Accepted Manuscript Title: Electroinduced release of recombinant ␤-galactosidase from Saccharomyces cerevisiae Author: Valentina Ganeva Debora Stefanova Boyana Angelova Bojidar Galutzov Isabel Velasco Miguel Ar´evalo-Rodr´ıguez PII: DOI: Reference:

S0168-1656(15)30050-X http://dx.doi.org/doi:10.1016/j.jbiotec.2015.06.418 BIOTEC 7163

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

21-1-2015 29-5-2015 25-6-2015

Please cite this article as: Ganeva, Valentina, Stefanova, Debora, Angelova, Boyana, Galutzov, Bojidar, Velasco, Isabel, Ar´evalo-Rodr´iguez, Miguel, Electroinduced release of recombinant rmbeta-galactosidase from Saccharomyces cerevisiae.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2015.06.418 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Electroinduced release of recombinant β-galactosidase from Saccharomyces cerevisiae Valentina Ganevaa*, Debora Stefanovaa, Boyana Angelovaa, Bojidar Galutzova, Isabel Velascob, Miguel Arévalo-Rodríguezb a

Sofia University, Dept. Biophysics and Radiobiology, 8 Dragan Tzankov blvd, 1164 Sofia,

Bulgaria b

Biomedal S.L., Avda. Américo Vespucio 5E. 1◦ M12, 41092 Seville, Spain

*Corresponding author: Valentina Ganeva, Sofia University, Dept. Biophysics and Radiobiology, 8 Dragan Tzankov blvd, 1164 Sofia, Bulgaria, tel. (359) 2 8167 237, mobile: (359) 885109316 e-mail: [email protected]

Highlights

►

► The present study evaluates the effect of pulsed electric field, with intensity of 4.3–5.4

kV/cm, on the release of recombinant LITAG-β-galactosidase fusion protein from S. cerevisiae. ► Maximal β-galactosidase release, approximately 45 % of the total activity was obtained at field intensity of 5.2 kV/cm and 1.25 ms pulse duration. ► At these electrical conditions 97% of the cells were irreversibly permeabilised, but the vacuoles remained to a large degree preserved (intact). ► The addition of lyticase (1-2 U/ml) to the electropermeabilised cells accelerates the release of the recombinant protein and increases the yield without provoking a significant cell lysis. ► PEF treatment and subsequent incubation with lyticase have a synergistic effect on β-

galactosidase liberation ► A protocol was developed allowing the recovery of approximately 70% of the recombinant protein with a factor of purification 2.6. Abstract

Yeasts are one of the most commonly used systems for recombinant protein production. When the protein is intracelullarly expressed the first step comprises a cell lysis, achieved usually by a mechanical disintegration. This leads to non-selective liberation of the cytoplasmic content, which complicates the following downstream process. Here, we present a new approach suitable for more selective and efficient recovery of large intracellular proteins from yeast, based on the combination of electropermeabilisation and subsequent treatment with lytic enzyme. The experiments were performed with S. cerevisiae strains expressing LYTAG-β-galactosidase from E.coli. The permeabilzation of plasma membrane was induced by application of rectangular electric pulses, with 1.25 ms duration and field intensity of 4.3 – 5.4 kV/cm. In the presence of a reducing agent the cells released approximately 80% of the total protein 4h after electrical treatment. At the same conditions the release of the recombinant protein was very slow, reaching 45% from total activity 20 h after pulse application. The great difference in the release kinetics enabled to remove a part of the total protein, without significant loss of β-galactosidase activity, only by substituting the incubation buffer. The subsequent addition of lyticase (1-2 U/ml) led to recovery of approximately 70% from the recombinant enzyme, with a factor of purification 2.6, without provoking a significant cell lysis. The applicability of similar protocol for liberation of large recombinant and native proteins from yeast is discussed.

Keywords: pulsed electric field (PEF), recombinant protein, E. coli β-galactosidase, S. cerevisiae, electropermeabilisation, extraction, lyticase

1. Introduction Yeast-expression systems have been used for many years to produce large amounts of proteins for industrial and biopharmaceutical use. They have become some of the most utilised hosts for recombinant protein production, due to the good knowledge of their molecular biology, their

suitability for large-scale fermentation in a simplified medium and their high protein yields (Gerngross, 2004). Although several yeast species are being explored as sources of recombinant proteins, the recombinant pharmaceuticals so far approved by the FDA and/or European Medicine Agency (EMEA) have been obtained mainly with S. cerevisiae (Huang et al., 2010). A number of the recombinant proteins produced in S. cerevisiae can be efficiently secreted into the growth media, which is important for simplifying the downstream process (Gerngross, 2004 and Demain and Vaishnav, 2009). Other recombinant proteins accumulate in the cell cytoplasm or periplasm, and mechanical disintegration or chemical extraction is mainly applied for their recovery (Kim et al., 2009, Heim et al., 2007 and Bansal-Mutalik and Gaikar, 2006). Although applicable on a large scale, these procedures can lead to protein inactivation and complicate the downstream purification process, thus leading to higher production costs (Balasundaram et al., 2009). The treatment of cells with lytic enzyme is an attractive alternative for the recovery of intracellular and periplasmic proteins. It offers a degree of selectivity of the product release and is performed under very mild conditions, minimising the product damage. However, the susceptibility of various yeast species to lytic enzymes can vary greatly. Furthermore, the cultivation/induction conditions applied for the production of recombinant proteins often renders the cells very resistant to lytic enzymes. This imposes the utilisation of high lytic enzyme concentrations, thus making their application on a large scale prohibitively expensive. The development of new approaches combining the advantages of the existing conventional methods for protein recovery can reduce the cost and time associated with the purification of intracellularly expressed recombinant proteins in yeast. Pulsed electric field (PEF)-assisted extraction is a relatively new, highly promising method for the recovery of bioproducts from various types of cells (Vorobiev and Lebovka, 2011, Liu et al., 2013, and Mahnič-Kalamiza et al., 2014). It is based on the loss of the membrane barrier function induced by high-intensity electric field pulses known as electropermeabilisation or electroporation (Weaver and Chizmadzhev, 1996) and the subsequent leakage of the soluble cytosolic content out of the cells. Recently, the PEF treatment was successfully applied to recover homologous intracellular enzymes from various yeast species (Ganeva et al. 2003, 2004 and Oshima and Sato, 2004). The protein yield depended on the fraction of irreversibly permeabilised cells, and it was influenced by compounds that increase the cell wall porosity. The

