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 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.
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References
Abramson J, Smirnova I, Kasho V et al (2003) Structure and mechanism of the lactose permease of Escherichia coli. Science 301:610–615. doi:10.1126/science.1088196
Assenberg R, Wan PT, Geisse S, Mayr LM (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23:393–402. doi:10.1016/j.sbi.2013.03.008
Cavanagh J, Fairbrother WJ, Palmer AG III, Skelton NJ (1996) Protein NMR spectroscopy, principles and practice. Academic Press, Waltham
Daegelen P, Studier FW, Lenski RE et al (2009) Tracing ancestors and relatives of Escherichia coli B, and the derivation of B strains REL606 and BL21 (DE3). J Mol Biol 394:634–643. doi:10.1016/j.jmb.2009.09.022
Dickson RC, Abelson J, Barnes WM, Reznikoff WS (1975) Genetic regulation: the Lac control region. Science 187:27–35. doi:10.1126/science.1088926
Gardner KH, Kay LE (1998) The use of 2H, 13C, 15N multidimensional NMR to study the structure and dynamics of proteins. Annu Rev Biophys Biomol Struct 27:357–406. doi:10.1146/annurev.biophys.27.1.357
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.1998.2990
Hudson BG, McKenzie L, Ebner KE (1972) Enzymic synthesis of lactose-14C (galactosyl-14C) and lactose-3H (glucosyl-3H)1. Anal Biochem 48:524–528. doi:10.1016/0003-2697(72)90107-8
Jacob F, Monod J (1961) Genetic regulatory mechanisms in the synthesis of proteins. J Mol Biol 3:318–356. doi:10.1016/S0022-2836(61)80072-7
Kaback HR, Sahin-Toth M, Weinglass AB (2001) The kamikaze approach to membrane transport. Nat Rev Mol Cell Biol 2:610–620. doi:10.1038/35085077
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.8.8.1668
LeMaster DM (1989) Deuteration in protein proton magnetic resonance. Methods Enzymol 177:23–43. doi:10.1016/0076-6879(89)77004-X
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-z
McIntosh LP, Dahlquist FW (1989) Biosynthetic incorporation of 15N and 13C. Q Rev Biophys 23:1–38. doi:10.1017/S0033583500005400
Michnick SW, Rosen MK, Wandless TJ et al (1991) Solution structure of FKBP, a rotamase enzyme and receptor for FK506 and rapamycin. Science 252:836–839. doi:10.1126/science.1709301
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)77005-1
Peti W, Page R (2007) Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr Purif 51:1–10. doi:10.1016/j.pep.2006.06.024
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.2004.12.022
Studier FW (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234. doi:10.1016/j.pep.2005.01.016
Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113–130. doi:10.1016/0022-2836(86)90385-2
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.2004.12.024
Acknowledgments
We thank Patrick Graff for excellent support with mass spectrometry and Jean-Michel Rondeau for helpful discussions.
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The authors declare that they have no conflict of interest.
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Guthertz, N., Klopp, J., Winterhalter, A. et al. Auto-inducing media for uniform isotope labeling of proteins with 15N, 13C and 2H. J Biomol NMR 62, 169–177 (2015). https://doi.org/10.1007/s10858-015-9931-x
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DOI: https://doi.org/10.1007/s10858-015-9931-x