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Production of Fusion Protein SpA::EcoRI in Batch Culture in a 60‐L Airlift Tower Loop Reactor

机译:在 60 L 气升塔式环路反应器中批量培养生产融合蛋白 SpA::EcoRI

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AbstractEscherichia coliJM103 carrying the expression plasmid pMTC48, repressor plasmid pRK248, and protection plasmid pEcoR4 was grown in a 60‐L working volume airlift tower loop reactor on M9 minimal medium. Production of fusion protein SpA::EcoRI was induced by a temperature shift from 30 to 38 (optimum), 40, or 42°C. The following parameters were monitored: cell mass concentration (X), total cell counts (TCC), number of colony‐forming units (CFU), concentrations of glucose, acetate, ethanol, pyruvate, lactate, succinate, amino acids, and ammonia, and soluble and total protein content, as well as product concentration (enzyme activity of the fusion protein), dissolved oxygen concentration, oxygen utilization rate (OUR), CO2production rate (CPR), respiration quotient (RQ), and volumetric mass‐transfer coefficients (kLa). Product formation by temperature shift was only observed if LB concentrate was added to the culture at the same time the aeration rate was increased to avoid oxygen‐transfer limitation. No product accumulation was observed with glucose and ammonia supplementation. During gene expression, X and TCC increased, CFU decreased, acetate increased, and the primary metabolite (ethanol, pyruvate, lactate) concentrations as well as OUR and CPR passed a maximum while RQ changed only slightly. These facts indicate that, during gene expression, the metabolic activity of the cell passes a maximum, and after that it decreases. With increasing aeration rate, the volumetric productivity increased, but the specific productivity with respect to the cell concentration d
机译:摘要 携带表达质粒pMTC48、阻遏蛋白质粒pRK248和保护质粒pEcoR4的大肠杆菌JM103在M9最小培养基上于60-L工作容积的空运塔环反应器中生长。融合蛋白 SpA::EcoRI 的产生是由温度从 30°C 到 38(最佳)、40 或 42°C 的温度变化诱导的。 监测以下参数:细胞质量浓度 (X)、总细胞计数 (TCC)、集落形成单位数 (CFU)、葡萄糖、乙酸盐、丙酮酸、乳酸、琥珀酸盐、氨基酸和氨的浓度,以及可溶性和总蛋白含量,以及产物浓度(融合蛋白的酶活性)、溶解氧浓度、氧利用率 (OUR)、CO2 产生率 (CPR)、 呼吸商 (RQ) 和体积传质系数 (kLa)。只有当将LB浓缩物添加到培养物中时,才观察到温度变化的产物形成,同时增加曝气速率以避免氧气转移限制。补充葡萄糖和氨未观察到产物积累。在基因表达过程中,X和TCC升高,CFU降低,乙酸盐升高,初级代谢物(乙醇、丙酮酸、乳酸)浓度以及OUR和CPR达到最大值,而RQ仅略有变化。这些事实表明,在基因表达过程中,细胞的代谢活性通过最大值,然后降低。随着曝气速率的增加,体积生产率增加,但比生产率相对于细胞浓度 d

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