extraction efficiency, reaching approximately 90% of the total protein content, was achieved by using continuous PEF treatment, which enables the scaling of the process. The aim of the present work was to validate the applicability of the pulsed electric field treatment for the liberation of recombinant proteins from yeast. As a model system, we utilised the E. coli β-galactosidase expressed in the yeast S. cerevisiae. Here, we present a new approach for the selective recovery of large intracellular proteins from yeast, based on the combination of cell electropermeabilisation and subsequent treatment with a very low concentration of lytic enzyme.

2. Materials and methods 2.1 Plasmids The plasmids pMAB10-LacZ-5 and pBIVU02-1, harbouring E. coli β-galactosidase as a fusion to the LYTAG affinity tag (Biomedal S.L., Spain; http://www.biomedal.com/bls/en/clytag.html?id=/expresion-systems.html), were obtained by cloning the LYTAG-lacZ translational gene fusion under the control of the pGAL1 promoter into yeast-bacteria shuttle vectors pRS416 (URA3 CEN6) and pRS425 (LEU2 2- m) (Sikorski and Hieter, 1989).

2.2. Yeast strains The S. cerevisiae strain W303-1A (MATa leu2-3, 112 his3-11, 15 trp1-1 can1-100 ade2-1 ura31) (Thomas and Rothshtein, 1989), transformed with the plasmid pMAB10-LacZ-5 or pBIVU021, was used as the host strain for the recombinant expression of the LYTAG-β-galactosidase fusion protein (131.88 kDa) used in this work.

2.3. Media and culture conditions for the recombinant protein expression Yeast transformants were propagated on YNB medium (0.67% (w/v) yeast nitrogen base and 2% (w/v) glucose) supplemented with 20 mg·l−1 L-histidine, 20 mg·l-l L-adenine, 100 mg·l−1 Ltryptophan, and 20 mg·l−1 uracil for W303-1A [pBIVU02-1]; and 20 mg·l−1 L-histidine, 20 mg·l−1 L-adenine, 100 mg·l−1 L-tryptophan, and 100 mg·l−1 L-leucine for W303-1A [pMAB10LacZ-5]. The 30-ml cultures were incubated for 16 h at 30°C and 200 rpm in a Biosan ES-20/60 orbital shaker. For galactose-induced recombinant protein expression, 1 ml of the cell suspension from each culture was centrifuged and washed with distilled water. The cells were transferred

into 50 ml of the same medium prepared with 2% (w/v) galactose instead of glucose, and the mixture was further incubated in a 300-ml flask for 20-24 h at 30°C and 200 rpm.

2.4. Pulsed electric field treatment The electric field treatment in a continuous-flow chamber was performed with a generator of monopolar rectangular pulses, a Hydropulse mini (GBS-Elektronik, Germany). The chamber (0.3-ml volume) has two parallel stainless steel electrodes that are 0.3 cm apart (MEHEL, Bulgaria). Cells in the late exponential phase were washed twice and diluted in distilled water to a concentration corresponding to 40 mg wet weight/ml. The PEF treatment was performed at flow rate of 2.4 ml·min-1 with W303-1A [pBIVU02-1] and 4.8 ml·min-1 with W303-1A [pMAB10-LacZ-5], controlled by a peristaltic pump. During the passage through the chamber, the cells received 15 pulses with a duration of 1.25 or 1.5 ms at a frequency of 2 (4) Hz and field intensity range 4-6.5 kV/cm. After pulsation, the cell suspension was diluted twofold by adding the same volume of 200 mM potassium phosphate buffer (PPB) at pH 7.5, 560 mM glycerol, with or without 16 mM dithiothreitol (DTT), and incubated at room temperature. Samples of 0.5 ml were taken at various times after pulsation and were centrifuged for 2 min at 12 000 rpm in an Eppendorf centrifuge. The supernatant was analysed for β-galactosidase activity and total protein concentration. 2.5. Two-step protocols for the liberation and partial purification of β-galactosidase

Protocol A At 1-2 h after dilution in 200 mM potassium phosphate buffer at pH 7.5 with 560 mM glycerol containing 16 mM DTT, the cell suspension was centrifuged for 10 min at 1500 x g, the supernatant was removed and the cells were diluted in the same volume of 100 mM PPB with pH = 7.5 and 280 mM glycerol and incubated at room temperature. The 0.5-ml samples, taken at various times after pulsation, were centrifuged for 2 min at 12 000 rpm in an Eppendorf centrifuge, and the supernatant was analysed for its β-galactosidase activity and total protein concentration.

