Production of uniformly stable isotope labelled proteins from mammalian cells

Abstract

In order to implement a new strategy for determining the kinetics and metabolism of therapeutic proteins, we had to produce milligram quantities of a uniformly stable isotope labelled species. To produce the labelled protein, the rat pituitary tumor cell line GH3 was cultured in Celtone-M, a commercially prepared medium that contains all the necessary 13C,15N-labelled amino acids as well as a uniformly 13C-labelled carbohydrate source to produce uniformly 13C,15N-labelled rat growth hormone. The growth hormone secreted into the culture medium was purified using ultrafiltration followed by reversed-phase chromatography. The yield of labelled protein was approximately 10 mg/L. SDS-PAGE and western blot analyses confirmed that the labelled growth hormone migrated similarly to the unlabelled material, and that both labelled and unlabelled growth hormone were immunoreactive. Electrospray ionization mass spectrometry (ESI) of the purified material revealed that the molecular weight of the labelled material was 995 units higher (22804 Da) than its unlabelled counterpart (21809 Da). This indicated labelling at 81% of all possible carbon and nitrogen sites throughout the protein. An alternative strategy using HPLC/CRIMS indicated that the protein was 89% 13C-labelled. It is possible to quantitate this stable-isotope labelled protein following iv injection in a rat, using HPLC/CRIMS. These results show that once cells have been adapted to growth in Celtone-M, the preparation of highly and uniformly labelled proteins from mammalian cells appears no more difficult than production of their unlabelled counterparts. This will make a much wider range of proteins available for NMR and other applications that employ stable-isotope labelled proteins. © 1997 John Wiley & Sons, Ltd.