A practical method for uniform isotopic labeling of recombinant proteins in mammalian cells.
A method to obtain uniformly isotopically labeled (15N and 15N/13C) protein from mammalian cells is described. The method involves preparation of isotopically labeled media consisting of amino acids isolated from bacterial and algal extracts supplemented with cysteine and enzymatically synthesized glutamine. The approach is demonstrated by producing 15N-labeled and 15N/13C-labeled urokinase from Sp2/0 cells and successfully growing Chinese hamster ovary (CHO) cells on the labeled media. Thus, using the procedures described, isotopically labeled proteins that have been expressed in mammalian cells can be prepared, allowing them to be studied by heteronuclear multidimensional NMR techniques.
Hansen
A P
AP
Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, Illinois 60064.
Petros
A M
AM
Mazar
A P
AP
Pederson
T M
TM
Rueter
A
A
Fesik
S W
SW
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D016466:CHO Cells / Q000201:enzymology; D002247:Carbon Isotopes; D002478:Cells, Cultured; D006224:Cricetinae; D003470:Culture Media / Q000032:analysis; D003545:Cysteine; D005973:Glutamine; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator
D000596:Amino Acids; D002247:Carbon Isotopes; D003470:Culture Media; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D005973:Glutamine; D014568:Urokinase-Type Plasminogen Activator; D003545:Cysteine