Protocol B

2-4 hr after dilution in a buffer containing 6-8 mM DTT, the cell suspension was centrifuged for 10 min at 1500 x g, the supernatant was removed and the cells were diluted in 100 mM PPB pH = 7.5 with 280 mM glycerol to a final concentration of 40 mg wet weight/ml. Immediately afterward, lyticase was added to a final concentration of 1-10 U/ml, and the suspensions were further incubated at room temperature. The 0.5-ml samples taken at various intervals after the lyticase additions were centrifuged for 2 min at 12 000 rpm in an Eppendorf centrifuge, and the supernatant was analysed for its β-galactosidase activity and total protein content.

2.6. Preparation of the cell lysate The total protein from the yeast cells not exposed to electric pulsation was extracted by mixing 200 µl of the cell suspension in water with the same volume of 200 mM potassium phosphate buffer at pH 7.5 with 560 mM glycerol, 12.5 mM DTT and 28 µl lyticase (2 000 U/ml, Sigma). After 1.5 h of incubation at room temperature, the reaction mixture was diluted twofold with water, mixed with same volume of glass beads (diameter 0.42-0.6 mm, Sigma) and vortexed 12 times for 1 min each, with 30-s pause intervals with ice incubation. The combination of lyticase treatment and mechanical disruption resulted in 95% cell lysis, as determined by counting the cells in a Thoma chamber. The resulting lysate was centrifuged at 12 000 rpm for 2 min, and the supernatant was kept on ice until used for the β-galactosidase activity and total protein analysis. The enzyme activity and protein concentration values, after correction for the percent lysed cells, were used as the reference (100%) for calculating the protein (enzyme)-release yield in all experiments. A standard procedure for mechanical cell disruption was also applied. Cells resuspended in 100 mM PPB, pH =7,5, 280 mM glycerol were disrupted with a vibration homogenizer VHG1 (B Braun Melsungen AG, Melsungen, Germany) according to the protocol described by Nedeva et al. (2009). The homogenate was centrifuged at 12 000 rpm for 2 min, and protein content and βgalactosidase activity in supernatant were compared to the extract from electropulsed cells.

2.7. Determination of irreversible electropermeabilisation The membrane permeabilisation was assayed by loading the cells with 80 µM propidium iodide – final concentration (Sigma). The yeast plasma membrane recovery after pulsation requires 1530 min at room temperature (Pringle and Mor, 1975). To determine the fraction of cells with

irreversibly permeabilised membranes, PI was added 1 h after the pulse application and dilution of cells in buffer. The number of fluorescent cells was counted under an epifluorescent microscope (L3201 LED, Microscopesmall, China). The permeabilisation was expressed as a percentage of the number of fluorescent cells relative to the total cell number.

2.8. Lyticase test To detect electroinduced changes in the cell wall porosity, pulsed and control untreated cells from strain W303-1A [pBIVU02-1] were diluted twofold by adding the same volume of 200 mM potassium phosphate buffer at pH 7.5 with 560 mM glycerol, and 600 µl of the resulting cell suspension were incubated with 15 µl lyticase (2 000 U/ml) at room temperature. To monitor the cell lysis, 25-µl samples, taken every 15 min, were diluted in 975 µl distilled water, and the optical density (OD) at 660 nm was determined. The initial OD of all of the suspensions was under 0.3, where a linear relationship between the OD and cell number exists (Pringle and Mor, 1975). A decrease in the OD directly reflects the degree of cell lysis. The change in OD was calculated as a percent, with 100% being the OD for each sample before lyticase addition. 2.9. β-galactosidase and total protein determination The β-galactosidase activity was determined spectrophotometrically according to Mbuyi-Kalala et al. (1988), using o-nitrophenol-β-D-galactopyranoside as the substrate. The protein concentration was determined according to Bradford (1976). Bovine serum albumin (Sigma, Germany) was used as a standard. The results from three to five experiments are given as mean values. The error bars represent the sample standard deviation.

2.10. Protease activity determination The potease activity was determined by chemiluminescent detection method according to Zhang et al. (2013) with some modifications. The cell lysate and the extracts from electropermeabilised cells were concentrated by ultrafiltration (cutoff 10 kDa, Vivaspin, Sartorius), and equilibrated to 125 mM potassium phosphate buffer, pH=6. Cytochrom C was added to the probes (to final concentration of 0.1 mg/ml), and the probes were incubated for four hours at 30oC. After ten folds dilution with distilled water, 10 µl of the probes were added to 500 µl 50 mM borate buffer, pH=9, containing luminol (0.1 mM final concentration). The chemiluminescent reaction was

started by addition of hydrogen peroxide (1 mM final concentration) and registered on Luminometer 1250, LKB-Wallac, Sweden. The background (chemiluminescence of probes immediately after addition of cytochrome C) was subtracted from the maximal registered signal. 100% protease activity corresponds to the chemiluminescence of cell lysate, incubated with cytochrome C for four hours at 30oC. The results of three independent experiment in duplicates are given in the text. 2.11. Analytical gel-electrophoresis SDS-PAGE of the protein samples was performed on 10% polyacrylamide slab gels as described by Laemmli (1970). Protein was detected by silver staining as described by Nesterenko et al. (1994), and the protein molecular weight markers (Roti-Mark Standard, ROTH) were myosin (212 kDa), recombinant E. coli β-galactosidase (118 kDa), serum albumin (66 kDa), ovalbumin (43 kDa), trypsin inhibitor (20 kDa), and lysozyme (14 kDa). The molecular mass of β-galactosidase released from the electropermeabilised cells was determined using Ferguson plots after running the samples in nondenaturing electrophoreses at five different polyacrylamide concentrations (6,7,8,9, and 10% PAGE). The following standards (Sigma Chemical, USA) were used: bovine serum albumin monomer and dimer (66,000 and 132,000 Da), urease trimer and hexamer (272,000 and 545,000 Da). β-Galactosidase activity staining was performed by incubating native gels in a 50 mM sodium phosphate buffer containing 5 mM ONPG (o-nitrophenyl-β-d-galactopyranoside) at 30°C for several minutes until a yellow band appeared.

