title: Electroinduced release of recombinant β-galactosidase from Saccharomyces cerevisiae.
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 Saccharomyces cerevisiae strains expressing LYTAG-β-galactosidase from Escherichia coli. The permeabilzation of plasma membrane was induced by application of rectangular electric pulses, with 1.25ms duration and field intensity of 4.3-5.4kV/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 20h 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 (1-2U/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.
authors.affiliation: Sofia University, Department of Biophysics and Radiobiology, 8 Dragan Tzankov blvd, 1164 Sofia, Bulgaria. Electronic address: valentinaganeva@abv.bg.
authors.forename: Valentina
authors.initials: V
authors.lastname: Ganeva
authors.affiliation: Sofia University, Department of Biophysics and Radiobiology, 8 Dragan Tzankov blvd, 1164 Sofia, Bulgaria.
authors.forename: Debora
authors.initials: D
authors.lastname: Stefanova
authors.affiliation: Sofia University, Department of Biophysics and Radiobiology, 8 Dragan Tzankov blvd, 1164 Sofia, Bulgaria.
authors.forename: Boyana
authors.initials: B
authors.lastname: Angelova
authors.affiliation: Sofia University, Department of Biophysics and Radiobiology, 8 Dragan Tzankov blvd, 1164 Sofia, Bulgaria.
authors.forename: Bojidar
authors.initials: B
authors.lastname: Galutzov
authors.affiliation: Biomedal S.L., Avda. Américo Vespucio 5E. 1∘ M12, 41092 Seville, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Velasco
authors.affiliation: Biomedal S.L., Avda. Américo Vespucio 5E. 1∘ M12, 41092 Seville, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Arévalo-Rodríguez
mesh_terms: D002473:Cell Wall / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004560:Electricity; D004591:Electrophoresis, Polyacrylamide Gel; D018274:Electroporation / Q000379:methods*; D004693:Glucan Endo-1,3-beta-D-Glucosidase / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism; D016062:Porosity; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D001616:beta-Galactosidase / Q000302:isolation & purification*
chemical_list: D003470:Culture Media; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C025310:lyticase; D001616:beta-Galactosidase; D004693:Glucan Endo-1,3-beta-D-Glucosidase; D010447:Peptide Hydrolases; D004229:Dithiothreitol
keywords: E. coli β-Galactosidase; Electropermeabilisation; Extraction; Lyticase; Pulsed electric field (PEF); Recombinant protein; S. cerevisiae