University of Natural Resources and Life Sciences, Department of Biotechnology, Muthgasse 18, Vienna, Austria.
Microb Cell Fact. 2011 Jun 26;10:47. doi: 10.1186/1475-2859-10-47.
The budding yeast Pichia pastoris 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.
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-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 daughter cells.
A novel 34S labeling procedure that enables in vivo 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.
毕赤酵母是一种广泛应用于蛋白质生产的酵母。为了确定最适合的菌株改良策略，特别是针对高分泌水平，重组蛋白的细胞内通量的定量数据非常重要。特别是细胞内蛋白质形成、降解和分泌之间的平衡决定了生产系统的主要瓶颈。由于这些参数在无限生长（摇瓶）和碳限制生长（生物反应器）条件下是不同的，因此它们应该在“生产类似”的条件下确定。因此，标记程序必须与最小化生产培养基和生物反应器的使用兼容。无机且非放射性的 34S 标记的硫酸钠符合这两个要求。
我们使用了一种新的标记方法，使用稳定的硫同位素 34S，作为硫酸钠进行给药，该方法在恒化培养中进行。通过 HPLC-ICP-MS 测量纯化的重组蛋白、抗体片段 Fab3H6 的内外硫 32 至 34 比。这里描述的动力学模型是从连续样品的硫比计算动力学参数以及进行灵敏度分析所必需的。从细胞内产生的总蛋白量（每酵母干重和时间 143.1μg g-1 h-1 蛋白）中，约 58%在细胞内降解，35%分泌到细胞外，7%遗传给子细胞。
描述了一种新的 34S 标记程序，该程序可在“生产类似”条件下对重组蛋白的细胞内通量进行体内定量。随后使用 MATLAB 对通量进行灵敏度分析，表明了针对增加分泌的最有前途的菌株改良方法。
