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WO1996002629A2 - Procede de liberation de proteines heterologues de souches de saccharomyces cerevisiae - Google Patents

Procede de liberation de proteines heterologues de souches de saccharomyces cerevisiae Download PDF

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Publication number
WO1996002629A2
WO1996002629A2 PCT/ES1995/000088 ES9500088W WO9602629A2 WO 1996002629 A2 WO1996002629 A2 WO 1996002629A2 ES 9500088 W ES9500088 W ES 9500088W WO 9602629 A2 WO9602629 A2 WO 9602629A2
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Prior art keywords
proteins
protein
saccharomyces cerevisiae
release
cells
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Application number
PCT/ES1995/000088
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English (en)
Spanish (es)
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WO1996002629A3 (fr
WO1996002629B1 (fr
Inventor
César NOMBELA CANO
Pablo Alvarez Alvarez
Marta Sampedro Martinez
Jesús DE LA FUENTE CARRETERO
María Molina Martin
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Universidad Complutense De Madrid
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Application filed by Universidad Complutense De Madrid filed Critical Universidad Complutense De Madrid
Priority to AU30787/95A priority Critical patent/AU3078795A/en
Publication of WO1996002629A2 publication Critical patent/WO1996002629A2/fr
Publication of WO1996002629A3 publication Critical patent/WO1996002629A3/fr
Publication of WO1996002629B1 publication Critical patent/WO1996002629B1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • C12N1/063—Lysis of microorganisms of yeast