3. Results 3.1. Optimisation of the electrical conditions Recent data obtained with various nonrecombinant yeast systems confirmed that significant release of intracellular proteins occurs under electrical conditions, leading to irreversible plasma membrane permeabilisation (Ganeva et al., 2003, 2004). The loss of membrane integrity depends strongly on the field intensity, number, and duration of the pulses applied. Thus, similar effects can be obtained with various combinations of these pulse parameters. We have established that the maximal release of homologous intracellular enzymes from various yeast species occurs by

application of series of 10-15 pulses with 0.5 - 2 ms duration, for which the optimal field intensity varies in the range of 3-4.5 kV·cm-1. Insofar as there is no data on the utilisation of a pulsed electric field for the extraction of recombinant proteins from yeast, we decided to apply a similar combination of parameters. To simplify the procedure, the cells in the late exponential phase (OD660=3.8-4) were treated with 15 pulses of 1.25-1.5 ms duration, and the electropulsation protocol was optimised only by varying the field intensity in the range of 3.5 to 6 kV·cm-1. Maximal β-galactosidase release, approximately 45% of the total activity was obtained at 5.2 kV·cm-1 (Fig. 1). At this intensity, approximately 97% of the cells had suffered irreversible plasma membrane permeabilisation, but the vacuolar membranes are to a large degree preserved, as detected by PI labelling (Fig. 2).

At higher field intensities, the enzyme activity in the supernatant dropped, which could be a result of inactivation due to an increase in the temperature during pulsation. The total protein release from intact control cells was approximately 1% after 20 h of incubation at room temperature in the same buffer. Although the membrane permeabilisation occurs during the electric pulse application, the cells continue to release proteins (enzymes) for 2-10 h after pulsation due to the limited cell wall porosity (Ganeva et al., 2003). Previous investigations demonstrated that the passage of native enzymes with molecular masses in the range 30-250 kDa through the permeabilised cell envelope is faster and more efficient than the release of the total protein. As a result, the specific activity of enzymes, such as phosphoglycerate kinase, glyceraldehyde 3-phosphate dehydrogenase, hexokinase, and β-galactosidase, liberated after electrical treatment was 1.8-2 times higher than that in the cell lysates, and a recovery of 85-90% of the total activity was obtained, even from cells in the stationary phase. Here at all tested field intensities, we found the opposite trend: the liberation of the total protein was considerably more efficient than that of the recombinant enzyme (Fig. 1). We analysed the release of the total protein and the recombinant β-galactosidase at various time intervals after pulse application and found that approximately 70% of the total protein was

liberated during the first 4 h after dilution of the permeabilised cells in buffer (Fig. 3). On the other hand, the release of β-galactosidase was very slow, and even after 20 h of incubation, the enzyme yield did not exceed 50% of the total.

To check the possibility for eventual inactivation of the recombinant enzyme during the electrical treatment, the electroporated cells were subjected to lysis, as described for control cells in the Materials and methods section. The electrical treatment itself did not induce more than 5 % loss of activity. This led us to conclude that a significant part of the recombinant enzyme was retained inside the cell because of its large mass, leading to very slow diffusion through the cell wall. The E. coli β-galactosidase in its native form is a tetramer with a molecular mass of 456 kDa, and the recombinant enzyme is even larger (528 kDa) because of the LYTAG. In fact, this is the largest protein we have ever liberated using a pulsed electric field. 3.2. Influence of dithiothreitol on β-galactosidase and the total protein liberation The yeast cell wall, with a thickness of approximately 100-200 nm, has a layered structure. The inner layer is composed mainly of β-1,3-glucan and is responsible for the mechanical strength of the wall, and an outer mannoprotein layer determines the cell wall porosity (Zlotnik et al., 1984). This cell wall property varies between the various yeast strains and depends strongly on the growth phase and culture conditions (Klis et al., 2002). An increase in the wall porosity can be achieved by breaking the disulphide bridges between the mannoproteins in the outer layer with a reducing agent (Zlotnik et al., 1984). Recently, we demonstrated that low concentrations of DTT in the postpulse incubation buffer significantly accelerate the release of larger proteins such as yeast β-galactosidase (250 kDa) (Ganeva et al., 2003). Here, we applied the same approach to enhance the release of the recombinant enzyme. After testing DTT concentrations between 1 and 10 mM (data not shown), we found that the maximal effect on the total protein and the β-galactosidase release can be obtained with concentrations in the range of 6 to 8 mM. DTT significantly accelerated the liberation of total protein and further increased its efficiency, leading to yields over 80%. A positive effect on the enzyme release during the first four hours was also observed, but overall, the reducing agent did not change the enzyme yield.

