The Journal of Membrane Biology

, Volume 248, Issue 5, pp 893–901 | Cite as

Protein Extraction by Means of Electroporation from E. coli with Preserved Viability

  • Sasa Haberl Meglic
  • Tilen Marolt
  • Damijan Miklavcic
Article

Abstract

Extracting proteins by means of electroporation from different microorganisms is gaining on its importance, as electroporation is a quick, chemical-free, and cost-effective method. Since complete cell destruction (to obtain proteins) necessitates additional work, and cost of purifying the end-product is high, pulses have to be adjusted in order to prevent total disintegration. Namely, total disintegration of the cell releases bacterial membrane contaminants in the final sample. Therefore, our goal was to study different electric pulse parameters in order to extract as much proteins as possible from E. coli bacteria, while preserving bacterial viability. Our results show that by increasing electric field strength the concentration of extracted proteins increases and viability reduces. The correlation is reasonable, since high electric field destroys bacterial envelope, releasing all intracellular components into surrounding media. The strong correlation was also found with pulse duration. However, at longer pulses we obtained more proteins, while bacterial viability was not as much affected. Pulse number and/or pulse repetition frequency at our conditions have no or little effect on concentration of extracted proteins and/or bacterial viability. We can conclude that the most promising pulse protocol for protein extraction by means of electroporation based on our experience would be longer pulses with lower pulse amplitude assuring high protein yield and low effect on bacterial viability.