Definitions

  • the present invention falls within Biotechnology and specifically in the field of expression, production and release of heterologous proteins in yeast.
  • the invention provides a method for carrying out said release, based on simple treatments of certain strains of Saccharomyces cerevisiae, carriers of a mutation that leads to the release of intracellular content, thus obtaining protein preparations that are easily separated from the greater part of the undesirable cellular materials present in said preparations, such as cell walls. Protein preparations thus obtained can be subjected to the corresponding extraction and purification processes.
  • the mutation of which Saccharomyces cerevisiae cells are used in the process object of this invention affects the gene called SLT2; as a consequence of said mutation, the cells lose their integrity under certain conditions which leads to the release of their soluble protein content.
  • the existing knowledge about the function of the aforementioned gene largely developed in our laboratory, allows us to take advantage of the characteristics of this type of mutant cells to optimize said protein release.
  • the process object of this invention represents an alternative to other methods of releasing heterologous yeast proteins such as mechanical, chemical or enzymatic breakage of cellular integrity.
  • yeast Saccharomyces cerevisiae is a well known organism and widely used in Biotechnology for the expression and production of heterologous proteins. Several characteristics of this species stand out in relation to its usefulness:
  • Yeasts are used to produce a series of heterologous proteins. of high added value, as a consequence of a process of expression of the corresponding genes, in processes in which the protein accumulates at the intracellular level.
  • these are the surface antigen of the hepatitis B virus (HBV) that has given rise to the development of a recombinant vaccine already on the market, human proinsulin, human superoxide dismutase, VLP proteins as presenters of HIV1 virus antigens and the antigen that has allowed to develop a vaccine against malaria.
  • HBV hepatitis B virus
  • human proinsulin human superoxide dismutase
  • VLP proteins as presenters of HIV1 virus antigens and the antigen that has allowed to develop a vaccine against malaria.
  • the possibilities in this regard are widely collected in Romanos, M.A., Scorer, C.A. and Clare, J.J. 1992, Foreign gene expression in yeast: a review. Yeast 8: 423-488.
  • cell breakage is usually used.
  • the traditional methods described for achieving said cell rupture are drastic due to the hardness of the Saccharomyces cerevisiae cell wall and may consist of: - mechanical rupture with pressure homogenizers (Scdazzling, H. and
  • VLPs recombinant proteins particles
  • SUBSTITUTED SHEET Protein extraction is not always easy to eliminate and, in the case of the use of lithic enzymes.
  • Protease contamination (Asenjo, JA, Ventom.AM. Huang, RB and Andrews. BA 1993. Selective relay of recombinant proteins particles (VLPs) fro yeast using a puree lytic glucanase enzyme. Bio / echnology 11: 214-217) that They are usually present in the aforementioned complexes of lytic enzymes.
  • the alternative of carrying out a purification to eliminate proteases increases the value of the lithic complex that is already expensive in itself.
  • Saccharomyces cerevisiae carrying the sltl mutation that is to say it affects the gene
  • the SLT2 gene encodes a protein with phosphorylating activity of proteins, which is part of the group called "Protein kinase type MAP" (Mitogen Activated Protein) (Mart ⁇ n, H., Arroyo, J., Sánchez, M .. Molina, .M. And Nombela. C. 1993. Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37 ° C. Mol. Gen. GeneL 241: 177-184) and whose biological action seems to be framed in a route of signal transmission activated by the protein kinase Cl, the signal that triggers the activation of the route as well as the final substrate thereof is currently unknown.
  • Protein kinase type MAP Mitogen Activated Protein
  • yeast proteases Another factor to consider when addressing the expression of heterologous proteins in yeasts is the possibility of nonspecific proteolytic degradation of the corresponding protein, produced by the action of yeast proteases, especially those of vacuolar origin such as those encoded by PEP4 and -P ⁇ -S7 genes (Wingfield, JM and Dickinson, JR 1993. Increased activity of a model heterologous protein in Saccharomyces cerevisiae strains with reduced vacuolar proteinases. Appl. Microbiol. Biotechnol. 39: 211-215; Jones, EW 1991 Tackling the protease problem in Saccharomyces cerevisiae Meth in Enzymol 194: 428-453;
  • strains more suitable for our invention that is, carriers of the lithic character (slt2), but which are also deficient in vacuolar proteases.
  • the first studies of heterologous protein release according to the procedure of this invention were performed by inducing cell lysis by thermal shock, consisting of raising the temperature of the culture from 24 ° C to 37 ° C, reaching 6 to 8 hours maximum protein release, as that is the time required to achieve a culture lysis.
  • the patent process consists in the use of different strains of Saccharomyces cerevisiae, deficient in the function of the SLT2 gene, whose phenotype is characterized by:
  • -Thermosensitive growth so that the cells develop well at 24 ° C but stop growing and lysate when the temperature rises to 37 ° C, producing cell lysis with release of intracellular content to the external medium.
  • -sltl mutant cells are protected against the deficiencies indicated above, ie thermosensitivity and lysis at 37 "C, by osmotic stabilization of the medium with 0.5M sorbitol or 1.5% NaCl, which is indicative of the protein encoded by the referred gene is necessary for the formation of an osmotically stable cell wall at the temperature of 37 ° C.
  • the objective of the invention is to take advantage of these phenotypic characteristics to achieve the release of intracellularly produced heterologous proteins. Said release is based on the cell lysis that occurs under certain conditions and that leads to the release of intracellular content that said mutant strains present when the growth temperature rises from 24 * C to 37 * C or when, after growing at 37 * C but in an osmotically stabilized medium, they are subjected to an osmotic shock by simple resuspension of the cells in a medium lacking said osmotic stabilizer. It is obtained in this way
  • SUBSTITUTED SHEET a protein preparation, which is separated from the remains of the cellular material by simple centrifugation, and in which the intracellular heterologous proteins are present in amounts greater than 50% of the total intracellular protein produced.
  • strains of Saccharomyces cerevisiae developed to carry out the invention are:
