Untersuchung von Membranadsorbersystemen in der Proteinaufreinigung und Entwicklung Aptamer-basierter Affinitätschromatografie

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Transcriptional network analysis identifies key elements governing the recombinant protein production provoked reprogramming of carbon and energy metabolism in Escherichia coli BL21 (DE3)

The impact of recombinant protein production on carbon and energy metabolism in Escherichia coli BL21 (DE3) was studied through transcriptome and proteome analysis of cells induced in carbon-limited fed-batch cultures during either fast or slow growth. Production of human basic fibroblast growth factor (pET expression system, T7 promoter) during fast growth leads to a macroscopically observable response classifiable into two consecutive steps: i. apparently unperturbed growth and respiration with concomitant formation of pyruvate and acetate followed by ii. inhibition of growth, respiratory activity and glucose uptake. Down-regulation of genes involved in sugar and acetate uptake, tricarboxylic acid (TCA) cycle, and respiratory energy generation started already during apparently unperturbed growth with the exceptions of up-regulated genes encoding the less energy efficient NADH dehydrogenase and terminal oxidases. A transcription factor target gene network analysis revealed that observed changes are mainly attributable to the vanishing influence of the transcription factor CRP-cAMP but also to a strong down-regulation of AcrA-P repressed genes. Moreover, down-regulation of MalT activated and up-regulation of PdhR repressed genes contribute among others to the reorganization of the transcriptome. The main drivers were identified as accumulating metabolites, for example, pyruvate, which affect transcription factor activity. The resulting restructured proteome leads to reduced glucose uptake, TCA cycle, and respiratory capacities this way decreasing catabolic carbon breakdown and metabolite accumulation. At slow growth, the production provoked transcriptome rearrangements are more subtle not leading to a macroscopically evident response. In summary, the transcriptomic response towards recombinant gene expression mimics a carbon or nutrient up-shift response aiming to match catabolic carbon processing with compromised anabolic capacities of induced cells. It is not the reason for growth inhibition and the metabolic burden but the cellular attempt to attenuate the “toxic effect” of recombinant gene expression by reducing carbon catabolism

Downstream processing of high chain length polysialic acid using membrane adsorbers and clay minerals for application in tissue engineering

Polysialic acid (polySia) is a carbohydrate polymer of varying chain length. It is a promising scaffold material for tissue engineering. In this work, high chain length polySia was produced by an Escherichia coli K1 strain in a 10-L bioreactor in batch and fed-batch mode, respectively. A new downstream process for polySia is presented, based on membrane adsorber technology and use of inorganic anion exchanger. These methods enable the replacement of precipitation steps, such as acetone, cetavlon, and ethanol precipitation of the already established purification process. The purification process was simplified, while process efficiency and product qualities were improved. The overall yield of polySia from a 10-L batch cultivation process was 61% and for 10-L fed-batch cultivation process the yield was 40% with an overall purity of 98%. The endotoxin content was determined to be negligible (14 EU mg-1). The main advantage of this new downstream process is that polySia with high chain length of more than 130 degree of polymerization can be obtained. In fed-batch cultivation, chain lengths up to 160 degree of polymerization were obtained.DFG/FOR/54

Numerical modelling of contracted sharp-crested weirs and combined weir and gate systems

Discharge measurement and control structures are widely employed in hydraulic engineering applications. The objective of this study is to numerically investigate the modelling of two different structures, namely sharp-crested weirs as Problem 1 and combined weir and gate systems as Problem 2. The research methodology herein is based on the comparison of results of numerical simulations with experimental data for both problems separately. For the purpose of performing numerical simulations, the Reynolds-averaged Navier-Stokes (RANS) equations are solved by finite volume formulation using commercially available Flow-3D software. Assessment of empirical data and numerical findings for both problems reveals that discharge rates agree reasonably well. In addition, using the capabilities of numerical modelling, weir and gate discharge coefficients in the combined system are calculated separately which were not easy to obtain in experimental studies. It is seen that gate and weir discharge coefficients of the combined system are different and higher than the corresponding coefficients of the individual systems. (c) 2020 John Wiley & Sons, Ltd