Advertisement

Simple defined autoinduction medium for high-level recombinant protein production using T7-based Escherichia coli expression systems

  • Zhaopeng Li
  • Wolfgang Kessler
  • Joop van den Heuvel
  • Ursula RinasEmail author
Methods and Protocols

Abstract

Protein production under the control of lac operon regulatory elements using autoinduction is based on diauxic growth of Escherichia coli on lactose after consumption of more preferred carbon substrates. A novel simple and cost-effective defined autoinduction medium using a mixture of glucose, glycerol, and lactose as carbon substrate and NH 4 + as sole nitrogen source without any supplementation of amino acids and vitamins was developed for T7-based E. coli expression systems. This medium was successfully employed in 96-well microtiter plates, test tubes, shake flasks, and 15-L bioreactor cultivations for production of different types of proteins achieving an average yield of 500 mg L−1 product. Cell-specific protein concentrations and solubility were similar as during conventional isopropyl β-d-1-thiogalactopyranoside induction using Luria-Bertani broth. However, the final yield of target proteins was about four times higher, as a higher final biomass was achieved using this novel defined autoinduction broth.

Keywords

Escherichia coli Recombinant protein production Autoinduction Defined medium 

Notes

Acknowledgements

Special thanks are given to Wolf-Dieter Schubert, Torsten Lührs, and Konrad Büssow for providing recombinant E. coli strains for testing general medium applicability. Partial support of the FORSYS Partner Project “Dynamics and regulation of the metabolic balance in Escherichia coli” is greatly acknowledged. We also thank Antonio Villaverde for “smart” suggestions.

Supplementary material

253_2011_3407_MOESM1_ESM.pdf (229 kb)
ESM1 (PDF 228 kb)

