Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure

<p>Abstract</p> <p>Background</p> <p>The budding yeast <it>Pichia pastoris </it>is widely used for protein production. To determine the best suitable strategy for strain improvement, especially for high secretion, quantitative data of intracellular fluxes of recombinant protein are very important. Especially the balance between intracellular protein formation, degradation and secretion defines the major bottleneck of the production system. Because these parameters are different for unlimited growth (shake flask) and carbon-limited growth (bioreactor) conditions, they should be determined under "production like" conditions. Thus labeling procedures must be compatible with minimal production media and the usage of bioreactors. The inorganic and non-radioactive ³⁴S labeled sodium sulfate meets both demands.</p> <p>Results</p> <p>We used a novel labeling method with the stable sulfur isotope ³⁴S, administered as sodium sulfate, which is performed during chemostat culivations. The intra- and extracellular sulfur 32 to 34 ratios of purified recombinant protein, the antibody fragment Fab3H6, are measured by HPLC-ICP-MS. The kinetic model described here is necessary to calculate the kinetic parameters from sulfur ratios of consecutive samples as well as for sensitivity analysis. From the total amount of protein produced intracellularly (143.1 μg g^-1h^-1protein per yeast dry mass and time) about 58% are degraded within the cell, 35% are secreted to the exterior and 7% are inherited to the daughter cells.</p> <p>Conclusions</p> <p>A novel ³⁴S labeling procedure that enables <it>in vivo </it>quantification of intracellular fluxes of recombinant protein under "production like" conditions is described. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most promising approaches for strain improvement towards increased secretion.</p

Maleimide-functionalised platinum(IV) complexes as synthetic platform for targeted drug delivery

Maleimide-functionalised Pt(IV) complexes with highly selective binding properties to thiol groups were synthesised as precursors for binding of thiol-containing tumour targeting molecules like human serum albumi

Arabidopsis thaliana ?1,2-xylosyltransferase: an unusual glycosyltransferase with the potential to act at multiple stages of the plant N-glycosylation pathway

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Rhizosphere characteristics of the arsenic hyperaccumulator Pteris vittata L. and monitoring techniques for its use in phytoextraction.

Recently discovered As-hyperaccumulator ferns hold promise for phytoremediation of As-polluted soils. We investigated changes in the rhizosphere characteristics of Pteris vittata (Chinese Brake fern) relevant for its use in phytoextraction. Plants were grown in rhizoboxes filled with soil containing 2270 mg kg-1 As. Dissolved organic carbon (DOC) concentrations in rhizosphere soil solution were increased by 86% and appeared to enhance total Fe solubility due to complexation reactions. Despite substantial removal of As by the fern, As was not significantly decreased in the rhizsophere soil solution after one cropping, apparently due to the large buffer capacity of the soil and possibly because of ion competition with DOC. However, the difference between 0.05 M (NH4)2SO4-extractable labile As in bulk and rhizosphere soil accounted for 8.9% of total As accumulated in the fern, indicating that As was mainly acquired from less available pools. Moreover, As depletion in the rhizosphere and limited resupply from less available pools were indicated by a 19.3% decreased As flux, measured using the technique of diffusive gradients in thin films (DGT). Modeling of the DGT−soil system was able to show that the rate of release from solid phase to solution in the rhizosphere was one-third of that in the bulk soil. Applying the remedial strategy of bioavailable contaminant stripping, which aims at diminishing the phytoavailable pollutant fraction, DGT can be used as a monitoring tool to evaluate the efficiency of phytoextraction and to study the potential resupply of bioavailable pools after phytoextraction has ceased

Quantitative profiling of in vivo generated cisplatin-DNA adducts using different isotope dilution strategies

Platinum compounds are the major group of metal-based chemotherapeutic drug used in current practice and still a topic of intense investigation. The relative contribution of structurally defined cisplatin adducts with DNA to induce apoptosis and the cellular processing of these lesions is still poorly understood mostly due to the lack of sensitive and accurate analytical tools for in vivo studies. In this regard, two novel sensitive and selective strategies are proposed here to quantify cisplatin−DNA adducts generated in Drosophila melanogaster larvae and in head and neck squamous cell carcinoma cultures. The methods involve the isolation and enzymatic digestion of the DNA in the samples exposed to cisplatin and further quantification by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection (HPLC−ICPMS). Two different strategies, based on isotope dilution analysis (IDA), have been attempted and evaluated for quantification: species-unspecific (the postcolumn addition of a 194Pt-enriched solution) and the species-specific (by means of a synthesized isotopically enriched cisplatin (194Pt) adduct). For the second approach, the synthesis and characterization of the cisplatin adduct in a custom oligonucleotide containing the sequence (5′-TCCGGTCC-3′) was necessary. The adducted oligo was then added to the DNA samples either before or after enzymatic hydrolysis. The results obtained using these two strategies (mixing before and after enzymatic treatment) permit to address, quantitatively, the column recoveries as well as the efficiency of the enzymatic hydrolysis. Species-specific spiking before enzymatic digestion provided accurate and precise analytical results to clearly differentiate between Drosophila samples and carcinoma cell cultures exposed to different cisplatin concentrations