Keywords

Electroporation Escherichia coli Protein extraction Bacterial inactivation 

References

  1. Asavasanti S, Ristenpart W, Stroeve P, Barrett DM (2011) Permeabilization of plant tissues by monopolar pulsed electric fields: effect of frequency. J Food Sci 76:E98–E111CrossRefPubMedGoogle Scholar
  2. Assenberg R, Wan PT, Geisse S, Mayr LM (2013) Advances in recombinant protein expression for use in pharmaceutical research. Curr Opin Struct Biol 23:393–402CrossRefPubMedGoogle Scholar
  3. Bermudez-Aguirre D, Dunne CP, Barbosa-Canovas GV (2012) Effect of processing parameters on inactivation of Bacillus cereus spores in milk using pulsed electric fields. Int Dairy J 24:13–21CrossRefGoogle Scholar
  4. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. Cooper GM (2000) The cell: a molecular approach, 2nd edn. ASM press, Washington, USAGoogle Scholar
  6. Coustets M, Al-Karablieh N, Thomsen C, Teissié J (2013) Flow process for electroextraction of total proteins from microalgae. J Membr Biol 246:751–760CrossRefPubMedGoogle Scholar
  7. Coustets M, Ganeva V, Galutzov B, Teissie J (2015) Millisecond duration pulses for flow-through electro-induced protein extraction from E.coli and associated eradication. Bioelectrochemistry 103:82–91CrossRefPubMedGoogle Scholar
  8. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, Munster PN, Sullivan DM, Ugen KE, Messina JL, Heller R (2008) Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 26:5896–5903PubMedCentralCrossRefPubMedGoogle Scholar
  9. Flisar K, Haberl Meglic S, Morelj J, Golob J, Miklavcic D (2014) Testing a prototype pulse generator for a continuous flow system and its use for E. coli inactivation and microalgae lipid extraction. Bioelectrochemistry 100:44–51CrossRefPubMedGoogle Scholar
  10. Ganeva V, Galutzov B, Teissié J (2003) High yield electroextraction of proteins from yeast by a flow process. Anal Biochem 315:77–84CrossRefPubMedGoogle Scholar
  11. Garcia D, Gomez N, Manas P, Raso J, Pagan R (2007) Pulsed electric fields cause bacterial envelopes permeabilization depending on the treatment intensity, the treatment medium pH and the microorganism investigated. Int J Food Microbiol 113:219–227CrossRefPubMedGoogle Scholar
  12. Geciova J, Bury D, Jelen P (2002) Methods for disruption of microbial cells for potential use in the dairy industry—a review. Int Dairy J 12:541–553CrossRefGoogle Scholar
  13. Gurung N, Ray S, Bose S, Rai V (2013) A broader view: microbial enzymes and their relevance in industries, medicine, and beyond. BioMed Res Int 2013:329121PubMedCentralCrossRefPubMedGoogle Scholar
  14. Haberl S, Jarc M, Strancar A, Peterka M, Hodžić D, Miklavčič D (2013a) Comparison of alkaline lysis with electroextraction and optimization of electric pulses to extract plasmid DNA from Escherichia coli. J Membr Biol 246:861–867CrossRefPubMedGoogle Scholar
  15. Haberl S, Kandušer M, Flisar K, Hodžić D, Bregar VB, Miklavčič D, Escoffre JM, Rols MP, Pavlin M (2013b) Effect of different parameters used for in vitro gene electrotransfer on gene expression efficiency, cell viability and visualization of plasmid DNA at the membrane level. J Gene Med 15:169–181CrossRefPubMedGoogle Scholar
  16. Kargi AY, Merriam GR (2013) Diagnosis and treatment of growth hormone deficiency in adults. Nat Rev Endocrinol 9:335–345CrossRefPubMedGoogle Scholar
  17. Kotnik T, Pucihar G, Miklavcic D (2010) Induced transmembrane voltage and its correlation with electroporation-mediated molecular transport. J Membr Biol 236:3–13CrossRefPubMedGoogle Scholar
  18. Matos T, Senkbeil S, Mendonca A, Queiroz JA, Kutter JP, Bulow L (2013) Nucleic acid and protein extraction from electropermeabilized E. coli cells on a microfluidic chip. Analyst 138:7347–7353CrossRefPubMedGoogle Scholar
  19. Meacle FJ, Lander R, Shamlou PA, Titchener-Hooker NJ (2004) Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations. Biotechnol Bioeng 87:293–302CrossRefPubMedGoogle Scholar
  20. Miklavcic D, Mali B, Kos B, Heller R, Sersa G (2014) Electrochemotherapy: from the drawing board into medical practice. Biomed Eng Online 13:29PubMedCentralCrossRefPubMedGoogle Scholar
  21. Naglak TJ, Hettwer DJ, Wang HY (1990) Chemical permeabilization of cells for intracellular product release. Bioprocess Technol 9:177–205PubMedGoogle Scholar
  22. Neumann E, Rosenheck K (1972) Permeability changes induced by electric impulses in vesicular membranes. J Membr Biol 10:279–290CrossRefPubMedGoogle Scholar
  23. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH (1982) Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1:841–845PubMedCentralPubMedGoogle Scholar
  24. Niyonzima FN, More SS (2014) Detergent-compatible bacterial amylases. Appl Biochem Biotechnol 174:1215–1232CrossRefPubMedGoogle Scholar
