Protein Extraction by Means of Electroporation from E. coli with Preserved Viability
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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.
KeywordsElectroporation Escherichia coli Protein extraction Bacterial inactivation
This research was supported under various grants by the Slovenian Research Agency (ARRS) and was conducted within the scope of the EBAM European Associated Laboratory (LEA). This research was possible as a result of networking efforts within COST Action TD1104 (www.electroporation.net). Experimental work was performed in the infrastructure center ‘Cellular Electrical Engineering’ at University of Ljubljana. We would like to thank Dr. Rok Kostanjšek, from Biotechnical faculty, University of Ljubljana for providing SEM pictures of our samples and giving the explanations.
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Conflict of interest
The authors declare that there are no conflicts of interest.
- Cooper GM (2000) The cell: a molecular approach, 2nd edn. ASM press, Washington, USAGoogle Scholar
- 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