Human cells for human proteins: Isotope labeling in mammalian cells in suspension for functional NMR studies

In biological and biomedical research, the focus progressively moves towards difficult human proteins, which often can only be expressed in higher eukaryotic cells. Nuclear magnetic resonance (NMR) could contribute significantly to the understanding of important proteins as it is one of the most information-rich methods. However, to exploit the full potential of NMR, proteins must be isotope labeled. Although expression protocols in, for example, HEK293 cells are often established, isotope labeling is difficult and very expensive, and published protocols are mostly limited to adherent cells in plates. To resolve this disparity, we have developed a comprehensive suite of protocols for isotope labeling in HEK293 cells in suspension. We demonstrate uniform ¹⁵N and ¹³C labeling, as well as specific labeling, with special focus on methyl bearing amino acids, including the popular ILV ¹³C-methyl labeling pattern. In addition, several innovative practices are introduced to deal with special cases. Labeling is achieved with a simple laboratory setup and affordable labeling media. These are based on either labeled amino acids, their precursors or amino extracts from microorganisms, like yeast, algae or bacteria. We demonstrate how these protocols enable NMR studies of proteins that are considered to be especially difficult to produce, for example, different human membrane proteins and nuclear receptors. We therefore expect that these new methods make many highly important proteins accessible to NMR studies and allow exploiting the high information content of this method for accelerating biological and pharmaceutical research.