![]() ![]() The production of glycoproteins by transient expression in mammalian cells. Production of cell surface and secreted glycoproteins in mammalian cells. Seiradake, E., Zhao, Y., Lu, W., Aricescu, A.R. Transient expression of proteins using COS cells. High-level protein expression in scalable CHO transient transfection. A time- and cost-efficient system for high-level protein production in mammalian cells. Human superoxide dismutase 1 (hSOD1) maturation through interaction with human copper chaperone for SOD1 (hCCS). Sequential protein expression and selective labeling for in-cell NMR in human cells. Direct structural evidence of protein redox regulation obtained by in-cell NMR. Mercatelli, E., Barbieri, L., Luchinat, E. Protein interaction patterns in different cellular environments are revealed by in-cell NMR. In-cell NMR reveals potential precursor of toxic species from SOD1 fALS mutants. Structural insights of proteins in sub-cellular compartments: in-mitochondria NMR. Visualization of redox-controlled protein fold in living cells. High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system. Structure of proteins in eukaryotic compartments. Pruning the ALS-associated protein SOD1 for in-cell NMR. Investigation of quadruplex structure under physiological conditions using in-cell NMR. Hänsel, R., Foldynová-Trantírková, S., Dötsch, V. Protein nuclear magnetic resonance under physiological conditions. NMR observation of Tau in Xenopus oocytes. In situ observation of protein phosphorylation by high-resolution NMR spectroscopy. Structural disorder of monomeric α-synuclein persists in mammalian cells. Observation of NMR signals from proteins introduced into living mammalian cells by reversible membrane permeabilization using a pore-forming toxin, streptolysin O. Quantitative NMR analysis of the protein G B1 domain in Xenopus laevis egg extracts and intact oocytes. Selenko, P., Serber, Z., Gadea, B., Ruderman, J. Quinary structure modulates protein stability in cells. Monteith, W.B., Cohen, R.D., Smith, A.E., Guzman-Cisneros, E. Probing protein quinary interactions by in-cell nuclear magnetic resonance spectroscopy. Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR). Residue level quantification of protein stability in living cells. In-cell NMR characterization of the secondary structure populations of a disordered conformation of α-synuclein within E. Protein structure determination in living cells by in-cell NMR spectroscopy. Atomic-resolution monitoring of protein maturation in live human cells by NMR. ![]() High-resolution multi-dimensional NMR spectroscopy of proteins in human cells. Looking into live cells with in-cell NMR spectroscopy. The entire procedure takes 4 d from cell culture seeding to NMR data collection. Uniform 15N labeling and amino-acid-specific (e.g., cysteine, methionine) labeling schemes are possible. 1H and 1H– 15N correlation NMR experiments (for example, using band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC)) can be carried out in <2 h, ensuring cell viability. The cDNA is transiently transfected as a complex with a cationic polymer (DNA:PEI (polyethylenimine)), and protein expression is carried on for 2–3 d, after which the NMR sample is prepared. This protocol describes the necessary steps to overexpress one or more proteins of interest inside human embryonic kidney 293T (HEK293T) cells, and it explains how to set up in-cell NMR experiments. Recent progress in NMR instruments and sample preparation methods allows functional processes, such as metal uptake, disulfide-bond formation and protein folding, to be analyzed by NMR in living, cultured human cells. In-cell NMR spectroscopy is a unique tool for characterizing biological macromolecules in their physiological environment at atomic resolution. ![]()
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