A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy

Details

• Alternative Title:
  J Biomol NMR
• Personal Author:
  Subedi, Ganesh P. ; Roberts, Elijah T. ; Davis, Alexander R. ; Kremer, Paul G. ; Amster, I. Jonathan ; Barb, Adam W.
• Description:
  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-[|N]- 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 |N-labeling in HEK293 cells.
• Subjects:
  Amino Acids HEK293 Cells Humans Isotope Labeling Nitrogen Isotopes Nuclear Magnetic Resonance, Biomolecular Protein Processing, Post-Translational
• Keywords:
  CD16a FcγRIIIa Glycoprotein Post-translational Modification
• Source:
  J Biomol NMR. 78(2):125-132
• Pubmed ID:
  38407675
• Pubmed Central ID:
  PMC11178438
• Document Type:
  Journal Article
• Funding:
  U01 AI148114/AI/NIAID NIH HHSUnited States/ ; S10 OD025118/CD/ODCDC CDC HHSUnited States/ ; S10 OD025118/CD/ODCDC CDC HHSUnited States/ ; U01 AI148114/AI/NIAID NIH HHSUnited States/
• Volume:
  78
• Issue:
  2
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:5bae604a5f9a668e2455087a347a0925e87740d53441e070a2d8400d8c80f972
• Download URL:
  https://stacks.cdc.gov/view/cdc/157981/cdc_157981_DS1.pdf
• File Type:
  [PDF - 731.77 KB ]