  • - LD1 is a diploid strain carrying the slt2 mutation with the phenotypic characteristics listed above. Its genotype is: MATa MATa, slt2D-35 / slt2D-35, Ieu2-3.U2 / Ieu2-3.U2, his4 ⁇ 34 / his4A34.
  • - LHDP1 MAT a, slt2D-35, ade2-101, leu2-3.112, pep4 :: HIS3, pbrlA1.6R. It shows the same phenotypic characteristics related to lysis, being also deficient in vacuolar proteases Pep4 and Prbl responsible for the nonspecific proteolysis.
  • strains are deposited in the Spanish Type Culture Collection (CECT) located at the University of Valencia.
  • CECT Spanish Type Culture Collection
  • the access numbers are: 10815 for strain LD1 and 10816 for strain LHDP1.
  • SUBSTITUTED SHEET B Protein release by thermal shock.
  • the phases of the process are:
  • the main advantage over the similar system based on the use of the srbl mutation is the greater sensitivity to osmotic shock of the slt2 mutation in which 60-70% of the cells are lysed against 20% of the cells mutated in - yrW (Broker, M. 1994. Isolation of recombinant proteins from Saccharomyces cerevisiae by use of osmoücally fragüe mutant strains. Biotechniques 16: 604-610).
  • Figure I shows the phases of the process of releasing intracellular heterologous proteins in strains of Saccharomyces cerevisiae mutated in the SLT2 gene by means of an osmotic shock.
  • the four panels in Figure II describe the release of homologous (alkaline phosphatase) and heterologous (chloramphenicol acetyl transferase) proteins from the strains of Saccharomyces cerevisiae LDl (A, C) and LHDPl (B, D) transformed with the plasmid pCHIOOL under conditions of expression of the slt2 mutation. Crops made in a 101-fermentor incubated at 24 ° C throughout the crop (A and B) or changed at 37 ° C (C and D) (T, temperature change).
  • the parameters measured were: +, viability measured as a percentage of IP cells (-),, Optical Density at 600 nm (OD); X, Total protein in the culture medium (Prot.); *, chloramphenicol acetyl transferase (CAT) activity in cell extracts (EC) and •, culture medium (M) as units per milliliter; *, alkaline phosphatase (PA) activity in cell extracts (EC) and A, culture medium (M) as units per milliliter.
  • CAT chloramphenicol acetyl transferase
  • EC chloramphenicol acetyl transferase
  • M culture medium
  • PA alkaline phosphatase
  • strains of Saccharomyces cerevisiae used are those described previously in the section "Explanation and Description of the Invention";
  • an optional wild strain can be used for the lithic character that serves as a negative control in the experiment, for example we have used:
  • - BJ5461 It is a wild strain for the SLT2 gene accessible to the public in the collection called Yeast Genetic Stock Center (Mortimer, R.K. and Contopoulou, R. Yeast Genetic Stock Center Catalog. Seventh edition 1991). However, any non-lyric strain can be used as a control strain for the experiments.
  • the three strains (LDl, LHDPl and BJ5461) are transformed with the plasmid pCHIOOL (Hadfield, C, Cashmore, AM and Meacock, PA 1986.
  • pCHIOOL Hadfield, C, Cashmore, AM and Meacock, PA 1986.
  • An efficient chloramphenicol resistance marker for Saccharomyces cerevisiae and Escherichia coli. Gene 45: 149-158) modified in our laboratory by introducing a LEU2 marker (leucine) to select transformants.
  • the plasmid encodes the CAT (Chloramphenicol Acetyl Transferase) protein of bacterial origin and responsible for chloramphenicol resistance and is a heterologous protein for yeast and is also produced intracellularly and therefore will only be detectable in the culture medium as consequence of cell lysis.
  • CAT Chloramphenicol Acetyl Transferase
  • the transformed strains are grown in liquid YEPD medium supplemented with an osmotic stabilizer (0.5M sorbitol, 1.5% NaCl) at a temperature of 37 ° C on a thermostated stirrer until half of the exponential phase.
  • an osmotic stabilizer 0.5M sorbitol, 1.5% NaCl
  • SHEET Cells to separate them from the culture medium are taken samples of supernatant and cells to obtain reference values about the release of CAT to the external medium (they are negative because the absence of lysis causes that there is no release of intracellular content to the external medium ).
  • the values to be measured are the activity of CAT in the cell extract and in the supernatant before and after the osmotic shock to establish the amount of protein released from the cells to the supernatant, following a colorimetric method described by Hadfield, C, Cashmore , AM and Meacock, PA 1986.
  • the cells are separated by centrifugation at 5000rpm for 5 ', then they are resuspended in water, at which time another sample is taken again by centrifuging the cell debris at 5000rpm for 10 min and analyzing the amount of CAT present in the cells and in the supernatant showing the data in table I, this preparation obtained after the second centrifugation leaves a protein preparation ready to undertake a process of purification of the protein of interest.
  • the wild strain in this case BJ5461, is not osmotically sensitive and there is no occurrence of CAT activity in the supernatant, unlike the other two strains that are sensitive to osmotic shock and have a 60-70 % of its activity in the supernatant.
  • Highlights the higher productivity of the LHDPl strain in accordance with what has been published in other studies (Romanos, MA, Scorer, CA and Clare, JJ 1992, Foreign gene expression in yeast: a review. Yeast 8: 423-488; Wingfield, JM and Dickinson, JR 1993. Increased activity of a model heterologous protein in Saccharomyces cerevisiae strains with reduced vacuolar proteins. Appl. Microbiol.
  • Table I Release of the heterologous CAT protein by osmotic shock of strains slt2. Samples of equal cell density were taken and the CAT activity was detected in: cell extract (before and after the osmotic shock), culture medium or water supernatant in which the cells were resuspended after the osmotic shock.
  • SUBSTITUTED SHEET B Protein release by thermal shock. The data obtained in type experiments are shown in Figure II.
  • the inoculum was obtained by culturing the cells at 24 ° C in minimal medium without leucine to ensure 100% plasmid carrying cells. Its volume was 500ml and corresponded to a stationary phase culture.
  • the crop was batch type. -the stirring speed was 300rpm, the pH ranged between 4 and 6, the air inlet flow 5 1 / min.
  • the two strains present protein release to the external environment represented by the increase observed in total proteins, measured by the Bradford method (Bradford, MM 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein using the principie of protein -dye binding. Analyt. Biochem. 72: 248-254), and in the enzymatic activity CAT (Hadfield, C, Cashmore, AM and Meacock, PA 1986. An efficient chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli. Gene 45 : 149-158).
  • the stability of the CAT enzyme proves to be superior in the LHDP1 strain than in the LDl, probably due to not being exposed to the action of the vacuolar proteases Pep4 and Prbl in the case of the LHDP1 strain.

