EP1392727A1

EP1392727A1 - Production of a stress protein

Info

Publication number: EP1392727A1
Application number: EP02741520A
Authority: EP
Inventors: Govardus Adrianus Hubertus De Jong; Johannes Wilhelmus Leonardus Boumans
Original assignee: Alfa Biogene International BV
Current assignee: Alfa Biogene International BV
Priority date: 2001-06-05
Filing date: 2002-06-04
Publication date: 2004-03-03
Also published as: WO2002098910A1; NL1018211C2

Abstract

The present invention relates to an economically attractive method for increasing the stress protein content (as percentage of the total protein content) in a liquid. Also, the invention relates to a stress protein product and to use of a stress protein in applications such as food products and pharmaceutical preparations for man and animal.

Description

Title: Production of a stress protein.

The invention relates to a method for increasing the stress protein content in a liquid, to a stress protein product and to the use thereof.

Stress proteins (also called chaperone proteins) are formed by microorganisms, plants and animals when, as a result of a change in the environment such as exposure to heat, radiation or chemicals, so-called stress- susceptible genes are expressed. According to current insights, such proteins can contribute to a protection against detrimental effects resulting from such environmental changes. For that reason, the use of stress proteins is in the center of interest of, inter alia, medicine, molecular biology, the cosmetic industry and the industry producing plant protection products.

It is known that stress proteins have a number of important natural therapeutic functions in plants and animals, people included. These proteins are involved as regulator in protein synthesis and protein folding. In addition, an important function lies in the regulation of gene function during growth, but also during cell death. The stress proteins are capable of stimulating both human and animal immune systems, and clinical research has shown that the chaperone proteins contribute positively in the fight against cancer.

However, the availability of stress proteins such as HSP 70 is a problem as they are often isolated from bovine brain. In addition to the slight amounts that can be isolated therefrom and the accompanying extreme high cost price, the possible presence of BSE in brain is a large impediment to the use of these proteins. HSP 70 can also be produced from microorganisms and cancer cells, but these production methods too are expensive and only give a low yield. Alternatives to the production of the stress proteins, in particular for medicaHises, lie in the field of the production of fusion proteins with the aid of expression systems. This relatively involved in the use of bovine brain. Possible problems that may continue to exist are the high cost price and the small amounts that can be produced.

Stress proteins of vegetable origin could offer an alternative. As the production of stress proteins depends on the condition the cell is in, a change in the circumstances outside the cell can tremendously increase the synthesis of the stress proteins in the plant cell.

In WO 00/70931, a method is described for the preparation of stress proteins from vegetable material. By giving the plants a heat treatment, the amount of stress protein can be increased. This publication further describes a method wherein the sap of the plant is heated to 95°C. However, it has been found that stress proteins such as HSP 70 already denaturate in a temperature treatment of only 40 - 60°C (see Example 3), so that such a method is not desirable for the isolation of such stress proteins.

A disadvantage of the use of plants as a source of stress proteins is the large amount of other proteins (bulk proteins) and other bulk compounds present. As a result, the purification of stress proteins is difficult and also difficult to scale up, because the standard purification techniques such as ultrafiltration and column chromatography are hindered by the high concentrations of other proteins. Generally, the stress proteins / bulk proteins ratio is so low that a thorough separation of bulk proteins from the stress protein fraction is required to obtain a product with a suitable amount of stress protein without obtaining an excess of bulk proteins. When using an unpurified stress protein product in a food product or pharmaceutical use, the amount of product to be administered to obtain a desired amount of stress protein must be so high that an excess of bulk proteins and other bulk compounds such as polyphenols would be introduced into the food product or pharmaceutical preparation. Moreover, bulk proteins can have a negative effect on. the activity of the stress protein, for instance because the stress protein binds to a bulk protein. Also, bulk proteins (such as enzymes) can reduce the stability of a stress protein product. It is an object of the present invention to provide an economically attractive method for increasing the content of stress protein (as percentage of the total solid matter content, preferably as percentage of the total protein content) in a liquid containing protein. It has now been found that it is possible to selectively separate inconvenient bulk compounds such as bulk proteins from a liquid containing proteins by means of precipitation, which is realized by a pH-reduction. Therefore, the present invention relates to a method for increasing the stress protein content (as percentage of the total solid matter content, preferably as percentage of the total protein content) in a liquid that contains at least one stress protein and at least one bulk compound, wherein

- bulk compound is precipitated from the liquid by reducing the pH of the liquid, and

- the precipitate formed is separated from the supernatant containing the stress protein.

It has been found that a method according to the invention is particularly suitable for removing inconvenient bulk compounds and in particular inconvenient bulk proteins in a simple and industrially attractive manner. Thus a method is particularly suitable for the purification of a stress protein product, optionally in combination with one or more other purification steps. It has also been found that it is possible by means of a method according to the invention to remove bulk compounds while maintaining a high nativity and/or activity of one or more stress proteins.

