title: A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks.
abstract: We present a simple, convenient and robust protocol for expressing perdeuterated proteins in E. coli BL21(DE3) cells in shaker flasks that reduces D2O usage tenfold and d7-glucose usage by 30 %. Using a modified M9 medium and optimized growth conditions, we were able to grow cells in linear log phase to an OD600of up to 10. Inducing the cells with isopropyl β-D-1-thiogalactopyranoside at an OD600of 10, instead of less than 1, enabled us to increase the cell mass tenfold per unit volume of cell culture. We show that protein expression levels per cell are the same when induced at an OD600between 1 and 10 under these growth conditions. Thus, our new protocol can increase protein yield per unit volume of cell culture tenfold. Adaptation of E. coli from H2O-based to D2O-based medium is also key for ensuring high levels of protein expression in D2O. We find that a simple three-step adaptation approach-Luria-Bertani (LB) medium in H2O to LB in D2O to modified-M9 medium in D2O is both simple and reliable. The method increases the yield of perdeuterated proteins by up to tenfold using commonly available air shakers without any requirement for specialized fermentation equipment.
authors.affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
authors.forename: Mengli
authors.initials: M
authors.lastname: Cai
authors.affiliation: Laboratories of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Laboratories of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA.
authors.forename: Renbin
authors.initials: R
authors.lastname: Yang
authors.affiliation: Laboratories of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA. robertc@niddk.nih.gov.
authors.forename: Robert
authors.initials: R
authors.lastname: Craigie
authors.affiliation: Laboratories of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892-0520, USA. mariusc@mail.nih.gov.
authors.forename: G M
authors.identifier: 0000-0003-3809-1027
authors.initials: GM
authors.lastname: Clore
mesh_terms: D061249:Batch Cell Culture Techniques; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins
keywords: Cell adaptation; Modified M9 medium; NMR; Perdeuterated proteins; Protein expression; Shaker flask