A method for efficient isotopic labeling of recombinant proteins.
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.
Marley
J
J
Department of Biochemistry, Weill Medical College of Cornell University, New York, NY 10021, USA.
Lu
M
M
Bracken
C
C
D042702:Angiopoietin-2; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D002352:Carrier Proteins / Q000737:chemistry; D003470:Culture Media; D004926:Escherichia coli / Q000737:chemistry / Q000254:growth & development; D029968:Escherichia coli Proteins / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D007553:Isotope Labeling / Q000191:economics / Q000379:methods*; D008074:Lipoproteins; D009584:Nitrogen / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000302:isolation & purification; D025801:Ubiquitin / Q000737:chemistry
D042702:Angiopoietin-2; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D008074:Lipoproteins; C099932:Lpp protein, E coli; D011506:Proteins; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D002244:Carbon; D006859:Hydrogen; D009584:Nitrogen