Therefore, many tries have been designed to enhance the complex procedure for protein maturation, generally simply by co-overexpressing ER resident foldases or chaperons like BiP / Kar2, Pdi1 or calnexin [14-16]. fluxes of recombinant proteins are very essential. The total amount between intracellular proteins development Specifically, secretion and degradation defines the main bottleneck from the creation program. Because these variables will vary for unlimited development (tremble flask) and carbon-limited growth (bioreactor) conditions, they should be decided 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. Results 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 explained 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 g g-1 h-1 protein 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 child cells. Conclusions A novel 34S labeling process that enables em in vivo /em quantification of intracellular fluxes of recombinant protein under “production like” conditions is usually explained. Subsequent sensitivity analysis of the fluxes by using MATLAB, indicate the most encouraging approaches for strain improvement towards increased secretion. Background The production of recombinant proteins in yeast has to compete with other host organisms, mainly bacteria and mammalian Sivelestat sodium salt cell lines. Strain improvement therefore is an essential step between the discovery of a new protein and its large scale production. Yeasts like em Pichia pastoris /em grow faster and to a higher cell density compared to mammalian cells, however the low specific productivity (the amount of secreted protein per unit biomass and time) is usually their major drawback [1]. A lot of efforts have already been made to find and overcome specific bottlenecks in the cellular protein production and secretory system [examined by [2]]. At genomic level increasing the gene copy number as well as the promoter strength leads to higher productivities [3-5]. The overload of the endoplasmic reticulum (ER) with recombinant protein may induce the unfolded protein response (UPR) [6-8] followed by enhanced ER-associated degradation (ERAD) [9,10]. Among many other points, UPR reduces overall translation velocity [11] and enforces ERAD via the Ire1 signaling cascade [12]. ERAD causes proteolytic digestion of malfolded protein in the cytosolic proteasome [13]. Thus, reduced ER-stress can be beneficial for recombinant protein production. Therefore, many attempts have been made to improve the complex process of protein maturation, mainly by co-overexpressing ER resident chaperons or foldases like BiP COL3A1 / Kar2, Pdi1 or calnexin [14-16]. Furthermore the transport from your ER to the Golgi and finally into the exterior can be improved by co-overexpression of proteins involved in this pathways. Examples are Sso1 and Sso2, both coding for plasma membrane t-SNARE proteins [17] or Cog6, Coy1 and Bmh2, all coding for proteins involved in vesicular transport [18]. In the strain improvement process by cell engineering it is required to accomplish high yields in short time. A focused and systematic approach therefore would be to identify the most important bottleneck in recombinant protein synthesis being the one which modification has the highest impact on protein titers. Kinetic models are a useful tool in this regard, as they give insights into Sivelestat sodium salt intracellular fluxes. The formal kinetic description of the processing and transport of secreted proteins are already known for quite a while [19,20]. However, the challenge is the experimental determination of the parameters needed in those models. Furthermore it is necessary to make as few assumptions as you possibly can so that a production process can still be explained. In this regard the experiments have to be carried out under carbon limited, production “comparable”, growth in bioreactors under defined and controlled conditions instead of using shake flask cultivations. This is usually not possible when labeling is performed with radioactive isotopes or when protein kinetics is measured with microscopic tools, like fluorescence microscope imaging. Handling of large volumes of radioactive material is not feasible for risk of contamination. Sivelestat sodium salt Microscopic imaging on the other side quantifies the protein fluxes by comparing.
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