Heavy labeling of recombinant proteins.
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.
Rodriguez
Eric
E
Danone Vitapole, Cedex, France.
D000818:Animals; D002247:Carbon Isotopes; D004586:Electrophoresis; D007370:Interferon Type I / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010843:Pichia; D011257:Pregnancy Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000032:analysis* / Q000737:chemistry* / Q000302:isolation & purification; D012756:Sheep
D002247:Carbon Isotopes; D007370:Interferon Type I; D009587:Nitrogen Isotopes; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau