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

## TL;DR

A new, cost-effective method for producing isotopically labelled proteins in E. coli uses less expensive materials and simplifies the process.

## Contribution

A counterintuitive and cost-effective method for isotopic labelling of proteins in E. coli that reduces 13C-D-glucose consumption.

## Key findings

- An optimized method achieves high isotopic labelling with reduced 13C-D-glucose usage.
- The method is simple and does not require monitoring culture growth.
- The approach is cost-effective amid rising 13C-D-glucose prices.

## Abstract

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 15N and 13C. One of the most widely used methods to obtain these labelled proteins is heterologous expression of the proteins in E. coli using 13C-D-glucose and 15NH4Cl as the sole nutrient sources. In recent years, the price of 13C-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 13C-D-glucose, and an optimized method was developed to obtain labelled proteins with high labelling and low 13C-D-glucose consumption. Surprisingly, the optimized method is also simple and does not require monitoring of culture growth.

The online version contains supplementary material available at 10.1007/s10858-025-00461-2.