Selective isotopic labeling of recombinant proteins using amino acid auxotroph strains.
Labeling proteins with stable isotopes is important for many analytical and structural techniques, including NMR spectroscopy and mass spectrometry. Nonselective labeling, which uniformly labels all amino acids in the protein, may be accomplished with readily available wild-type expression hosts. However, there are often advantages to labeling a specific amino acid, and residue-selective labeling generally requires the use of an expression strain that is auxotrophic for the amino acid in order to efficiently incorporate the isotopic label. The behavior of an auxotrophic strain may be complicated by the regulatory properties of the biosynthetic pathway, by secondary nutritional requirements resulting from disruption of a biosynthetic pathway, and from acquired sensitivity to environmental factors resulting from build-up of metabolic intermediates. As a result, it is important to characterize the phenotype of the each auxotrophic strain in order to optimize its performance as an expression host for selective labeling of proteins. The application of aromatic auxotroph strains of Pichia pastoris to labeling tyrosines in a recombinant protein (galactose oxidase) will be used to illustrate selective-labeling methods.
Whittaker
James W
JW
Department of Environmental and Biomolecular Systems, Oregon Health Sciences University, Beaverton, OR, USA.
D000596:Amino Acids / Q000096:biosynthesis / Q000378:metabolism*; D052818:Autotrophic Processes; D004578:Electron Spin Resonance Spectroscopy; D007553:Isotope Labeling / Q000379:methods*; D010843:Pichia / Q000254:growth & development; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism*; D014170:Transformation, Genetic
D000596:Amino Acids; D011994:Recombinant Proteins