A simple protocol for expression of isotope-labeled proteins in Escherichia coli grown in shaker flasks at high cell density

  • Mengli Cai
  • Ying Huang
  • Robert CraigieEmail author
  • G. Marius CloreEmail author


Protein expression in E. coli grown in shaker flasks is a routine and pivotal tool in many research laboratories. To maximize protein yields, cells are normally induced in the middle of the linear growth phase, typically at an OD600 of ≤ 1 for cells grown in Luria–Bertani (LB) medium at 37 °C. We recently showed that the E. coli linear growth phase can be extended to higher cell density when cells are cultured under less than optimal conditions such as in minimal medium and/or at lower temperatures. Maximizing the yield of protein per unit volume of culture is important for reducing the costs, especially when isotopically labeling is required. Here, we present a modified minimal medium and a simple protocol that can increase the protein yield up to fourfold in a pH-stabilized LB medium and up to sevenfold in a modified M9+ medium (M9++). When M9++ medium coupled with the high density (OD600 ~ 6) cell growth protocol are used to express uniformly 15N- or 15N/13C-labeled proteins, the amount of 15NH4Cl and 13C6-glucose for a given cell mass is reduced by 50% and ~ 65%, respectively, relative to the traditional low density (OD600 ~ 1) cell growth protocol with M9 medium; the inclusion of 0.1% LB in the minimal medium permits a reduction in the concentration of both the trace element solution and MgCl2, which can cause precipitation. Mass data indicate that inclusion of 0.1% LB does not significantly affect the isotope enrichment level.


Protein expression NMR Shaker flask Modified M9 medium Oxygen transfer Oxygen consumption 



This work was supported by the Intramural Program of the National Institute of Diabetes and Digestive Diseases of the National Institutes of Health (to G.M.C. and R.C.).


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Copyright information

© This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2019

Authors and Affiliations

  1. 1.Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaUSA
  2. 2.Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney DiseasesNational Institutes of HealthBethesdaUSA

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