Strain specific properties of Escherichia coli can prevent non-canonical amino acid misincorporation caused by scale-related process heterogeneities.

Background: Escherichia coli is one of the most important hosts for production of recombinant proteins in biopharmaceutical industry. However, when selecting a suitable production strain, it is often not considered that a lot of different sub-species exist, which can differ in their genotypes and phenotypes. Another important development step is the scale-up of bioprocesses with the particular challenge that heterogeneities and gradients occur at production scale.

Determination of true ratios of different N-glycan structures in electrospray ionization mass spectrometry.

An ideal method for the analysis of N-glycans would both identify the isomeric structure and deliver a true picture of the relative, if not absolute, amounts of the various structures in one sample. Porous graphitic carbon chromatography coupled with electrospray ionization mass spectrometry (ESI-MS) detection has emerged as a method with a particularly high potential of resolving isomeric oligosaccharides, but little attention has so far been paid to quantitation of the results obtained. In this work, we isolated a range of structures from Man5 to complex type N-glycans with zero to four sialic acids and blended them into an equimolar "glyco tune mix".

The coordinated action of the MVB pathway and autophagy ensures cell survival during starvation.

The degradation and recycling of cellular components is essential for cell growth and survival. Here we show how selective and non-selective lysosomal protein degradation pathways cooperate to ensure cell survival upon nutrient limitation. A quantitative analysis of starvation-induced proteome remodeling in yeast reveals comprehensive changes already in the first three hours.

Inhibition of IgE binding to cross-reactive carbohydrate determinants enhances diagnostic selectivity.

Background: Allergy diagnosis by determination of allergen-specific IgE is complicated by clinically irrelevant IgE, of which the most prominent example is IgE against cross-reactive carbohydrate determinants (CCDs) that occur on allergens from plants and insects. Therefore, CCDs cause numerous false-positive results. Inhibition of CCDs has been proposed as a remedy, but has not yet found its way into the routine diagnostic laboratory.

Monitoring the production process of selenized yeast by elemental speciation analysis.

Elemental speciation analysis was implemented as an essential tool set addressing optimum fermentation conditions for the production of selenized yeast feed supplements. Accordingly, the study addressed intracellular levels of (1) total selenium and sulfur, (2) seleno methionine (SeMet), (3) cysteine (Cys) and methionine (Met) and (4) selenite and selenate. Dedicated sample preparation- and LC-ICP-MS methods were implemented and validated using the reference material Selm-1.

Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method.

Labeling of oligosaccharides with fluorescent dyes is the prerequisite for their sensitive analysis by high-performance liquid chromatography (HPLC). In this work, we present a fast new postlabeling cleanup procedure that requires no device other than the reaction vial itself. The procedure can be applied to essentially all labeling reagents.

Dissecting individual steps of nitrogen transcription factor cooperation in the Aspergillus nidulans nitrate cluster.

In the ascomycete fungus Aspergillus nidulans, the transcriptional activation of nitrate assimilating genes (niiA, niaD) depends on the cooperativity between a general nitrogen status-sensing regulator (the GATA factor AreA) and a pathway-specific activator (the Zn-cluster regulator NirA). Because nitrate assimilation leads to intracellular ammonium formation, it is difficult to determine the individual contributions of NirA and AreA in this complex activation/inactivation process. In an attempt to find a suitable marker for the nitrogen status sensed by AreA, we determined the intracellular free amino acid levels on different nitrogen growth conditions.

Molecular cloning and characterization of a plant alpha1,3/4-fucosidase based on sequence tags from almond fucosidase I.

Our work with almond peptide N-glycosidase A made us interested also in the alpha1,3/4-fucosidase which is used as a specific reagent for glycoconjugate analysis. The enzyme was purified to presumed homogeneity by a series of chromatographic steps including dye affinity and fast-performance anion exchange chromatography. The 63 kDa band was analyzed by tandem mass spectrometry which yielded several partial sequences.

Characterisation of peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A and its N-glycans.

Peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase A (PNGase A) was purified from almonds (Prunus amygdalus var. dulcis). Contrary to previous results in the literature, the enzyme appeared to be a heterodimer with subunits of 55 and 27 kDa when analysed by SDS/PAGE and two-dimensional electrophoresis.

Functional purification and characterization of a GDP-fucose: beta-N-acetylglucosamine (Fuc to Asn linked GlcNAc) alpha 1,3-fucosyltransferase from mung beans.

An alpha 1,3-fucosyltransferase was purified 3000-fold from mung bean seedlings by chromatography on DE 52 cellulose and Affigel Blue, by chromatofocusing, gelfiltration and affinity chromatography resulting in an apparently homogenous protein of about 65 kDa on SDS-PAGE. The enzyme transferred fucose from GDP-fucose to the Asn-linked N-acetylglucosaminyl residue of an N-glycan, forming an alpha 1,3-linkage. The enzyme acted upon N-glycopeptides and related oligosaccharides with the glycan structure GlcNAc2Man3 GlcNAc2.

Processing of asparagine-linked oligosaccharides in insect cells. N-acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells.

The levels of beta 1,2-N-acetylglucosaminyltransferase (GlcNAc-T) I and II activities in cultured cells from Bombyx mori (Bm-N), Mamestra brassicae (IZD-Mb-0503) and Spodoptera frugiperda (Sf-9 and Sf-21) were investigated. Apart from initial experiments with Man alpha-3(Man alpha 1-6)-Man beta 1-O(CH2)8COOH3 and 3H-labelled UDP-GlcNAc as substrates, GlcNAc-T I activity was measured with a non-radioactive HPLC method using pyridylaminated Man3-GlcNAc2 and Man5GlcNAc2 as acceptor oligosaccharides. It was shown by reversed-phase HPLC, exoglycosidase digestion and methylation analysis that the product obtained with Man3GlcNAc2 contained a terminal GlcNAc residue linked beta 1,2 to the alpha 1,3 arm of the acceptor.