Advertisement

Increasing the buffering capacity of minimal media leads to higher protein yield

  • Stephan B. Azatian
  • Navneet Kaur
  • Michael P. LathamEmail author
Communication

Abstract

We describe a general and simple modification to the standard M9 minimal medium recipe that leads to an approximate twofold increase in the yield of heterologously expressed proteins in Escherichia coli BL21(DE3) bacteria. We monitored the growth of bacteria transformed with plasmids for three different test proteins in five minimal media with different concentrations of buffering salts and/or initial media pH. After purification of the over-expressed proteins, we found a clear correlation between the protein yield and change in media pH over time, where the minimal media that were the most buffered and therefore most resistant to change in pH produced the most protein. And in all three test protein cases, the difference in yield was nearly twofold between the best and worst buffering media. Thus, we propose that increasing the buffering capacity of M9 minimal media will generally lead to a similar increase for most of the proteins currently produced by this standard protein expression protocol. Moreover, we have qualitatively found that this effect also extends to deuterated M9 minimal media growths, which could lead to significant cost savings in these preparations.

Keywords

Protein expression Modified M9 media Buffer capacity Cell growth 

Notes

Acknowledgements

We would like to thank Marella D. Canny for critically reading this manuscript, and other members of the Latham laboratory for help with bacterial growths and stimulating discussions. This work was supported by grant D-1798 from the Welch Foundation (M.P.L.).

References

  1. Anderson EH (1946) Growth requirements of virus-resistant mutants of Escherichia coli strain “B”. Proc Natl Acad Sci USA 32:120–128ADSCrossRefGoogle Scholar
  2. Bolivar F, Rodriguez RL, Greene PJ et al (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2:95–113.  https://doi.org/10.1016/0378-1119(90)90328-O CrossRefGoogle Scholar
  3. Cai M, Huang Y, Yang R et al (2016) A simple and robust protocol for high-yield expression of perdeuterated proteins in Escherichia coli grown in shaker flasks. J Biomol NMR 66:85–91.  https://doi.org/10.1007/s10858-016-0052-y CrossRefGoogle Scholar
  4. Dumon-Seignovert L, Cariot G, Vuillard L (2004) The toxicity of recombinant proteins in Escherichia coli: a comparison of overexpression in BL21(DE3), C41(DE3), and C43(DE3). Protein Expr Purif 37:203–206.  https://doi.org/10.1016/j.pep.2004.04.025 CrossRefGoogle Scholar
  5. 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.  https://doi.org/10.1146/annurev.biophys.27.1.357 CrossRefGoogle Scholar
  6. Gardner KH, Rosen MK, Kay LE (1997) Global folds of highly deuterated, methyl-protonated proteins by multidimensional NMR. Biochemistry 36:1389–1401.  https://doi.org/10.1021/bi9624806 CrossRefGoogle Scholar
  7. Kay LE, Gardner KH (1997) Solution NMR spectroscopy beyond 25 kDa. Curr Opin Struct Biol 7:722–731CrossRefGoogle Scholar
  8. Klopp J, Winterhalter A, Gébleux R et al (2018) Cost-effective large-scale expression of proteins for NMR studies. J Biomol NMR.  https://doi.org/10.1007/s10858-018-0179-0 Google Scholar
  9. Minton NP (1984) Improved plasmid vectors for the isolation of translational lac gene fusions. Gene 31:269–273.  https://doi.org/10.1016/0378-1119(84)90220-8 CrossRefGoogle Scholar
  10. Miroux B, Walker JE (1996) Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol 260:289–298.  https://doi.org/10.1006/jmbi.1996.0399 CrossRefGoogle Scholar
  11. O’Brien ES, Lin DW, Fuglestad B et al (2018) Improving yields of deuterated, methyl labeled protein by growing in H2O. J Biomol NMR.  https://doi.org/10.1007/s10858-018-0200-7 Google Scholar
  12. Roe AJ, McLaggan D, Davidson I et al (1998) Perturbation of anion balance during inhibition of growth of Escherichia coli by weak acids. J Bacteriol 180:767–772Google Scholar
  13. Rosano GL, Ceccarelli EA (2014) Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol 5:1–17.  https://doi.org/10.3389/fmicb.2014.00172 Google Scholar
  14. Sahdev S, Khattar SK, Saini KS (2008) Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies. Mol Cell Biochem 307:249–264.  https://doi.org/10.1007/s11010-007-9603-6 CrossRefGoogle Scholar
  15. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Lab, New YorkGoogle Scholar
  16. Studier F (2005) Protein production by auto-induction in high-density shaking cultures. Protein Expr Purif 41:207–234.  https://doi.org/10.1016/j.pep.2005.01.016 CrossRefGoogle Scholar
  17. Venters RA, Huang CC, Farmer BT et al (1995) High-level 2H/13C/15N labeling of proteins for NMR studies. J Biomol NMR 5:339–344.  https://doi.org/10.1007/BF00182275 CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Chemistry and BiochemistryTexas Tech UniversityLubbockUSA

Personalised recommendations