An economical method for 15N/13C isotopic labeling of proteins expressed in Pichia pastoris

We report a new and cost-effective approach to prepare N-15/C-13 labeled proteins for NMR using the Pichia pastoris expression system. Four protocols (P1 to P4) were defined and compared using recombinant Ovine interferon-tau (rOvIFN-tau). Our results demonstrate that in. order to get full incorporation of N-15 and C-13, the isotopes are not totally required during the initial growth phase of P, pastoris culture. The addition of small amounts of N-15 and C-13 compounds 6 h prior to the methanol induction phase is sufficient to obtain 99% incorporation of heavy isotopes into the protein. Our optimized protocol P4 is two-thirds less costly than the classical method using N-15 and C-13 isotopes during the entire growth phase.