A novel isotope labeling protocol for bacterially expressed proteins.
A novel protocol for isotopically labeling bacterially expressed proteins is presented. This method circumvents problems related to poor cell growth, commonly associated with the use of minimal labeled media, and problems with protein induction encountered, less commonly, when using enriched labeled media. The method involves initially growing the bacterial cells to high optical density in a commercially available enriched labeled medium. Following a suitable growth period, the cells are transferred to a different (minimal) labeled medium, appropriate for induction. The method is demonstrated using the protein melanoma growth stimulating activity (MGSA).
Department of Cell Culture, Genentech Inc., South San Francisco, CA 94080-4990.
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Reilly
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Fairbrother
D001419:Bacteria; D002247:Carbon Isotopes; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D003001:Cloning, Molecular; D006133:Growth Substances / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009363:Neoplasm Proteins / Q000737:chemistry; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification
C475829:CXCL1 protein, human; D002247:Carbon Isotopes; D054360:Chemokine CXCL1; D019743:Chemokines, CXC; D002630:Chemotactic Factors; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins