Optimised fermentation strategy for 13C/15N recombinant protein labelling in Escherichia coli for NMR-structure analysis

Abstract A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98–100%) incorporation of [ 13 C] and [ 15 N] into labelled recombinant protein expressed in Escherichia coli , allowing better assignment of the resonances for NMR studies. Isotope labelling of the culture was performed throughout the complete process, starting from preculture. Sufficient biomass is first generated in a batch phase. Upon consumption of glucose, identified by a sharp drop of on-line monitored oxygen consumption, expression is induced and cultivation is continued under glucose-limited conditions as fed-batch process. Thereby a quantitative utilisation of the most expensive component [ 13 C]-glucose is achieved, while the approximate amount of the [ 15 N]-ammonium chloride to be incorporated is calculated from the scheduled biomass. The usefulness of the strategy is demonstrated with production of uniformly [ 13 C/ 15 N]-labelled tryparedoxin of Crithidia fasciculata . Ideal isotope incorporation and product quality is documented by MALDI-TOF mass spectrometry and two- and three-dimensional NMR spectra.