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. These in turn can affect the production organism and can have negative impact on the process and the product quality. Therefore, researchers developed scale-down reactors, which are used to mimic manufacturing conditions in laboratory scale. The main objectives of this study were to determine the extent to which scale-related process inhomogeneities affect the misincorporation of non-canonical amino acids into the recombinant target protein, which is an important quality attribute, and whether strain specific properties may have an impact. RESULTS:We investigated two industrially relevant E. coli strains, BL21(DE3) and HMS174(DE3), which produced an antigen binding fragment (Fab). The cells were cultivated in high cell density fed-batch mode at laboratory scale and under scale-down conditions. We demonstrated that the two host strains differ significantly with respect to norleucine misincorporation into the target protein, especially under heterogeneous cultivation conditions in the scale-down reactor. No norleucine misincorporation was observed in E. coli BL21(DE3) for either cultivation condition. In contrast, norleucine incorporation into HMS174(DE3) was already detectable in the reference process and increased dramatically in scale-down experiments. Norleucine incorporation was not random and certain positions were preferred over others, even though only a single codon exists. Differences in biomass and Fab production between the strains during scale-down cultivations could be observed as well. CONCLUSIONS:This study has shown that E. coli BL21(DE3) is much more robust to scale-up effects in terms of norleucine misincorporation than the K12 strain tested. In this respect, BL21(DE3) enables better transferability of results at different scales, simplifies process implementation at production scale, and helps to meet regulatory quality guidelines defined for biopharmaceutical manufacturing.

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”. When subjected to liquid chromatography–ESI-MS in positive and negative modes, the glyco tune mix clearly demonstrated the futility of quantitation of N-glycans of different overall composition, different number of sialic acids, and strongly differing size without compensation for their very different molar responses. Relative quantitation of human plasma N-glycans was performed with correction factors deduced from this external glyco tune mix. Addition of just one isotope-coded internal standard with enzymatically added 13C-galactose led to absolute quantification in the same experiment.

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. In this period, many different integral plasma membrane proteins undergo endocytosis and degradation in vacuoles via the multivesicular body (MVB) pathway. Their degradation becomes essential to maintain critical amino acids levels that uphold protein synthesis early during starvation. This promotes cellular adaptation, including the de novo synthesis of vacuolar hydrolases to boost the vacuolar catabolic activity. This order of events primes vacuoles for the efficient degradation of bulk cytoplasm via autophagy. Hence, a catabolic cascade including the coordinated action of the MVB pathway and autophagy is essential to enter quiescence to survive extended periods of nutrient limitation.

For the first time, quantitative analysis of the proteinogenic sulfur containing amino acids methionine and cysteine was performed by LC-ICP-MS. A dedicated sample preparation procedure was implemented consisting of two steps, i.e. (1) protection of the redox sensitive amino acids by controlled oxidation of methionine, cystine and cysteine to methionine sulfone and cysteic acid, respectively, and (2) subsequent protein hydrolysis. Anion exchange chromatography enabled the separation of all relevant sulfur species within 10 min. Sulfur was detected on m/z32S16O using O2 as the reaction gas. Absolute limits of detection in the pmol range were achieved for methionine sulfone and cysteic acid. The method offered the possibility of protein quantification. Absolute amounts of 2 μg of hydrolyzed protein (on column) were investigated by LC-ICP-MS. Both oxidized forms of amino acids showed excellent recoveries from lysozyme and myoglobin standards, enabling accurate quantification. A repeatability of <10% (n = 6 independently prepared samples) was found without the application of isotope dilution strategies. The limits of detection of <1 μM protein were comparable to the limits of detection achieved by spectroscopy based protein quantification assays. Moreover, the validity of the approach was shown by implementing HPLC in combination with fluorescence detection as a reference method for the quantification of proteinogenic amino acids in yeast. Both methods were in good agreement and met the theoretical value in a yeast reference material certified for the methionine content.

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. Excellent repeatability precisions <10% (n = 4 biological replicates) could be obtained for all parameters. The study comprised fermentation monitoring over 72 hours (6 different time points) for a Saccharomyces cerevisiae strain under different selenite feed conditions. It was observed that for this strain an increase in the selenium concentration in the fermentation feed by 50% did not result in enhanced selenium accumulation. Fermentation monitoring of three different Saccharomyces cerevisiae strains under the same conditions showed strain specific selenium uptake after 72 hours. The strain with the lowest cell viability of 60% showed the lowest SeMet content. After 47 h of fermentation, all strains reached a critical point, at which seleno methionine accounted for approximately 100% of the total selenium and cell viability started to decrease. This could be explained by sulfur limitation and/or excess of the seleno methionine storage capacity. Strains showing cell viability of approx. 90% after 72 hours of fermentation revealed SeMet concentrations up to 3000 μg g(-1). In the final product, an apparent threshold level for Met/SeMet of approx. 1 was observed for all strains.

SummaryIn 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. We show that the amount of glutamine (Gln) inversely correlates with all known AreA activities. We find that AreA mediates chromatin remodelling by increasing histone H3 acetylation, a process triggered by transcriptional activation and, independently of transcription, by nitrogen starvation. NirA also participates in the chromatin opening process during nitrate induction but its function is not related to histone acetylation. This chromatin remodelling function of NirA is dispensable only in nitrogen‐starved cells, conditions that lead to elevated AreA chromatin occupancy and histone H3 hyperacetylation. Continuous nitrate assimilation leads to self‐nitrogen metabolite repression but nitrate‐activated NirA is partially compensating for lowered AreA activities under these conditions.

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. We also compare the performance of 15 different labels for N-glycan analysis in various analytical procedures. We took special care to prevent obscuring influences from incomplete derivatization and signal quenching by impurities. Procainamide emerged as more sensitive than anthranilic acid for normal-phase HPLC, but its chromatographic performance was not convincing. 2-aminopyridine was the label with the lowest retention on reversed-phase and graphitic carbon columns and, thus, appears to be most suitable for glycan fractionation by multidimensional HPLC. Most glycan derivatives performed better than native sugars in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray ionization-MS (ESI-MS), but the gain was small and hardly sufficient to compensate for sample loss during preparation.

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 GlcNAc(2)Man(3) GlcNAc(2). Fucose in alpha 1,6-linkage to the asparagine-linked GlcNAc had no effect on the activity. No transfer to N-glycans was observed when the terminal GlcNAc residues were either absent or substituted with galactose. N-acetyllactosamine, lacto-N-biose and N-acetylchito-oligosaccharides did not function as acceptors for the alpha 1,3-fucosyltransferase.The transferase exhibited maximal activity at pH 7.0 and a strict requirement for Mn2+ or Zn2+ ions. The enzyme's activity was moderately increased in the presence of Triton X-100. It was not affected by N-ethylmaleimide.