Isotopic labeling of recombinant proteins from the methylotrophic yeast Pichia pastoris.
The methylotrophic yeast Pichia pastoris is now an established expression system for the production of recombinant protein for nuclear magnetic resonance (NMR) studies. It is capable of expressing high levels of heterologous proteins and possesses the ability to perform many of the posttranslational modifications of higher eukaryotes. Here, we describe efficient methods for the production of uniformly 13C,15N-labeled proteins in shake flasks and of uniformly 13C,15N-labeled and 2H,13C,15N-labeled proteins in fermenters. We also provide details of two chromatographic procedures, cation exchange and concanavalin A lectin affinity, that have proved useful in purifying P. pastoris-expressed proteins for NMR studies.
Pickford
Andrew R
AR
Department of Biochemistry, University of Oxford, UK.
O'Leary
Joanne M
JM
D002247:Carbon Isotopes / Q000737:chemistry; D002412:Cations / Q000737:chemistry; D002845:Chromatography / Q000379:methods; D002846:Chromatography, Affinity; D003208:Concanavalin A / Q000737:chemistry; D015870:Gene Expression; D007553:Isotope Labeling / Q000379:methods*; D037102:Lectins / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D014316:Tritium / Q000737:chemistry
D002247:Carbon Isotopes; D002412:Cations; D037102:Lectins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014316:Tritium; D003208:Concanavalin A