Rodriguez Eric
Danone Vitapole, Cedex, France.
Methods Mol Biol. 2007;389:151-64. doi: 10.1007/978-1-59745-456-8_11.
Because of the cost of isotopic chemicals and heterologous proteins to produce, an economical 15N/13C isotopic labeling method is critically needed. Four protocols have been tested for the expression of Ovine interferon-tau in Pichia pastoris. 13C-glucose in place of 13C-glycerol as well as the need for 15N/13C-sources were evaluated during the growth phase. Sequential addition of 15NH4Cl and 13C-methanol were also evaluated at different ratio. Our results demonstrate that 15N/13C isotopes are not required throughout the initial growth period but are necessary at low concentration a few hours prior to the methanol induction period. We have evaluated the cost of the use of isotopes 15NH4Cl, 13C-glucose and 13C-methanol in our optimised P4 protocol conditions. The cost was one-third that of the standard method using 15NH4Cl and 13C-glucose throughout the entire growth period and was even lower using 13C-glycerol.
由于生产同位素化学品和异源蛋白质的成本高昂，因此迫切需要一种经济的15N/13C同位素标记方法。已经对四种在毕赤酵母中表达绵羊干扰素-tau的方案进行了测试。在生长阶段评估了用13C-葡萄糖代替13C-甘油以及对15N/13C源的需求。还对不同比例下依次添加15NH4Cl和13C-甲醇进行了评估。我们的结果表明，在整个初始生长期间不需要15N/13C同位素，但在甲醇诱导期前几小时以低浓度添加是必要的。我们已经评估了在优化的P4方案条件下使用同位素15NH4Cl、13C-葡萄糖和13C-甲醇的成本。该成本是在整个生长期间使用15NH4Cl和13C-葡萄糖的标准方法成本的三分之一，而使用13C-甘油时成本甚至更低。
