An economical method for (15)N/(13)C isotopic labeling of proteins expressed in Pichia pastoris.
We report a new and cost-effective approach to prepare (15)N/(13)C 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 (15)N and (13)C, the isotopes are not totally required during the initial growth phase of P. pastoris culture. The addition of small amounts of (15)N and (13)C 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 (15)N and (13)C isotopes during the entire growth phase.
Rodriguez
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Department of Biochemistry and Molecular Genetics and Comprehensive Cancer Center, 1530-3rd Ave. South, University of Alabama at Birmingham, AL 35294-2041, USA. erodrigu@danone.com
Krishna
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D000818:Animals; D001671:Biochemistry / Q000191:economics* / Q000379:methods*; D002247:Carbon Isotopes; D002455:Cell Division; D004591:Electrophoresis, Polyacrylamide Gel; D007370:Interferon Type I / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000432:Methanol / Q000494:pharmacology; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010843:Pichia / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D011257:Pregnancy Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002247:Carbon Isotopes; D007370:Interferon Type I; D009587:Nitrogen Isotopes; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; C020947:interferon tau; D000432:Methanol