and time series data.

The implementation of a design of experiments strategy to increase recombinant protein yields in yeast (review)

Biological processes are subject to the influence of numerous factors and their interactions, which may be non-linear in nature. In a recombinant protein production experiment, understanding the relative importance of these factors, and their influence on the yield and quality of the recombinant protein being produced, is an essential part of its optimisation. In many cases, implementing a design of experiments (DoE) approach has delivered this understanding. This chapter aims to provide the reader with useful pointers in applying a DoE strategy to improve the yields of recombinant yeast cultures.

THOMAS NIEHR AND KARIN BÖKE (eds), Einwanderungsdiskurse. Vergleichende diskurslinguistische Studien. [Immigration Discourses: Comparative Discourse Studies.] Opladen: Westdeutscher Verlag, 2000. 241 pp. DM 56.00 (pbk). ISBN 3–5311–3307–1

Book review

Pyrolysis of Napier grass in a fixed bed reactor:effect of operating conditions on product yields and characteristic

This study presents a report on pyrolysis of Napier grass stem in a fixed bed reactor. The effects of nitrogen flow (20 to 60 mL/min), and reaction temperature (450 to 650 degrees C) were investigated. Increasing the nitrogen flow from 20 to 30 mL/min increased the bio-oil yield and decreased both bio-char and non-condensable gas. 30 mL/min nitrogen flow resulted in optimum bio-oil yield and was used in the subsequent experiments. Reaction temperatures between 450 and 600 degrees C increased the bio-oil yield, with maximum yield of 32.26 wt% at 600 degrees C and a decrease in the corresponding bio-char and non-condensable gas. At 650 degrees C, reductions in the bio-oil and bio-char yields were recorded while the non-condensable gas increased. Water content of the bio-oil decreased with increasing reaction temperature, while density and viscosity increased. The observed pH and higher heating values were between 2.43 to 2.97, and 25.25 to 28.88 MJ/kg, respectively. GC-MS analysis revealed that the oil was made up of highly oxygenated compounds and requires upgrading. The bio-char and non-condensable gas were characterized, and the effect of reaction temperature on the properties was evaluated. Napier grass represents a good source of renewable energy when all pyrolysis products are efficiently utilized.