Millisecond duration pulses for flow-through electro-induced protein extraction from E. coli and associated eradication.

Pulsed electric fields are used to induce membrane permeabilization on cells. In the case of species with cell wall (yeasts, microalgae), it was previously shown that when the pulse duration was several ms long, this resulted in a cytoplasmic soluble protein slow leakage. In this work, we show that a similar consequence can be obtained with different strains of E. coli. Experimental evidences of a resulting wall alteration are described. Pre-industrial flow process pilots are used. As the membrane electropermeabilization can be irreversible by applying a proper choice of the pulse parameters, this approach is used for bacterial inactivation in flow process. It is observed that sub-millisecond pulse trains are more cost effective than longer ones.

Irreversibly electropermeabilized yeast retains the capability for ATP synthesis via oxidative phosphorylation.

ATP synthesis in irreversibly electropermeabilized yeast Kluyveromyces lactis was studied by using different respiratory substrates. The permeabilization itself provoked a dramatic decrease of the total ATP level and the cells lost their ability to synthesize ATP via glycolysis. The addition of exogenous NADH supported ATP synthesis in irreversibly permeabilized cells for up to 4-6 h after substrate addition when the total ATP level became twice that of intact cells incubated for the same period with lactose.

Flow process for electroextraction of intracellular enzymes from the fission yeast, Schizosaccharomyces pombe.

Flow treatment of the yeast, Schizosaccharomyces pombe, with high intensity electric field pulses released intracellular enzymes such as glyceraldehyde-3-phosphate dehydrogenase and phosphoglycerate kinase. Over 70% of the total activity was liberated within 4 h after pulse application. The optimal field intensities were considerably higher than that needed for irreversible plasma membrane permeabilization.

High yield electroextraction of proteins from yeast by a flow process.

High yields of intracellular enzymes from yeast can be obtained by application of a series of electric field pulses with a flow process. Up to 80-90% of the total activity can be liberated without any further or previous treatment of cells. The method is based on electroinduced changes in the cell envelope leading to a leakage of part of the intracellular proteins without formation of debris and permits treatment of large volumes. Field parameters require a limited electrical power. Treatment of at least 20% wet weight suspensions is possible. The optimal field conditions must be adjusted to the suspension concentration. Maximal yield is obtained within 4h at 30 degrees C for enzymes from Saccharomyces cerevisiae such as hexokinase, 3-phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase. The extraction of beta-D-galactosidase from Kluyveromyces lactis lasts 10h but can be accelerated by adding dithiothreitol in the postpulse medium. The specific activities of the electroextracted enzymes are higher than those obtained by mechanical disintegration or enzymatic lysis.

Recent biotechnological developments of electropulsation. A prospective review.

During the last 25 years, basic research has improved our knowledge on the molecular mechanisms triggered at the membrane level by electric pulses. Applied aspects may now be used under safe conditions. Electropulsation is known as a very efficient tool for obtaining gene transfer in many species to produce genetically modified organisms (GMO). This is routinely used for industrial purposes to transfer exogenous activities in bacteria, yeasts and plants. The method is simple and of a low cost. But electropulsation is not limited to this application for biotechnological purposes. It is known that the field-associated membrane alterations can be irreversible. The pulsed species cannot recover after the treatment. Their viability is strongly affected. This appears as a very promising technology for the eradication of pathogenic microorganisms. Recent developments are proposed for sterilization purposes. New flow technologies of field generation allow the treatment of large volumes of solution. When high flow rates are used, microorganisms are submitted both to a hydromechanical and to an electrical stress. The synergy of the two effects may be present when suitable pulsing conditions are chosen. Several examples for the treatment of domestic water and in the food industry are described. Walled microorganisms are affected not only at the membrane level. We observed that alterations are present on the cell wall. A very promising technology is the associated controlled leakage of the cytoplasmic soluble proteins. Large dimeric proteins such as beta-galactosidases can be extracted at a high yield. High volumes can be treated by using a flow process. Extraction of proteins is obtained with many systems including mammalian cells.

Electroinduced extraction of beta-galactosidase from Kluyveromyces lactis.

A new methodology for the extraction of beta-galactosidase from the yeast Kluyveromyces lactis was obtained by electropulsation. The application of a series of electric pulses (2 ms duration, 1 Hz frequency, and 4-4.5 kV/cm field strength) to fresh cells suspended in deionized water, followed by incubation in PBS, led to a spontaneous slow release of enzyme at a yield of 75-80% without any further treatment. Most of the enzyme was extracted within 8 h after electropulsation. This release was dependent on the growth phase. The specific activity of beta-galactosidase in the supernatant of pulsed cells was higher by a factor of 1.5-1.7 in comparison with crude extract.

