title: SILAC compatible strain of Pichia pastoris for expression of isotopically labeled protein standards and quantitative proteomics.
abstract: The methylotrophic yeast Pichia pastoris is a powerful eukaryotic platform for the production of heterologous protein. Recent publication of the P. pastoris genome has facilitated strain development toward biopharmaceutical and environmental science applications and has advanced the organism as a model system for the study of peroxisome biogenesis and methanol metabolism. Here we report the development of a P. pastoris arg-/lys- auxotrophic strain compatible with SILAC (stable isotope labeling by amino acids in cell culture) proteomic studies, which is capable of generating large quantities of isotopically labeled protein for mass spectrometry-based biomarker measurements. We demonstrate the utility of this strain to produce high purity human serum albumin uniformly labeled with isotopically heavy arginine and lysine. In addition, we demonstrate the first quantitative proteomic analysis of methanol metabolism in P. pastoris, reporting new evidence for a malate-aspartate NADH shuttle mechanism in the organism. This strain will be a useful model organism for the study of metabolism and peroxisome generation.
authors.affiliation: Institute for Systems Biology, Seattle, WA 98109, USA.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Austin
authors.forename: Rolf E
authors.initials: RE
authors.lastname: Kuestner
authors.forename: Debbie K
authors.initials: DK
authors.lastname: Chang
authors.forename: Knut R
authors.initials: KR
authors.lastname: Madden
authors.forename: Daniel B
authors.initials: DB
authors.lastname: Martin
mesh_terms: D000595:Amino Acid Sequence; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D006801:Humans; D007553:Isotope Labeling; D000432:Methanol / Q000378:metabolism; D008969:Molecular Sequence Data; D020675:Peroxisomes / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D040901:Proteomics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000592:standards; D012015:Reference Standards; D022142:Secretory Vesicles / Q000378:metabolism; D012709:Serum Albumin / Q000096:biosynthesis* / Q000378:metabolism / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000592:standards; D015854:Up-Regulation
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D012709:Serum Albumin; D000432:Methanol