New optimised method for obtaining maximum yields of labelled protein that minimises nutrient consumption and the need for active intervention by the researcher.

NMR is a powerful tool for the structural and dynamic study of proteins. In order to obtain the maximum information from these proteins, it is necessary to obtain them isotopically labeled with 15N and 13C. For this purpose, they are usually obtained by heterologous expression in E. coli using 13C-D-glucose and 15NH4CI as the only nutrient sources. In recent years, the price of 13C-D-glucose has almost tripled, making it more expensive to obtain NMR samples. In this work, different parameters have been studied to achieve the most rational use of 13C-D-glucose and the maximum yield in protein production. As a result, an optimized method has been developed to obtain labeled proteins with a high labeling percentage and a low consumption of 13C-D-glucose. The method also has additional advantages since it eliminates the need to monitor cell culture growth and the active time consumed by the researcher. https://doi.org/10.1007/s10858-025-00461-2. Direct link: https://rdcu.be/ebRtq