Optimised fermentation strategy for 13C/15N recombinant protein labelling in Escherichia coli for NMR-structure analysis.
A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98-100%) incorporation of [13C] and [15N] 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 [13C]-glucose is achieved, while the approximate amount of the [15N]-ammonium chloride to be incorporated is calculated from the scheduled biomass. The usefulness of the strategy is demonstrated with production of uniformly [13C/15N]-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.
Ross
A
A
Department of Biochemical Engineering, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Kessler
W
W
Krumme
D
D
Menge
U
U
Wissing
J
J
van den Heuvel
J
J
Flohé
L
L
D002247:Carbon Isotopes; D003470:Culture Media; D004926:Escherichia coli / Q000737:chemistry*; D005285:Fermentation; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry
D002247:Carbon Isotopes; D003470:Culture Media; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; C114110:tryparedoxin; D013879:Thioredoxins