References

  1. Albano CR, Randers-Eichhorn L, Chang Q, Bentley WR, Rao G (1996) Quantitative measurement of green fluorescent protein expression. Biotechnol Tech 10:953–958. doi: 10.1007/BF00180401 CrossRefGoogle Scholar
  2. Bitto E, Bingman CA, Bittova L, Kondrashov DA, Bannen RM, Fox BG, Markley JL, Phillips GN Jr (2008) Structure of human J-type co-chaperone HscB reveals a tetracysteine metal-binding domain. J Biol Chem 283:30184–30192. doi: 10.1074/jbc.M804746200 CrossRefGoogle Scholar
  3. Bitto E, Bingman CA, Bittova L, Frederick RO, Fox BG, Phillips GN Jr (2009) X-ray structure of Danio rerio secretagogin: a hexa-EF-hand calcium sensor. Proteins Struct Funct Bioinf 76:477–483. doi: 10.1002/prot.22362 CrossRefGoogle Scholar
  4. Blommel PG, Becker KJ, Duvnjak P, Fox BG (2007) Enhanced bacterial protein expression during auto-induction obtained by alteration of lac repressor dosage and medium composition. Biotechnol Prog 23:585–598. doi: 10.1021/bp070011x CrossRefGoogle Scholar
  5. Bollag DM, Rozycki MD, Edelstein SJ (1996) Protein methods, 2nd edn. Wiley-Liss, Inc., New York, pp 107–151Google Scholar
  6. Bublitz M, Polle L, Holland C, Heinz DW, Nimtz M, Schubert WD (2009) Structural basis for autoinhibition and activation of Auto, a virulence-associated peptidoglycan hydrolase of Listeria monocytogenes. Mol Microbiol 71:1509–1522. doi: 10.1111/j.1365-2958.2009.06619.x CrossRefGoogle Scholar
  7. Candiano G, Bruschi M, Musante L, Santucci L, Ghiggeri GM, Carnemolla B, Orecchia P, Zardi L, Righetti PG (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333. doi: 10.1002/elps.200305844 CrossRefGoogle Scholar
  8. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene expression. Science 263:802–805. doi: 10.1126/science.8303295 CrossRefGoogle Scholar
  9. Cronet P, Petersen JF, Folmer R, Blomberg N, Sjoblom K, Karlsson U, Lindstedt EL, Bamberg K (2001) Structure of the PPARα and -γ ligand binding domain in complex with AZ 242; ligand selectivity and agonist activation in the PPAR family. Structure 9:699–706. doi: 10.1016/S0969-2126(01)00634-7 CrossRefGoogle Scholar
  10. Faessel HM, Levasseur LM, Slocum HK, Greco WR (1999) Parabolic growth patterns in 96-well plate cell growth experiments. In Vitro Cell Dev Biol Anim 35:270–278. doi: 10.1007/s11626-999-0071-z CrossRefGoogle Scholar
  11. Fox BG, Blommel PG (2009) Autoinduction of protein expression. Curr Protoc Protein Sci Chapter 5:Unit 5.23. doi: 10.1002/0471140864.ps0523s56
  12. Frederick RO, Bergeman L, Blommel PG, Bailey LJ, McCoy JG, Song J, Meske L, Bingman CA, Riters M, Dillon NA, Kunert J, Yoon JW, Lim A, Cassidy M, Bunge J, Aceti DJ, Primm JG, Markley JL, Phillips GN Jr, Fox BG (2007) Small-scale, semi-automated purification of eukaryotic proteins for structure determination. J Struct Funct Genomics 8:153–166. doi: 10.1007/s10969-007-9032-5 CrossRefGoogle Scholar
  13. Hoffmann F, Rinas U (2000) Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli. Biotechnol Prog 16:1000–1007. doi: 10.1021/bp0000959 CrossRefGoogle Scholar
  14. Hoffmann F, van den Heuvel J, Zidek N, Rinas U (2004) Minimizing inclusion body formation during recombinant protein production in Escherichia coli at bench and pilot plant scale. Enzyme Microb Technol 34:235–241. doi: 10.1016/j.enzmictec.2003.10.011 CrossRefGoogle Scholar
  15. Ingraham JL, Maaloe O, Neidhardt FC (1983) Composition, organization, and structure of the bacterial cell. In: Growth of the bacterial cell. Sinauer Associates Inc, Sunderland, pp 1–48Google Scholar
  16. Kayser A, Weber J, Hecht V, Rinas U (2005) Metabolic flux analysis of Escherichia coli in glucose-limited continuous culture. I. Growth-rate-dependent metabolic efficiency at steady state. Microbiology 151:693–706. doi: 10.1099/mic.0.27481-0 CrossRefGoogle Scholar
  17. Korz DJ, Rinas U, Hellmuth K, Sanders EA, Deckwer WD (1995) Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol 39:59–65. doi: 10.1016/0168-1656(94)00143-Z CrossRefGoogle Scholar
  18. Letzelter M, Sorg I, Mota LJ, Meyer S, Stalder J, Feldman M, Kuhn M, Callebaut I, Cornelis GR (2006) The discovery of SycO highlights a new function for type III secretion effector chaperones. EMBO J 25:3223–3233. doi: 10.1038/sj.emboj.7601202 CrossRefGoogle Scholar
  19. Machner MP, Isberg RR (2006) Targeting of host Rab GTPase function by the intravacuolar pathogen Legionella pneumophila. Dev Cell 11:47–56. doi: 10.1016/j.devcel.2006.05.013 CrossRefGoogle Scholar
  20. Oppenheimer M, Poulin MB, Lowary TL, Helm RF, Sobrado P (2010) Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus. Arch Biochem Biophys 502:31–38. doi: 10.1016/j.abb.2010.06.035 CrossRefGoogle Scholar
  21. Rinas U, Hoffmann F, Betiku E, Estape D, Marten S (2007) Inclusion body anatomy and functioning of chaperone-mediated in vivo inclusion body disassembly during high-level recombinant protein production in Escherichia coli. J Biotechnol 127:244–257. doi: 10.1016/j.jbiotec.2006.07.004 CrossRefGoogle Scholar
  22. Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, Thirdth edn. Cold Spring Harbor Laboratory Press, New York, p A 2.7Google Scholar
  23. Schmidt M, Viaplana E, Hoffmann F, Marten S, Villaverde A, Rinas U (1999) Secretion-dependent proteolysis of heterologous protein by recombinant Escherichia coli is connected to an increased activity of the energy-generating dissimilatory pathway. Biotechnol Bioeng 66:61–67. doi: 10.1002/(SICI)1097-0290(1999)66:1<61::AID-BIT6>3.0.CO;2-G CrossRefGoogle Scholar
  24. Sivashanmugam A, Murray V, Cui C, Zhang Y, Wang J, Li Q (2009) Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci 18:936–948. doi: 10.1002/pro.102 CrossRefGoogle Scholar
  25. Sobrado P, Goren MA, James D, Amundson CK, Fox BG (2008) A protein structure initiative approach to expression, purification, and in situ delivery of human cytochrome b5 to membrane vesicles. Protein Expr Purif 58:229–241. doi: 10.1016/j.pep.2007.11.018 CrossRefGoogle Scholar
  26. Sreenath HK, Bingman CA, Buchan BW, Seder KD, Burns BT, Geetha HV, Jeon WB, Vojtik FC, Aceti DJ, Frederick RO, Phillips GN Jr, Fox BG (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 CrossRefGoogle Scholar
  27. 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 CrossRefGoogle Scholar
  28. 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 CrossRefGoogle Scholar
  29. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544. doi: 10.1146/annurev.biochem.67.1.509 CrossRefGoogle Scholar
  30. Tyler RC, Sreenath HK, Singh S, Aceti DJ, Bingman CA, Markley JL, Fox BG (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 CrossRefGoogle Scholar
  31. Zahn R, von Schroetter C, Wuthrich K (1997) Human prion proteins expressed in Escherichia coli and purified by high-affinity column refolding. FEBS Lett 417:400–404. doi: 10.1016/S0014-5793(97)01330-6 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Zhaopeng Li
    • 1
    • 3
  • Wolfgang Kessler
    • 2
  • Joop van den Heuvel
    • 1
  • Ursula Rinas
    • 1
    • 3
    Email author
  1. 1.Helmholtz Centre for Infection Research (SB)BraunschweigGermany
  2. 2.Helmholtz Centre for Infection Research (MWIS)BraunschweigGermany
  3. 3.Technical Chemistry – Life ScienceLeibniz University of HannoverHannoverGermany

Personalised recommendations