WO1996002629A2

WO1996002629A2 - Process for releasing heterolog proteins of saccharomyces cerevisiae strains

Info

Publication number: WO1996002629A2
Application number: PCT/ES1995/000088
Authority: WO
Inventors: César NOMBELA CANO; Pablo Alvarez Alvarez; Marta Sampedro Martinez; Jesús DE LA FUENTE CARRETERO; María Molina Martin
Original assignee: Universidad Complutense De Madrid
Priority date: 1994-07-14
Filing date: 1995-07-14
Publication date: 1996-02-01
Also published as: WO1996002629A3; AU3078795A; ES2092439B1; ES2092439A1

Abstract

The invention relates to a process for releasing intracellular heterolog proteins in the Saccharomyces Cerevisiae yeast based on the osmotic sensitivity which confers the mutation slt2 at the temperature of 37 °C. Said osmotic sensitivity may be used for releasing the intracellular content either by change of temperature of the culture from 24° to 37° C, or by transfer of grown cells in an osmotically stabilized medium at said second temperature to a non-stabilized medium. In both cases, the cells undergo a lysis and release their intracellular content and, after centrifugation to eliminate the cellular rests, a preparation of proteins is obtained which is appropriate to initiate a process of purification of the protein of interest. This process results in a cost reduction since the use of equipment is avoided and the preparation of proteins which is obtained contains less impurities than that which is obtained through other processes.

Description

TITLE

Heterologous protein release procedure of Saccharomyces cerevisiae strains.

OBJECT OF THE INVENTION

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.

BACKGROUND

The 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:

SUBSTITUTED SHEET - It is a eukaryotic organism.

- it is an agency of the GRAS type ("Generally Recognized As Safe") according to the Food and Drug Administration (FDA) of the United States. that is to say generally recognized as safe, due to the absence of possible viral contaminants and pyrogens; This makes it a suitable host for the production of proteins with food and therapeutic use.

-There is a broad knowledge of its behavior on a large scale in fermenter for its use in traditional fermentations.

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. Among 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. 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.

For the recovery of these intracellularly produced heterologous proteins, 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 (Schütte, H. and

Kula, M.R. 1990. Pilot and process-scale techniques for cell disruption. Biotechnol Appl. Biochem 12: 599-620).

- chemical breakage using chemical detergents (Breddam, K. and Beenfeldt, T. 1991. Acceleration of yeast autolysis by chemical methods for production of intracellular enzymes. Appl. Microbiol. Biotechnol. 35: 323-329).

- Enzymatic breakdown using complex mixtures of lithic enzymes (Asenjo, JA, Ventom, AM Huang, RB and Andrews. BA 1993. Selective relay of recombinant proteins particles (VLPs) from yeast using a puree lytic glucanase enzyme. Bio / Technology 11: 214-217).

These methods have as disadvantages the possible degradation of the polypeptides due to the high pressures they may suffer in the homogenizers. the possible contamination that the chemical compounds used for the

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.

The technical problem whose resolution we pose is to achieve the release of heterologous proteins produced intracellularly in yeast, without having to perform a process of mechanical, chemical or enzymatic breakage of the cells. For this we rely on the use of certain strains of

Saccharomyces cerevisiae carrying the sltl mutation, that is to say it affects the gene

SLT2 discovered and cloned in our laboratory (Torres, L., Martín, H., García-Saez, MI, Arroyo, J., Molina, M., Sánchez, M. and Nombela, C. 1991. A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants. Mol. Microbiol. 5: 2845-2854; Fuente, JM, Vázquez, A., González, M.,

Sánchez, M., Molina, M. and Nombela, C. 1993 Expression of mutations and protein relée by yeast conditional autolytic mutants in batch and continuous cultures. Appl. Microbiol Biotechnol 38: 763-769).

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) (Martín, 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.

