The Effect of Temperature on the Proteome of Recombinant <i>Pichia pastoris</i>

Dragosits, Martin; Stadlmann, Johannes; Albiol, Joan; Baumann, Kristin; Maurer, Michael; Gasser, Brigitte; Sauer, Michael; Altmann, Friedrich; Ferrer, Pau; Mattanovich, Diethard

doi:10.1021/pr8007623

Cited by 179 publications

(196 citation statements)

References 52 publications

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“…In later purifications, Amb a 4 was identified via its N-terminal peptide KLCEKPSVTWSGK by tryptic digestion of 1% of each fraction and subsequent reversed phase-HPLC-ESI-MS as described (12). Pooled fractions were subjected to size exclusion chromatography (Sephacryl S100 HR, GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

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A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

Léonard

Wopfner

Pabst

et al. 2010

Journal of Biological Chemistry

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Art v 1, the major pollen allergen of the composite plant mugwort (Artemisia vulgaris) has been identified recently as a thionin-like protein with a bulky arabinogalactan-protein moiety. A close relative of mugwort, ragweed (Ambrosia artemisiifolia) is an important allergen source in North America, and, since 1990, ragweed has become a growing health concern in Europe as well. Weed pollen-sensitized patients demonstrated IgE reactivity to a ragweed pollen protein of apparently 29 -31 kDa. This reaction could be inhibited by the mugwort allergen Art v 1. The purified ragweed pollen protein consisted of a 57-amino acidlong defensin-like domain with high homology to Art v 1 and a C-terminal proline-rich domain. This part contained hydroxyproline-linked arabinogalactan chains with one galactose and 5 to 20 and more ␣-arabinofuranosyl residues with some ␤-arabinoses in terminal positions as revealed by high field NMR. The ragweed protein contained only small amounts of the single hydroxyproline-linked ␤-arabinosyl residues, which form an important IgE binding determinant in Art v 1. cDNA clones for this protein were obtained from ragweed flowers. Immunological characterization revealed that the recombinant ragweed protein reacted with >30% of the weed pollen allergic patients. Therefore, this protein from ragweed pollen constitutes a novel important ragweed allergen and has been designated Amb a 4.The pollen of common ragweed (Ambrosia artemisiifolia) is a major cause of hay fever and associated asthma in Northern America. During the past few decades, ragweed has started to spread in many parts of central Europe, where it has become a serious health problem in the sensitized population. Several initiatives have formed to prevent further spread in e.g. France, Austria or southern Germany. The Compositae (or Asteraceae) family is one of the largest families of flowering plants, but only a few are important allergenic sources. These include Ambrosia (ragweed), Artemisia (mugwort), Helianthus (sunflower), and Parthenium (feverfew). It was further demonstrated that sera of mugwort allergic patients show considerable cross-reactivity with ragweed pollen extracts (1, 2). IgE-binding to mugwort allergens in immunoblots was inhibited effectively by ragweed pollen extract (1), which indicates close homology of the essential allergens in ragweed and mugwort pollen. So far, six groups of allergens have been identified in ragweed pollen. Most patients were classified as ragweed allergic if they reacted with the pectate lyases of the Amb a 1/2 group (3, 4). The homologous pectate lyase Art v 6 in mugwort has been reported to play only a minor role in allergic disease. Amb a 6 (lipid transfer protein), Amb a 8 (profilin), Amb a 9 and Amb a 10 (both calcium-binding proteins) are small proteins belonging to the group of well known cross-reactive pan-allergens (1, 4 -8). Amb a 7 and the fragment Amb a 3 are plastocyanins and are described only as minor ragweed allergens (9).In mugwort pollen, the major allergen is Art v 1, a protein w...

