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
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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.).
Anderson EH (1946) Growth requirements of virus-resistant mutants of Escherichia coli Strain-B. Proc Natl Acad Sci USA 32:120–128ADSCrossRefGoogle Scholar
Azatian SB, Kaur N, Latham MP (2019) Increasing the buffering capacity of minimal media leads to higher protein yield. J Biomol NMR 73:11–17CrossRefGoogle Scholar
Cai M, Huang Y, Sakaguchi K, Clore GM, Gronenborn AM, Craigie R (1998) An efficient and cost-effective isotope labeling protocol for proteins expressed in Escherichia coli. J Biomol NMR 11:97–102CrossRefGoogle Scholar
Cai M, Huang Y, Yang R, Craigie R, Clore GM (2016) A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks. J Biomol NMR 66:85–91CrossRefGoogle Scholar
Collins T, Azevedo-Silva J, da Costa A, Branca F, Machado R, Casal M (2013) Batch production of a silk-elastin-like protein in E. coli BL21 (DE3): key parameters for optimization. Microb Cell Fact 12:21CrossRefGoogle Scholar
Duff AP, Wilde KL, Rekas A, Lake V, Holden PJ (2015) Robust high-yield methodologies for 2H and 2H/15N/13C labeling of proteins for structural investigations using neutron scattering and NMR. Meth Enzymol 565:3–25CrossRefGoogle Scholar
Li M, Jurado KA, Lin S, Emgelman A, Craigie R (2014) Engineered hyperactive integrase for concerted HIV-1 DNA integration. PLoS ONE 9:e105078ADSCrossRefGoogle Scholar
Sezonov G, Joseleau-Petit D, D’Ari R (2007) Escherichia coli physiology in Luria–Bertani broth. J Bacteriol 189:8746–8749CrossRefGoogle Scholar
Yin Z, Lapkowski M, Yang W, Craigie R (2012) Assembly of prototype foamy virus strand transfer complexes on product DNA bypassing catalysis of integration. Protein Sci 21:1849–1857CrossRefGoogle Scholar