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 -PΛ-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;

REPLACED Pohlig, G .. Zimmermann, W. and Heim, J. 1991. Influence of yeast proteases on hirudin expression in Saccharomyces cerevisiae. Biomed Biochim Minutes 50: 711-716). Therefore, we have also developed 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.

EXPLANATION AND DESCRIPTION OF THE INVENTION

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.

The 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, his4Δ34 / 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.

Both strains are deposited in the Spanish Type Culture Collection (CECT) located at the University of Valencia. The access numbers are: 10815 for strain LD1 and 10816 for strain LHDP1.

The protein release procedure can be carried out as indicated in Figures I and II by one of the following alternatives:

A) Protein release by osmotic shock. The phases of the process are described in Figure I.

-growth of the mutant strain in the SLT2 gene at the restrictive temperature of 37 ^* C in the presence of an osmotic stabilizer up to half of the exponential phase.

-separation of the cells from the culture medium by centrifugation keeping the cells.

- Osmotic shock of the cells by resuspension of them in water, which leads to the lysis of them with the release of intracellular proteins, including the heterologous protein of interest.

-elimination of cell debris by centrifugation obtaining a crude preparation of intracellular proteins among which is the protein of interest, and preparation that is suitable for initiating purification processes.

SUBSTITUTED SHEET B) Protein release by thermal shock. The phases of the process are:

-growth of the mutant strain in the SLT2 gene at the permissible temperature of 24 ° C until half of the exponential phase.

- thermal shock of the culture by raising the growth temperature to 37 ° C, which causes lysis of the cells after 6-8 hours of incubation at that temperature, with release of the intracellular content to the culture medium.

-separation of cell debris from the culture medium (containing the proteins released as a result of cell lysis) by centrifugation, obtaining as in the previous case a crude protein preparation, carrier of the protein of interest, suitable for addressing purification processes

The parameters that indicate that there is lysis of the culture with release of intracellular content to the culture medium are explained in Figure II.

The main advantages of the invention are the following:

- allows to achieve a smooth and rapid release of proteins avoiding the use of high pressures in the homogenizers (Schütte, H. and Kula, MR 1990. Pilot and process-scale techniques for cell disruption. Biotechnol. Appl. Biochem. 12: 599 -620), of chemical products (Breddam, K. and Beenfeldt, T. 1991. Acceleration of yeast autolysis by chemical methods for production of intracellular enzymes. Appl. Microbiol. Biotechnol. 35: 323-329) and lithic enzymes (Asenjo JA, Ventom, AM, Huang, RB and Andrews, BA 1993. Selective relée of recombinant proteins particles (VLPs) from yeast using a puree lytic glucanase enzyme Bio / Technology 11: 214-217) to digest the wall; which also translates into a cheaper process.

- prevents contamination of the protein preparation that can occur in the case of extraction with chemicals that add impurities to protein preparations not always easy to remove (Breddam. K. and Beenfeldt, T. 1991. Acceleration of yeast autolysis by chemical methods for production of intracellular enzymes. Appl. Microbiol. Biotechnol. 35: 323-329) or of protease contamination in the case of lithic enzyme extraction that results in degradation of the polypeptides (Asenjo, JA, Ventom. AM, Huang, RB and Andrews, BA 1993. Selective relay of recombinant proteins particles (VLPs) from yeast using a puree lytic glucanase enzyme. Bio Technology 11: 214-217).

- the possibility of concentrating the initial culture volume, producing the osmotic shock together with the fact that heterologous proteins can be

SUBSTITUTED SHEET expressed under the control of strong promoters which means that their relative proportion can be high which facilitates the subsequent purification thereof.

- 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).

BRIEF DESCRIPTION OF THE DRAWINGS

To facilitate the understanding of the features of the invention, two explanatory figures are attached. 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.

SUBSTITUTED SHEET DETAILED EXHIBITION OF AN EMBODIMENT OF THE INVENTION

We will now explain by way of an example, not limiting its scope, an embodiment of the invention in the two modalities set forth above.

The strains of Saccharomyces cerevisiae used are those described previously in the section "Explanation and Description of the Invention"; In addition, 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. 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.

