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 15N, 13C and/or 2H 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 13C, 15N of 96.6 % and 2H, 15N of 98.8 %. With the present protocol, the NMR community could profit from the many advantages that auto-inducing media offer.
Auto-induction Protein expression Isotope labeling NMR Deuteration 13C-labeling Lactose
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We thank Patrick Graff for excellent support with mass spectrometry and Jean-Michel Rondeau for helpful discussions.
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Geoghegan KF, Dixon HBF, Rosner PJ et al (1999) Spontaneous alpha-N-6-phosphogluconoylation of a “His Tag” in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins. Anal Biochem 267:169–184. doi:10.1006/abio.1998.2990CrossRefGoogle Scholar
Kapust RB, Waugh DS (1999) Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused. Protein Sci 8:1668–1674. doi:10.1110/ps.8.8.1668CrossRefGoogle Scholar
Li Z, Kessler W, van den Heuvel J, Rinas U (2011) Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems. Appl Microbiol Biotechnol 91:1203–1213. doi:10.1007/s00253-011-3407-zCrossRefGoogle Scholar
Muchmore DC, McIntosh LP, Russel CB et al (1989) Expression and nitrogen-15 labeling of proteins for proton and nitrogen-15 nuclear magnetic resonance. Methods Enzymol 177:44–73. doi:10.1016/0076-6879(89)77005-1Google Scholar
Sreenath HK, Bingman CA, Buchan BW et al (2005) Protocols for production of selenomethionine-labeled proteins in 2-L polyethylene terephthalate bottles using auto-induction medium. Protein Expr Purif 40:256–267. doi:10.1016/j.pep.2004.12.022CrossRefGoogle Scholar
Tyler RC, Sreenath HK, Singh S et al (2005) Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination. Protein Expr Purif 40:268–278. doi:10.1016/j.pep.2004.12.024CrossRefGoogle Scholar