We performed similar experiments with the strain W303 [pMAB10-LacZ-5]. The cells in the late exponential phase (OD660=3.8-4.2) were subjected to pulses of a 1.5-ms duration, 4 Hz, and a flow rate of 4.8 ml/min. At these conditions, the optimal field intensity for β-galactosidase extraction was 4.5 kV/cm. The kinetics of the β-galactosidase and total protein release in the presence of 8 mM DTT were very similar to that observed with strain W303 [pBIVU02-1], and the maximal liberated activity was approximately 45% of the total, after 20 h of postpulse incubation. In view of the difference in the total protein and β-galactosidase release kinetics, we tested the possibility of performing a partial purification of the recombinant protein by simply replacing the buffer after 1-2 h of the initial incubation: the two-step incubation of protocol A (see the Materials and methods section for details).

As one can see in Fig. 4, after 1.5 h of incubation in the presence of DTT, approximately 50% of the total protein and only 5% of total β-galactosidase activity was released. During the next 18.5 h after substitution of the medium, a release of approximately 40% of the total β-galactosidase activity and 30% of the total protein was detected. Comparison of these results (Fig. 4B) with the yields obtained without substitution of the medium (Fig. 4C) shows that, as expected, this approach led to a significant enrichment of the recombinant protein in the medium. Nevertheless, the enzyme yield remained twofold lower than the yields obtained in our previous explorations (Ganeva et al., 2003), which could be due to the limited cell wall porosity. 3.3. Lyticase test for determination of the cell wall porosity. The susceptibility of yeasts to lytic enzymes (lyticase, zymolyase) is often utilised to compare the cell wall porosity between various strains and to reveal changes in the wall structure, in dependence on the physiological state, growth conditions, chemical agents, or mutations (DeNobel et al, 2000, Simoes et al., 2003). The activity of lyticase is due to the synergistic action of two enzymes: β-1,3-glucanase and alkaline protease (Salazar and Asenjo, 2007). In the first stage of the enzyme lysis, the protease binds to the wall surface and opens holes in the outer mannoprotein layer, thus exposing the

glucan network below the glucanase. Chemical agents perturbing the structure of the mannoprotein layer, such as reducing or oxidative compounds, detergents, high salt concentrations and others, also greatly facilitate the action of the glucanase (Ganeva et al., 2004 and Pringle and Mor, 1975). The application of PEF with parameters suitable for the efficient recovery of intracellular proteins leads to an increase in the yeast wall porosity, thus making the cells more susceptible to lyticase digestion (Ganeva et al., 2014). Here, we compared the sensitivity to lyticase of the control cells and cells subjected to electrical pulses with various field intensities. As shown in Fig. 5, the PEF significantly changed the sensitivity of the cells to lyticase. The maximal effect was observed at 5.2 kV·cm-1, the optimal intensity for protein and βgalactosidase liberation. At higher field intensities, a degree of reversion of this effect was observed, the reason for which is not clear yet. These results confirm that the electrical treatment induced an increase in the cell wall porosity of the recombinant strain, as already observed with other yeast systems (Ganeva et al., 2014). However, it was obviously insufficient to allow an effective liberation of the recombinant β-galactosidase.

3.4. Influence of lyticase on the enzyme liberation from electrically treated cells Recently, we found that the addition of a very low concentration (1-2 U/ml) of lyticase to electropermeabilised yeast cells enhances the rate of protein liberation without inducing significant cell lysis. This effect was observed even with cells with very strong and impermeable cell walls. Here, we decided to verify if a combination of electrical and enzyme treatment could increase the efficiency of liberation of the recombinant protein. The effect of lyticase on βgalactosidase liberation was studied using a two-step incubation similar to that described above, but with some changes (protocol B – see the Materials and methods section for details). To reduce the β-gal liberation during the first step, these experiments were performed with cells in the late exponential phase (OD660=4.6-6), and the duration of the first incubation was extended to remove more of the total protein. After 2-3 h of incubation in the presence of 6-8 mM DTT, the cells liberated approximately 60% of the total protein and only 7% from the beta-galactosidase activity. After removal of the liberated protein, lyticase was added in various final

concentrations, and the cells were further incubated for 2-16 h. As shown on Fig. 6A, even in at a very low concentration (1 U/ml final concentration), lyticase led to release of 50% of the total βgalactosidase activity after incubation for 6 h, and the yield attained 70 % from total activity after incubation for 16 h. At higher lyticase concentrations, the liberation became faster and more efficient (Fig. 6B). The specific activity of liberated β-galactosidase was 6.6 ± 0.9 U/mg, which is approximately 2.6 times higher than that in lysate (2.5 ± 0.2 U/mg). For comparing the yields obtained with the developed protocol, a standard procedure for mechanical cell disintegration was also applied. The cells were disrupted with a vibration homogenizer VHG1 and the total protein and β-galactosidase activity were determined in the clarified extracts. This procedure led to liberation of about 60 % from the total enzyme activity, and the specific activity was 1.7 ± 0.3 U/mg. This lower efficiency is not surprising taking into account that the galactose induction leads to a strong decrease of cell wall porosity (AguilarUscanga and Francois, 2003) and obviously to an increase of wall rigidity, which makes the cells more resistant to mechanical lysis. For obtaining a lysis of about 95% of the cells a combination of enzyme and mechanical treatment was necessary as described in Material and methods. The supernatants of the permeabilised cells were checked for protease activity under conditions where no significant cell lysis occurred. The liberated protease activity from permeabilised cells, after 6h incubation with 1 U/ml lyticase was 8 ± 2%, and after 4h incubation with 10 U/ml lyticase was 12 ± 3% from that of the lysate, which indicates that at these conditions the vacuoles intactness was largely preserved. We found that 6 h of incubation with 10 U/ml lyticase induced lysis of approximately 20% of the pulsed cells. This led us to conclude that the β-galactosidase liberation reaching nearly 70% of the total activity is due mainly to the increased wall permeability, but the cell lysis also contributed to this effect. Electrically treated cells do not efficiently release neither the recombinant enzyme nor the total protein without lyticase (Fig. 7). On the other hand, control cells incubated for 6 h with lyticase in concentrations of 1 or 10 U/ml liberate only 2% and 11%, respectively, of the total βgalactosidase activity, obviously as a result of cell lysis. These results unequivocally demonstrate that electrical treatment and lyticase have a synergistic effect.