Previous studies have demonstrated a good stability of the plasmid which allows working with a non-selective YEPD type (1% yeast extract, 2% peptone, 2% glucose) that allows faster growth and better expression of the lyric phenotype.

A) Protein release by osmotic shock. The process phases are summarized in Figure I, included in the "Explanation and description of the invention" section.

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. Before picking up the

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. An efficient chloramphenicol- resistance marker for Saccharomyces cerevisiae and Escherichia coli. Gene 45: 149-158). As indicated in Figure I, 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.

As expected, 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. Biotechnol. 39:21 1-215). These data are supported by parallel measures of total protein present in cell extracts and supernatants following the colorimetric method described by Bradford, M.M. 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, showing profiles similar to those of the CAT activity.

SUBSTITUTED SHEET TABLE I

CAT ACTIVITY (EU / ml)

YEPD + soibitol 37 * CH ₂ O

Strains Cell extract Supernatant Cell extract Supernatant% CAT §

BJ5461 100 n.d. 98 n-d. 0

LDl 75 n.d 23 50 70

LHDPl 220 n-d. 80 130 60

n.d .: not detected

§:% CAT released to the supernatant

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.

Experiments in this modality have been carried out using a 101-volume cutaway fermenter model BIOSTAT E (Braun); which gives the results an additional value since a fermenter of these characteristics represents a scale greater than that of the flask and approaches the pilot plant scale. The culture conditions were: -YEPD culture medium

-Temperature: initial of 24 ° C rising the same to 37 ° C in the middle of the exponential phase, to cause lysis and the consequent release of proteins to the culture medium.

-technical data:

-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.

-sampling: periodic shots were taken at 24 ° and 37 ° C in order to follow the evolution of the optical density and the viability of the culture. In addition, to evaluate the total protein and CAT enzymatic activity, one or two samples were taken before the thermal shock and after this at shorter intervals at the beginning (3 hours), increasing until 24-30 hours after the temperature change.

Under the conditions mentioned above the culture was maintained at 24 ° C to an optical density between 1 and 1.5; at that time the temperature of the culture was changed to 37 ° C to cause cell lysis. The above and the following refers to data shown in Figure II in its different sections. To follow the lysis the different parameters were evaluated:

Cellular analysis, which logically means cell death, was measured following a method developed in our laboratory consisting of measuring the percentage of cell population that is stained by fluorochrome propidium iodide using the flow cytometry technique (Source, JM, Alvarez , A., Nombela. C. and Sánchez, M. 1992. Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts. Yeast 8: 39-45) and equivalent to a profile of the viability of the crop.

SU SHEET -as in the case of osmotic shock protein release, the enzymatic activity of CAT and the total protein was measured. Protein released is considered to be the increase over the baseline value measured in the culture at 24 ^° C immediately before changing the temperature to 37 ° C.

The results presented in Figure II (page 15) correspond to type fermentations of the LDl and LHDPl strains. The graphs A (LDl) and C (LHDPl) to fermentations carried out at 24 ° C in order to follow the profile of these strains at their permissive temperature and to establish that at no time does lysis occur (no decrease in the viability profile) or, therefore, protein release to the culture medium (CAT activity is only detectable in the cell extract, that is inside the cells). It can be said that there are two fermentations that serve as a control for the comparison with the fermentations performed with thermal shock in the same strains that allow us to more accurately assess the effect of said thermal shock. In the graphs B (DL1) and D (LHDPl), corresponding to fermentations with thermal shock, the following phenomena are observed after the temperature change, represented by a continuous vertical line in the graph:

- in both of them a marked loss of viability is observed up to levels around 20 to 30% of viable cells, as a result of the expression of the phenotype of the mutation in the SLT2 gene at the temperature of 37 ° C.

-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). However, 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.

- As in the case of osmotic shock, the separation of cell debris by centrifugation leaves a crude preparation of proteins suitable for undertaking a purification process.