Counterintuitive method improves yields of isotopically labelled proteins expressed in flask-cultured Escherichia coli

NMR is a powerful tool for the structural and dynamic study of proteins. One of the necessary conditions for the study of these proteins is their isotopic labelling with 15 N and 13 C. One of the most widely used methods to obtain these labelled proteins is heterologous expression of the proteins in E. coli using 13 C-D-glucose and 15 NH 4 Cl as the sole nutrient sources. In recent years, the price of 13 C-D-glucose has almost tripled, making it essential to develop labelling methods that are as cost effective as possible. In this work, different parameters were studied to achieve the most rational use of 13 C-D-glucose, and an optimized method was developed to obtain labelled proteins with high labelling and low 13 C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.