Abstract
A large proportion of human proteins contain post-translational modifications that cannot be synthesized by prokaryotes. Thus, mammalian expression systems are often employed to characterize structure/function relationships using NMR spectroscopy. Here we define the selective isotope labeling of secreted, post-translationally modified proteins using human embryonic kidney (HEK)293 cells. We determined that alpha-[15N]- atoms from 10 amino acids experience minimal metabolic scrambling (C, F, H, K, M, N, R, T, W, Y). Two more interconvert to each other (G, S). Six others experience significant scrambling (A, D, E, I, L, V). We also demonstrate that tuning culture conditions suppressed V and I scrambling. These results define expectations for 15N-labeling in HEK293 cells.
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Acknowledgements
We thank Kelley Moremen (UGA) for suggesting this inquiry area and for use of the instrument to measure osmolality.
Funding
This work was supported by National Institutes of Health Award NIAID U01 AI148114 to AWB, NIH S10 OD025118 to IJA, and by funds from the Biochemistry and Molecular Biology Department at the University of Georgia and the Roy J. Carver Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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GPS, ARD, PGK and ETR performed experiments. GPS, AWB, ETR and IJA designed experiments. IJA and AWB acquired funding. ARD and AWB wrote the manuscript. All authors approved the final version of the manuscript.
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Subedi, G.P., Roberts, E.T., Davis, A.R. et al. A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy. J Biomol NMR (2024). https://doi.org/10.1007/s10858-023-00434-3
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DOI: https://doi.org/10.1007/s10858-023-00434-3