Ganesh P. Subedi, Elijah T. Roberts, Alexander R. Davis, Paul G. Kremer, I. Jonathan Amster, Adam W. Barb
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引用次数: 0
摘要
人类蛋白质中有很大一部分含有原核生物无法合成的翻译后修饰。因此，哺乳动物表达系统经常被用来利用核磁共振光谱鉴定结构/功能关系。在这里，我们利用人体胚胎肾脏（HEK）293 细胞对分泌的翻译后修饰蛋白质进行了选择性同位素标记。我们确定，来自 10 个氨基酸（C、F、H、K、M、N、R、T、W、Y）的α-[15N]- 原子的代谢扰乱最小。还有两个相互转化（G、S）。另外六种则出现了明显的混淆（A、D、E、I、L、V）。我们还证明，调整培养条件可抑制 V 和 I 的混淆。这些结果确定了对 HEK293 细胞中 15N 标记的预期。
A comprehensive assessment of selective amino acid 15N-labeling in human embryonic kidney 293 cells for NMR spectroscopy
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.
期刊介绍：
The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include:
Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR.
New NMR techniques for studies of biological macromolecules.
Novel approaches to computer-aided automated analysis of multidimensional NMR spectra.
Computational methods for the structural interpretation of NMR data, including structure refinement.
Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals.
New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.
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