Electropulsation as an alternative method for protein extraction from yeast.

The application of series of high intensity electric pulses to a yeast suspension provoked a considerable release of some cytoplasmic proteins, glutathione reductase, 3-phosphoglycerate kinase and alcohol dehydrogenase. A maximal yield was achieved 3-8 h after pulsation. The electro-induced protein efflux was accelerated by pretreatment with the reducing agent dithiothreitol and showed a strong dependence on the growth phase and the presence of monovalent ions in the post-pulse incubation medium. The results obtained for two strains of Saccharomyces cerevisiae, PV3 (diploid) and Y47 (wild haploid), showed that electropulsation can be used for the effective extraction of cytoplasmic proteins with a preserved functional activity.

Electric field mediated loading of macromolecules in intact yeast cells is critically controlled at the wall level.

The mechanism of electric field mediated macromolecule transfer inside an intact yeast cell was investigated by observing, under a microscope, the fluorescence associated to cells after pulsation in a buffer containing two different hydrophilic fluorescent dyes. In the case of a small probe such as propidium iodide, a long lived permeabilized state was induced by the field as classically observed on wall free systems. Penetration of a 70 kDa FITC dextran was obtained only by using drastic conditions and only a very limited number of yeast cells which took up macromolecules remained viable. Most dextrans were trapped in the wall. A dramatic improvement in transfer of dextrans was observed when the cells were treated by dithiothreitol before pulsation. A cytoplasmic protein leakage was detected after the electric treatment suggesting that an irreversible damage took place in the walls of many pulsed cells. Electroloading of macromolecules in intact yeast cells appears to be controlled by a field induced short lived alteration of the envelope organization.

Fast kinetic studies of plasmid DNA transfer in intact yeast cells mediated by electropulsation.

Intact yeast cell Electrotransformation process was investigated. It is a two step process. The plasmid must be pre-mixed and present in contact with the cells during the pulse. During the millisecond field pulse, plasmid DNA is associated to the envelope. It therefore crosses the membrane by a process which lasts several seconds as shown by its sensitivity to a post pulse addition of DNase. Electrotransformation is not supported by an electrophoretic transfer due to the external field nor by a free diffusion across the electropermeabilized envelope. DNA is first bound during the field pulse and then is transferred by a still unknown active process due to cell metabolism.

Influence of glucose and other substrates on electric field and polyethylene glycol-mediated transformation of intact yeast cells.

A prepulse incubation with 2% glucose (mannose, fructose) strongly inhibited electrotransformation of intact yeast cells. This inhibitory effect was not due to alterations of cell viability or to cell membrane electropermeabilization, and was not affected by the solution buffer properties of preincubation and pulsing media. The electrotransformation efficiency was not modified by non-metabolized substances, such as sorbitol or alose. The glucose inhibition of electrotransformation was fast, occurring after only a 3-5-min preincubation. The postpulse incubation with substrate also decreased transformation efficiency, but was pH-dependent. We observed a similar, strong inhibitory effect of glucose when cells were transformed chemically. A pH dependence of yeast electrotransformation was established.

Influence of pulsing and postpulsing media tonicity on electrotransformation of intact yeast cells.

The maximal transformation yield of intact yeast cells in hypotonic medium was obtained by application of nine pulses with a duration of 990 microseconds at 2.5 kV cm-1. Pulsation at the same electrical parameters in isotonic solution did not lead to any transformation and the electropermeability decreased by 50%. The transfer of cells, 1 min after pulsation in hypotonic medium, into media with different tonicity led to an increase of the number of transformed cells, depending on the sorbitol concentration of up to 250 mM. Further augmentation of the tonicity of postpulse medium in the range 330-1000 mM provoked strong decrease of transformation. This effect was present even when cells were resuspended in isotonic medium 30 min after pulsation.

Effect of n-alcohols on the electrotransformation and permeability of Saccharomyces cerevisiae.

The correlation between electrotransformation and electropermeability was studied in yeast cells following the modification of their membranes by treatment with n-alcohols. It was found that the number of transformed cells decreased with increase of chain length of the alcohols used as follows: methanol < ethanol < propanol < butanol. The electropermeability was unaffected by the prepulse n-alcohol treatment. The lack of a unidirectional link between permeability and transformation leads to the assumption that the mechanism of DNA introduction into the cell could not be interpreted solely as a result of the existence of pores.