3.5. Analysis of the liberated proteins by gel electrophoresis

The proteins liberated from the electroporated cells during the first and second steps of incubation in protocol B were analysed by SDS electrophoresis. Evidently, a large portion of the total protein is released during the first step of incubation. The most intense band in the second supernatant, appearing at approximately 118 kDa, likely corresponds to the recombinant protein.

The proteins released from electroporated cells at the second step of the protocol B were also separated on the native gel. The staining for β-galactosidase activity (Fig. 8B) revealed only one band in the running gel. The calculated molecular mass is approximately 530 kDa, which corresponds to the native tetramer form of the recombinant enzyme. A second, very intense band that appeared at the top of the stacking gel suggests that a portion of the liberated enzyme is in the form of aggregates. 4. Discussion The method utilised for the intracellular product release from cells has a great impact on the entire downstream process (Balasundaram et al., 2009 and Garcia, 2013). A higher selectivity at this stage can reduce and/or simplify the next purification steps, thus leading to lower manufacturing costs. Cell permeabilisation induced by physical or chemical treatments is of particular interest because the treatments are milder than mechanical disintegration, but most importantly, because it ensures selective product release and avoids contamination with small fragments of cell debris, hampering the further steps of extract clarification and purification. There are several examples of utilisation of chemical permeabilisation for protein recovery from yeast (Naglak et al., 1990, Shepard et al., 2002 and Garcia, 2013). Most often, the permeabilisation is achieved by treatment with solvents or detergents. These compounds destabilise the membranes nonselectively by influencing the lipid-lipid and lipid-protein interactions. After entering into the cell, they also destroy the intracellular membrane systems, including vacuoles, where the main proteolytic activity is concentrated. The efficiency of chemical treatment varies and depends on the yeast system and the molecular mass of the protein of interest (Naglak et al., 1990). Due to the high resistance of the yeast cell wall to chemical agents, a combination of different chemicals or chemical and mechanical treatments is often necessary for efficient protein recovery (Naglak et al., 1990, Bansal-Mutalik and Gaikar, 2006 and Ferrara et al., 2010).

The pulsed electrical treatment as a method for cell permeabilisation has several advantages over chemical methods. It can be easily adapted to various yeast strains and species, and at definite electrical conditions, it does not affect the protein activity. There are no contaminants that can additionally complicate the downstream process or impose a fireproof environment. A very important difference is that, by applying electrical pulses, one can selectively permeabilise the plasma membrane without changing the integrity of the vacuoles, as confirmed here for the recombinant yeast strains. This fact makes the electropermeabilisation suitable for the recovery of proteins that are very sensitive to proteolytic degradation. The yeast cell wall is a relatively thick and rigid structure that limits the passage of macromolecules into and out of cells. The conditions applied for the growth and induction of recombinant yeast strains often leads to further strengthening of the cell walls and to a decrease in the permeability. As a result, the cells become more resistant to mechanical disintegration, as well as to enzyme lysis and chemical permeabilisation, which complicates the extraction of intracellular and periplasmic proteins. The electrical treatment, leading to efficient protein release, not only permeabilised the plasma membrane but also induced an increase in the cell wall porosity. This facilitates the liberation of proteins, even from cells with very strong cell walls; additionally, it makes the cells more sensitive to lyticase treatment. We observed this effect for both recombinant strains. Depending on the growth phase, the electropermeabilised cells liberated a significant portion (60-80%) of the total protein (Figs. 3 and 4). However, most of the recombinant protein was retained inside, obviously due to its large molecular mass. As shown earlier, the addition of a very low concentration of lytic enzyme to the electrically treated cells greatly enhanced the protein release from yeasts with very strong cell walls. To improve the liberation of the recombinant β-galactosidase, we applied a similar approach here. We observed that even at a concentration of 1-2 U/ml, the lyticase leads to efficient release of the recombinant enzyme. Because most of the total protein was already removed, the specific activity of β-galactosidase obtained was 2.6 times higher than the specific activity of the cell lysate. The release of the recombinant enzyme at these lyticase concentrations is long lasting, but we did not observe any inactivation of the recombinant protein, even at longer (10 h and more) incubations. The high stability of the recombinant enzyme in the medium without protease inhibitors can be explained by the preserved vacuolar intactness. On other hand, because we

applied a very low lyticase concentration, the proteases presented in the preparation are obviously not detrimental to recombinant enzymes. Faster liberation of β-galactosidase can be obtained by applying higher lyticase concentrations (5-10 U/ml). PEF treatment and subsequent incubation with lyticase have a synergistic effect on βgalactosidase liberation. The electrical treatment itself has a limited efficiency for the recovery of large intracellular proteins, even in the presence of a reducing agent. On the other hand, the lytic enzyme concentrations (1-10 U/ml) that were very efficient with electropermeabilised cells do not induce significant liberation of the recombinant enzyme nor the total protein from the intact control cells. For efficient lysis of the control cells, five to ten times higher enzyme concentrations were required. In conclusion, the protocol developed in this study allows the liberation and partial purification of large intracellular proteins from yeast. It is based on the electropermeabilisation of the plasma membrane and on an increase in the cell wall porosity by electric field pulses and lyticase, treatments that have a synergistic effect. As the electric field treatment, performed in the flow mode, can be easily scaled and the lytic enzyme concentration applied is relatively low, this procedure could be useful for the production of large recombinant and native proteins from yeast.

