Heavy Labeling of Recombinant Proteins

Because of the cost of isotopic chemicals and heterologous proteins to produce, an economical ¹⁵ N/¹³ C isotopic labeling method is critically needed. Four protocols have been tested for the expression of Ovine interferon-τ in Pichia pastoris . ¹³ C-glucose in place of ¹³ C-glycerol as well as the need for ¹⁵ N/¹³ C-sources were evaluated during the growth phase. Sequential addition of ¹⁵ NH₄ Cl and ¹³ C-methanol were also evaluated at different ratio. Our results demonstrate that ¹⁵ N/¹³ C 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 ¹⁵ NH₄ Cl, ¹³ C-glucose and ¹³ C-methanol in our optimised P4 protocol conditions. The cost was one-third that of the standard method using ¹⁵ NH₄ Cl and ¹³ C-glucose throughout the entire growth period and was even lower using ¹³ C-glycerol.