Abstract
A rapid and efficient approach for preparing isotopically labeled recombinant proteins is presented. The method is demonstrated for 13C labeling of the C-terminal domain of angiopoietin-2, 15N labeling of ubiquitin and for 2H/13C/15N labeling of the Escherichia coli outer-membrane lipoprotein Lpp-56. The production method generates cell mass using unlabeled rich media followed by exchange into a small volume of labeled media at high cell density. Following a short period for growth recovery and unlabeled metabolite clearance, the cells are induced. The expression yields obtained provide a fourfold to eightfold reduction in isotope costs using simple shake flask growths.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Angiopoietin-2
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Bacterial Outer Membrane Proteins / chemistry
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Carbon / chemistry
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Carrier Proteins / chemistry
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Culture Media
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Escherichia coli / chemistry
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Escherichia coli / growth & development
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Escherichia coli Proteins / chemistry
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Hydrogen / chemistry
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Isotope Labeling / economics
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Isotope Labeling / methods*
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Lipoproteins*
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Nitrogen / chemistry
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Nuclear Magnetic Resonance, Biomolecular
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Proteins / chemistry
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Recombinant Fusion Proteins / chemistry*
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Recombinant Fusion Proteins / isolation & purification
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Ubiquitin / chemistry
Substances
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Angiopoietin-2
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Bacterial Outer Membrane Proteins
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Carrier Proteins
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Culture Media
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Escherichia coli Proteins
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Lipoproteins
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Lpp protein, E coli
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Proteins
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Recombinant Fusion Proteins
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Ubiquitin
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Carbon
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Hydrogen
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Nitrogen