Acknowledgements. This research received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreements n° 222220 and 312004.

References Aguilar-Uscanga. B., Francois, J. 2003. A study of yeast wall composition and structure in response to growth conditions and mode of cultivation. Lett. Appl. Microbiol. 37, 268-274. Balasundaram, B., Harrison, S., Bracewell. D.G., 2009. Advances in product release strategies and impact on bioprocess design. Trends Biotechnol. 27, 477-485. Bansal-Mutalik, R., Gaikar, V.G., 2006. Reverse micellar solutions aided permeabilisation of baker’s yeast. Process Biochem. 41, 133-141. Bradford, M., 1976. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

De Nobel, H., Ruiz, C., Martin, H., Morris, W., Brul, S., Molina, M., & Klis, F. M., 2000. Cell wall perturbation in yeast results in dual phosphorylation of the Slt2/Mpk1 MAP kinase and in an Slt2-mediated increase in FKS2–lacZ expression, glucanase resistance and thermotolerance, Microbiol-UK, 146, 2121–2132. Demain, A.L., Vaishnav, P., 2009. Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 27, 297-306. Ferrara, M.A., Bonomo Severino, N.M., Valente, R.H., Perales, J., Bon, E.P.S., 2010. Highyield extraction of periplasmic asparaginase produced by recombinant Pichia pastoris harbouring the Saccharomyces cerevisiae ASP3gene. Enzyme Microb. Tech. 47, 71-76. Ganeva, V., Galutzov, B., Teissie, J., 2003. High yield electroextraction of proteins by a flow process. Anal. Biochem. 315, 77-84. Ganeva, V., Galutzov, B., Teissie, J., 2004. Flow process for electroextraction of intracellular enzymes from the fission yeast Schizosaccharomyces pombe. Biotechnol. Lett 26, 933-937. Ganeva, V., Galutzov, B., Teissie, J., 2014. Evidence that Pulsed Electric Field Treatment Enhances the Cell Wall Porosity of Yeast Cells. Anal. Biochem. Biotechnol. 172, 1540-1552. Garcia, F.A.P., 2013. Cell wall disruption and lysis in Flickinger, M.C. (ed) Downstream Industrial Biotechnology: Recovery and Purification, First Edition, John Wiley & Sons, Inc pp. 81-94. Gerngross, T.U., 2004. Advances in the production of human therapeutic proteins in yeast and filamentous fungi. Nat. Biotechnol. 22, 1409-1414. Heim, A., Kaminowska, U., Solecki, M., 2007. The effect of microorganism concentration on yeast cell disruption in bed mill. J. Food Engin. 83, 121-128. Huang, C.J., Low, A.J., Batt, C.A., 2010. Recombinant immunotherapeutics: current state and perspectives regarding the feasibility and market. Appl. Microbiol. Biot. 87, 401- 410. Kim, E.-J., Pack, Y.-K., Lim, H.-K., Park, Y.-Ch., 2009. Expression of hepatitis B surface antigen S domain in recombinant S.cerevisiae using GAL 1 promoter. J. Biotechnol. 141, 155-159. Klis, F.M., Mol, P., Hellingwerf, K., Bral, S., 2002. Dynamics of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 26, 239-256. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.

Liu, D., Lebovka, N.I., Vorobiev, E., 2013. Impact of electric pulse treatment on selective extraction of intracellular compounds from Saccharomyces cerevisiae yeasts. Food Bioprocess Tech. 6, 576-584. Mahnič-Kalamiza, S., Vorobiev, E., Miklavčič, D., 2014. Electroporation in food processing and biorefinery. J. Membrane Biol. 247, 1279-1304. Mbuyi-Kalala, A., Schneck, A.G., Leonis, J., 1988. Separation and characterization of four enzyme forms of β-galactosidase from Saccharomyces lactis. Eur. J. Biochem. 178, 437-443. Naglak, T.J., Hettwer, D.J., Wang, H.Y., 1990. Chemical permeabilization of cells for intracellular product release, In: Asenjo A.S. (ed.) Separation processes in biotechnology, Marcel Dekker INC New York, pp. 177-205. Nedeva, T., Dolashka-Angelova, P., Moshtanska, V., Voelter, W., Petrova, V., Kujumdzieva, A., 2009. Purification and partial characterization of Cu/Zn superoxide dismutase from Kluyveromyces marxianus yeast. J. Chromatogr. B 877, 3529-3536. Nesterenko, M.V., Tilley, V., Upton, S.J., 1994. A simple modification of Blum’s silver staining method allows for 30 min detection of proteins in polyacrylamide gels. J. Biochem. Bioph.. Meth. 28, 239-242. Oshima, T., Sato, M., 2004. Bacterial sterilization and intracellular protein release by a pulsed electric field. Adv. Biochem. Eng. Biot. 90, 113-133. Pringle, J.R., Mor, J.-R., 1975. Methods for monitoring the growth of yeast cultures and dealing with the clamping problems. In Prescot, D. M. (ed), Methods in Cell Biology, XI Yeast Cells, Academic Press, pp. 131-169. Simões, T., Teixeira, M. C., Fernandes, A. R., & Sá-Correia, I., 2003. Adaptation of Saccharomyces cerevisiae to the herbicide 2, 4-dichlorophenoxyacetic acid, mediated by Msn2p-and Msn4p-regulated genes: important role of SPI1, Appl. Environ. Microb. 69, 4019–4028. Shepard, S.R., Stone, C., Cook, S., Bouvier, A., Boyd, G., Weatherly, G., Lydiard, D., Schrimsher, J., 2002. Recovery of intracellular recombinant proteins from the yeast Pichia pastoris by cell permeabilization. J. Biotechnol. 99, 149-160. Sikorski, R.S., Hieter, P., 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27. Thomas, B.J., Rothstein, R., 1989. Elevated recombination rates in transcriptionally active DNA. Cell, 56, 619-630.

