Chemical Synthesis of an Integral Membrane Enzyme — The Challenges of Diacylglycerol Kinase

Access to integral membrane proteins via chemical synthesis or semisynthesis can provide detailed insights into the assembly and function of this diverse class of proteins. About one‐quarter to one‐third of all eukaryotic proteins are membrane proteins, and they fulfill a multitude of different functions as receptors and transducers for extracellular signals, as ion channels, and as scaffolds for large multi‐component assemblies. Many of these are still enigmatic and could benefit from synthetic access to these proteins, which allows almost unlimited variations of the peptide chain in order to address highly specific questions regarding their folding and function. The technical and synthetic challenges encountered during synthesis of these macromolecules are tremendous and help to drive the field of chemical protein synthesis beyond current limitations with respect to the ability to synthesize “difficult” hydrophobic sequences, purify them, and handle them in subsequent transformations, such as chemoselective ligation reactions. Here we provide an account of the synthetic challenges that we encountered en route to the first total chemical synthesis of an integral membrane protein comprising three membrane‐spanning domains: the E. coli diacylglycerol kinase. We hope this detailed description of our efforts will be helpful to others in tackling challenging protein synthesis targets.