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Abstract

L'invention décrit un procédé permettant la libération de protéines hétérologues intracellulaires dans la levure Saccharomyces Cerevisiae, ce procédé se basant sur la sensibilité osmotique que confère la mutation slt2 à la température de 37 °C. Cette sensibilité osmotique peut être utilisée pour la libération du contenu intracellulaire soit par changement de température de la culture de 24 °C à 37 °C, soit par transfert de cellules développées en milieu stabilisé osmotiquement à cette dernière température vers un milieu non stabilisé. Dans les deux cas, les cellules subissent une lyse libérant leur contenu intracellulaire, ce qui permet d'obtenir, après centrifugation afin d'éliminer les restes cellulaires, une préparation de protéines qui permet de commencer un processus de purification de la protéine d'intérêt. Ce procédé se traduit par une réduction des coûts en évitant l'utilisation d'équipement, et la préparation de protéines qu'on obtient contient moins d'impuretés que celle obtenue par d'autres procédés.
PCT/ES1995/000088 1994-07-14 1995-07-14 Procede de liberation de proteines heterologues de souches de saccharomyces cerevisiae WO1996002629A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU30787/95A AU3078795A (en) 1994-07-14 1995-07-14 Process for releasing heterolog proteins of saccharomyces cerevisiae strains

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ESP9401536 1994-07-14
ES9401536A ES2092439B1 (es) 1994-07-14 1994-07-14 Procedimiento de liberacion de proteinas heterologas de cepas de saccharomyces cerevisiae.

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999060138A3 (fr) * 1998-05-16 2000-01-20 Univ Manchester Expression regulee de pkc et/ou de srb1/psa1 dans la levure
WO2005104649A3 (fr) * 2004-04-30 2006-05-11 Consejo Superior Investigacion Levures (bgs4delta) autolithiques, procede d'obtention et applications desdites levures dans des procedes d'expression de proteines

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 38, num. 6, páginas 763-769, JESŸS MANUEL DE LA FUENTE ET AL. 'Expression of mutations and protein release by yeast conditional autolytic mutants in batch and continuous cultures' citado en la solicitud *
BIOTECHNIQUES, vol. 16, num. 4, NATICK US, páginas 604-610, MICHAEL BRÖKER 'Isolation of recombinant proteins from Saccharomyces cerevisiae by use of osmotically fragile mutant strains' citado en la solicitud *
J. BIOTECHNOL. (1994), 38(1), 81-8 CODEN: JBITD4;ISSN: 0168-1656, ALVAREZ, PABLO ET AL 'A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity' *
MOLECULAR AND GENERAL GENETICS, vol. 241, num. 1-2, BERLIN DE, páginas 177-184, HUMBERTO MARTÍN ET AL. 'Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37ŸC' citado en la solicitud *
MOLECULAR MICROBIOLOGY, vol. 5, num. 11, páginas 2845-2854, L. TORRES ET AL. 'A protein kinasr gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants' citado en la solicitud *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999060138A3 (fr) * 1998-05-16 2000-01-20 Univ Manchester Expression regulee de pkc et/ou de srb1/psa1 dans la levure
GB2353798A (en) * 1998-05-16 2001-03-07 Univ Manchester Regulated expression of PKC and/or SRB1/PSA1 in yeast
GB2353798B (en) * 1998-05-16 2003-11-12 Univ Manchester Regulated expression of SRB1/PSA1 in yeast
WO2005104649A3 (fr) * 2004-04-30 2006-05-11 Consejo Superior Investigacion Levures (bgs4delta) autolithiques, procede d'obtention et applications desdites levures dans des procedes d'expression de proteines

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WO1996002629A3 (fr) 1996-04-04
AU3078795A (en) 1996-02-16
ES2092439B1 (es) 1997-07-01
ES2092439A1 (es) 1996-11-16

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