Vorobiev, E., Lebovka, N., 2011. Pulse electric field assisted extraction. In: Lebovka, N., Vorobiev, E., Chemat, F. (eds) Enhancing Extraction Processes in the Food Industry, CRC Press, Boca Raton, FL., pp.25-84. Weaver, J.C., Chizmadzhev Yu.A., 1996. Theory of electroporation: A review. Bioelectrochem. Bioenerg. 41, 135-150. Zhang, Q., Li, W., Chen, J., Wang, F., Wang, Y., Chen, Y., Yu, C., 2013. An ultrasensitive chemiluminescence turn-on assay for protease and inhibitor screening with a natural substrate. Chem. Commun. 49, 3137-3139. Zlotnik, H., Fernandez, M.P., Bowers, B., Cabib, E., 1984. Saccharomyces cerevisiae mannoproteins form an external layer that determinates wall porosity. J. Bacteriol. 159 (3), 1018-1026.

FIGURE CAPTIONS

Fig. 1. Influence of the field intensity on the release of total protein and β-galactosidase from

W303-1A [pBIVU02-1]. Experimental conditions: 15 pulses, 1.25 ms duration, 2 Hz, and flow rate of 2.4 ml·min-1. The cells were incubated in a potassium phosphate buffer for 20 hours after pulsation. 100% corresponds to the total protein content and β-galactosidase activity in lysate.

Fig. 2. Light and fluorescent micrographs of control and electropermeabilized yeast cells: А)

control cells; B) propidium iodide labeled electropermeabilized cells; C) micrograph of electropermeabilized cells. Experimental conditions: 15 pulses, 5.2 kV/cm, 1.25 ms duration, 2 Hz. Magnification – 40x.

Fig. 3. Electroinduced release of β-galactosidase and total protein at various periods of postpulse

incubation. Experimental conditions: 5.2 kV/cm, 15 pulses, 1.25 ms duration, 2 Hz, and a flow

rate of 2.4 ml·min-1. Final DTT concentration - 8 mM. 100% corresponds to the total protein content and β-galactosidase activity in lysate.

Fig. 4 Influence of media substitution on the protein and β-galactosidase release. Experimental

conditions: 15 pulses, 1.25 ms, 2 Hz, and 5.2 kV·cm-1. The total protein and β-galactosidase activity liberated during the first 1.5 h in a medium with DTT (A), after replacing the medium with fresh medium w/o DTT and further incubation for 18.5 h (B), or after 20 h without medium replacement (C). 100% corresponds to the total protein content and β-galactosidase activity in lysate.

Fig. 5. Influence of PEF on the lyticase sensitivity of strain W303-1A [pBIVU02-1].

Experimental conditions: 15 pulses, 1.25 ms in duration, 2 Hz, and 2.4 ml·min-1. The final concentration of the lyticase is 65 U/ml.

Fig. 6. Influence of lyticase on the β-galactosidase and protein liberation from electrically treated

cells from the strain W303 [pMAB10-LacZ-5]. Electrical conditions: pulse duration 1.5 ms, 4 Hz, field intensity 4.5 kV/cm, and flow rate 4.8 ml/min. After 3 h of incubation with DTT, the cells were washed to remove the released protein, diluted in buffer containing 1(A) or 10 U/ml (B) lyticase and incubated at room temperature. 100% corresponds to the total protein content and β-galactosidase activity in lysate.

Fig. 7. Influence of lyticase on β-galactosidase and the total protein release.

The control and electrically treated cells were incubated for 3 h in a buffer containing 8 mM DTT. Afterward, the supernatant was removed, and the cells were diluted in buffer with or without lyticase and incubated for 6 h at room temperature. 100% corresponds to the total protein content and β-galactosidase activity in lysate.

Fig. 8. Electrophoretic analysis of the proteins in the cell lysate and the supernatant of the

electrically treated cells. (A) 10% SDS electrophoresis: 1 – markers; 2 – cell lysate; 3 – supernatant after the first step of incubation; 4 – supernatant after incubation for 6 h with 10 U/ml lyticase; (B) – native 7 % gel electrophoresis of the proteins released after the second step of incubation, stained for beta-gal activity.

Fig. 1.

Fig. 2.

color required

Fig. 3.

Fig.4.

Fig. 5.

Fig. 6

Fig. 7.

Fig. 8. A

1

2

3

4

B

118 kDa

Color required 66 kDa

43 kDa

545 kDa 29 kDa 272 kDa

20 kDa