title: Auto-inducing media for uniform isotope labeling of proteins with (15)N, (13)C and (2)H.
abstract: Auto-inducing media for protein expression offer many advantages like robust reproducibility, high yields of soluble protein and much reduced workload. Here, an auto-inducing medium for uniform isotope labelling of proteins with (15)N, (13)C and/or (2)H in E. coli is presented. So far, auto-inducing media have not found widespread application in the NMR field, because of the prohibitively high cost of labeled lactose, which is an essential ingredient of such media. Here, we propose using lactose that is only selectively labeled on the glucose moiety. It can be synthesized from inexpensive and readily available substrates: labeled glucose and unlabeled activated galactose. With this approach, uniformly isotope labeled proteins were expressed in unattended auto-inducing cultures with incorporation of (13)C, (15)N of 96.6% and (2)H, (15)N of 98.8%. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer.
authors.affiliation: Division of Structural Biology, Institute of Cancer Research, London, UK.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Guthertz
authors.forename: Julia
authors.initials: J
authors.lastname: Klopp
authors.forename: Aurélie
authors.initials: A
authors.lastname: Winterhalter
authors.forename: César
authors.initials: C
authors.lastname: Fernández
authors.forename: Alvar D
authors.initials: AD
authors.lastname: Gossert
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005786:Gene Expression Regulation; D007553:Isotope Labeling / Q000379:methods*; D007785:Lactose / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D011506:Proteins; D007785:Lactose