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Section: Methodsmentioning

confidence: 99%

“…Internal peptide sequences were obtained from proteinase K peptides. The peptides were analyzed by reversed-phase HPLC coupled to an ESI source and a Q-TOF hybrid mass spectrometer operating in the MS/MS mode as described recently (12). C-terminal sequencing was attempted with carboxypeptidase Y (SigmaAldrich) in pyridine acetate buffer (22).…”

Section: Methodsmentioning

confidence: 99%

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

Léonard

Wopfner

Pabst

et al. 2010

Journal of Biological Chemistry

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“…Oxygen and carbon dioxide in the exiting gas were monitored during the whole process (BlueSense, Herten, Germany). Chemostat cultures were performed by adjusting the flow rate of chemostat medium [29] and the harvest rate to maintain a dilution rate D of 0.1/h after the end of the batch phase.…”

Section: Culture Conditionmentioning

confidence: 99%

Genome‐scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production

Sohn

Graf

Kim

et al. 2010

Biotechnology Journal

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114

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The methylotrophic yeast Pichia pastoris has gained much attention during the last decade as a platform for producing heterologous recombinant proteins of pharmaceutical importance, due to its ability to reproduce post-translational modification similar to higher eukaryotes. With the recent release of the full genome sequence for P. pastoris, in-depth study of its functions has become feasible. Here we present the first reconstruction of the genome-scale metabolic model of the eukaryote P. pastoris type strain DSMZ 70382, PpaMBEL1254, consisting of 1254 metabolic reactions and 1147 metabolites compartmentalized into eight different regions to represent organelles. Additionally, equations describing the production of two heterologous proteins, human serum albumin and human superoxide dismutase, were incorporated. The protein-producing model versions of PpaMBEL1254 were then analyzed to examine the impact on oxygen limitation on protein production.

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“…Anabolism is introduced through the pentose phosphate pathway and a general lumped biomass equation, according to which growth is assumed to depend exclusively on key biochemical precursors. Branch-point metabolites, such as NADH, NADPH, AcCoA, oxalacetate and pyruvate, are considered in compartmentalised cytosolic and mitochondrial pools [30]. …”

Section: Metabolic Network Reconstructionmentioning

confidence: 99%

“…The 40 scenarios under study show different uptake rates of the substrates glucose, glycerol and methanol (see Figure 2). Scenario A1 corresponds to a P. pastoris culture expressing the Fab fragment of the human anti-HIV antibody 3H6 [30]. Scenarios B1-B7 and C1-C2 correspond to cultures producing a lipase from Rhizopus oryzae (ROL) [31,32].…”

Section: P Pastoris Experimental Data Setmentioning

confidence: 99%

MCR-ALS on metabolic networks: Obtaining more meaningful pathways

Folch-Fortuny

Tortajada

Prats-Montalbán

et al. 2015

Chemometrics and Intelligent Laboratory Systems

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With the aim of understanding the flux distributions across a metabolic network, i.e. within living cells, Principal Component Analysis (PCA) has been proposed to obtain a set of orthogonal components (pathways) capturing most of the variance in the flux data. The problems with this method are (i) that no additional information can be included in the model, and (ii) that orthogonality imposes a hard constraint, not always reasonably. To overcome these drawbacks, here we propose to use a more flexible approach such as Multivariate Curve Resolution -Alternating Least Squares (MCR-ALS) to obtain this set of biological pathways through the network. By using this method, different constraints can be included in the model, and the same source of variability can be present in different pathways, which is reasonable from a biological standpoint. This work follows a methodology developed for Pichia pastoris cultures grown on different carbon sources, lately presented in [González Martínez et al. 2014].In this paper a different grey modelling approach, which aims to incorporate a priori knowledge through constraints on the modelling algorithms, is applied to the same case of study. The results of both models are compared to show their strengths and weaknesses.

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The Effect of Temperature on the Proteome of Recombinant Pichia pastoris

Cited by 179 publications

References 52 publications

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

A New Allergen from Ragweed (Ambrosia artemisiifolia) with Homology to Art v 1 from Mugwort

Genome‐scale metabolic model of methylotrophic yeast Pichia pastoris and its use for in silico analysis of heterologous protein production

MCR-ALS on metabolic networks: Obtaining more meaningful pathways

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