  25. Ohshima T, Hama Y, Sato M (2000) Releasing profiles of gene products from recombinant Escherichia coli in a high-voltage pulsed electric field. Biochem Eng J 5:149–155CrossRefPubMedGoogle Scholar
  26. Okino M, Mohri H (1987) Effects of a high-voltage electrical impulse and an anticancer drug on in vivo growing tumors. Jpn J Cancer Res Gann 78:1319–1321PubMedGoogle Scholar
  27. Olempska-Beer ZS, Merker RI, Ditto MD, DiNovi MJ (2006) Food-processing enzymes from recombinant microorganisms-a review. Regul Toxicol Pharmacol 45:144–158CrossRefPubMedGoogle Scholar
  28. Pataro G, Ferrentino G, Ricciardi C, Ferrari G (2010) Pulsed electric fields assisted microbial inactivation of S. cerevisiae cells by high pressure carbon dioxide. J Supercrit Fluids 54:120–128CrossRefGoogle Scholar
  29. Pataro G, Senatore B, Donsi G, Ferrari G (2011) Effect of electric and flow parameters on PEF treatment efficiency. J Food Eng 105:79–88CrossRefGoogle Scholar
  30. Pucihar G, Krmelj J, Rebersek M, Batista Napotnik T, Miklavcic D (2011) Equivalent pulse parameters for electroporation. IEEE Trans Biomed Eng 58:3279–3288CrossRefPubMedGoogle Scholar
  31. Reasoner DJ (2004) Heterotrophic plate count methodology in the United States. Int J Food Microbiol 92:307–315CrossRefPubMedGoogle Scholar
  32. Roff SR, Noon-Song EN, Yamamoto JK (2014) The significance of interferon-γ in HIV-1 pathogenesis, therapy, and prophylaxis. Front Immunol 4:498PubMedCentralCrossRefPubMedGoogle Scholar
  33. Rols MP, Teissie J (1990) Electropermeabilization of mammalian cells. Quantitative analysis of the phenomenon. Biophys J 58:1089–1098PubMedCentralCrossRefPubMedGoogle Scholar
  34. Rols MP, Teissie J (1998) Electropermeabilization of mammalian cells to macromolecules: control by pulse duration. Biophys J 75:1415–1423PubMedCentralCrossRefPubMedGoogle Scholar
  35. Salazar O, Asenjo JA (2007) Enzymatic lysis of microbial cells. Biotechnol Lett 29:985–994CrossRefPubMedGoogle Scholar
  36. Saulis G (2010) Electroporation of cell membranes: the fundamental effects of pulsed electric fields in food processing. Food Eng. Rev. 2:52–73CrossRefGoogle Scholar
  37. Schiavoni G, Mattei F, Gabriele L (2013) Type I interferons as stimulators of DC-mediated cross-priming: impact on anti-tumor response. Front Immunol 4:483PubMedCentralCrossRefPubMedGoogle Scholar
  38. Schütte H, Kula MR (1990) Pilot- and process-scale techniques for cell disruption. Biotechnol Appl Biochem 12:599–620PubMedGoogle Scholar
  39. Shiina S, Ohshima T, Sato M (2007) Extracellular production of alpha-amylase during fed-batch cultivation of recombinant Escherichia coli using pulsed electric field. J Electrost 65:30–36CrossRefGoogle Scholar
  40. Suga M, Hatakeyama T (2009) Gene transfer and protein release of fission yeast by application of a high voltage electric pulse. Anal Bioanal Chem 394:13–16CrossRefPubMedGoogle Scholar
  41. Suga M, Goto A, Hatakeyama T (2007) Electrically induced protein release from Schizosaccharomyces pombe cells in a hyperosmotic condition during and following a high electropulsation. J Biosci Bioeng 103:298–302CrossRefPubMedGoogle Scholar
  42. Toepfl S, Heinz V, Knorr D (2007) High intensity pulsed electric fields applied for food preservation. Chem Eng Process 46:537–546CrossRefGoogle Scholar
  43. Wolf H, Rols MP, Boldt E, Neumann E, Teissie J (1994) Control by pulse parameters of electric field-mediated gene transfer in mammalian cells. Biophys J 66:524–531PubMedCentralCrossRefPubMedGoogle Scholar
  44. Wong TK, Neumann E (1982) Electric field mediated gene transfer. Biochem Biophys Res Commun 107:584–587CrossRefPubMedGoogle Scholar
  45. Xie TD, Tsong TY (1992) Study of mechanisms of electric field-induced DNA transfection. III. Electric parameters and other conditions for effective transfection. Biophys J 63:28–34PubMedCentralCrossRefPubMedGoogle Scholar
  46. Zgalin MK, Hodzic D, Rebersek M, Kanduser M (2012) Combination of microsecond and nanosecond pulsed electric field treatments for inactivation of Escherichia coli in water samples. J Membr Biol 245:643–650CrossRefPubMedGoogle Scholar
  47. Zhan Y, Martin VA, Geahlen RL, Lu C (2010) One-step extraction of subcellular proteins from eukaryotic cells. Lab Chip 10:2046–2048PubMedCentralCrossRefPubMedGoogle Scholar
  48. Zhan Y, Sun C, Cao Z, Bao N, Xing J, Lu C (2012) Release of intracellular proteins by electroporation with preserved cell viability. Anal Chem 84:8102–8105CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sasa Haberl Meglic
    • 1
  • Tilen Marolt
    • 1
  • Damijan Miklavcic
    • 1
  1. 1.Laboratory of Biocybernetics, Faculty of Electrical EngineeringUniversity of LjubljanaLjubljanaSlovenia

Personalised recommendations