In particular, a method proves to be very suitable for separating chlorophyll (in the form of chlorophyll protein complex and/ or as free chlorophyll) and/or rubisco as precipitate from a liquid that contains vegetable proteins. By removing these two components, a next concentration or isolation of the stress protein proves to be highly facilitated. It appears, for instance, that after precipitation of chlorophyll protein complexes and/or rubisco, stress protein can be efficiently isolated from the supernatant or concentrated with conventional techniques such as, for instance, (preparative) column chromatography, which technique was unsuitable because of binding to the column material (for instance binding of chlorophyll), damage to and/or clogging of the column by chlorophyll particles and overloading because of the high concentration of protein (for instance a high concentration of rubisco). As referred to herein, a stress protein is a protein that under the influence of an exposure to a harmful environmental factor, not internally determined by the organism, ("stress") is expressed by a gene inducible by "stress". Very suitable examples of such a "stress" are exposure to temperature change (exposure to a warm or cold environment), to radiation, for instance UV-radiation, to a changed atmosphere such as hypoxia or anoxia, to an osmolarity change or a combination thereof. It is also possible to induce stress protein production by exposure to an agent inducing stress protein, for instance a transition metal ion (for instance silver or copper) or an organic xenobiotic compound (for instance paraquat).

Examples of stress proteins particularly suitable to be produced through the invention are the so-called "heat-shock" proteins (proteins induced by a heat treatment), such as HSP 20-30, HSP 40, HSP 60 (Chaperonin) HSP 70, HSP 90, HSP 130 and ubiquitin, which are described in S. Lewis et al., Ecotoxicology 8, 351-368 (1999). Other examples of stress proteins that can be produced according to the invention are cytochrome p450 proteins, metallothioneins and heme oxygenases.

As referred to herein, bulk compounds are compounds that are contained rri a large amount in the liquid from which the desired stress protein can be produced, such as proteins and other peptidic compounds and other contaminants tnaj; are not stress proteins. Typical examples of bulk compo nds are chlorophyll and proteins such as complexes of chlorophyll with "chlorophyll binding proteins", rubisco, enzymes (for instance proteases and polyphenol oxidases) and other proteins present in the liquid to such a degree that they impede efficient production of the stress protein. Inconvenient proteins, such as rubisco, "chlorophyll binding protein" and chlorophyll protein complexes are referred to herein as bulk proteins.

In a preferred embodiment, the bulk proteins rubisco, "chlorophyll binding protein", chlorophyll protein complexes and optionally also other inconvenient bulk compounds are removed from the liquid by a pH reduction and separation of the formed precipitate. The stress protein can be recovered from the resultant supernatant optionally by means of a next purification step, wherein a residue of the mentioned bulk proteins and/or other components such as polyphenols, water and/or organic solvents are removed from the stress protein fraction.

Very good results are achieved by increasing the stress protein content in a hquid, the stress protein coming from an organism in which one or more stress proteins have been induced by a heat treatment between 20 and 90°C, preferably between 30 and 60°C, and more preferably between 35 and 45°C. The duration of the treatment can be chosen within a wide range, preferably between 1 minute and 24 hours, more preferably 30 min. - 12 hours, and still more preferably between 1 — 4 hours.

Preferably, the invention is used for a hquid with stress proteins coming from a plant, such as for instance lucerne (alfalfa), a cereal (for instance barley), soy, a grass (for instance oat), beet, potato, clover or a water plant (for instance an alga). This technique can also be used for stress protein (such as HSPs) from yeasts, fungi and bacteria. Particularly good results are obtained with stress proteins from lucerne, clover, oat and barley.

Preferably, the leaf of the plant is used as source for one or more stress proteins. Particularly suitable are beet tops, lucerne leaves, barley leaves, oat leaves and potato tops. In addition to the particular suitability of such leaf material as source for stress proteins, their use also offers the possibility to use such leaf material, which is normally a waste product, in a useful manner. The plant can be grown in a conventional manner. Very good results have also been obtained with a plant grown in the dark in light- deficient conditions, or, conversely, in conditions rich in light. The choice of the light conditions can influence the formation of specific bulk proteins. By growing the plant, for instance barley, in the dark or under conditions rich in light, it is possible to obtain plant material with a relatively low chlorophyll content, which yields a favorable base material for the preparation of a stress protein-containing liquid. It has been found that in such a hquid not only the chlorophyll content can be very effectively reduced by means of a method according to the invention, but also the rubisco content, in some cases even below the limit of detection. It has been found that a plant grown in such a manner is a very good source for stress proteins, which can be produced in a simple and very efficient manner with a high degree of purity by a pH reduction according to the invention and/or by isolation or concentration in a different manner, for instance as herein described.

In principle, any liquid with vegetable proteins can be used as source from which a stress protein according to the invention can be produced. Particularly suitable is for instance the sap of the plant obtained by pressing vegetable material and an extract of a vegetable material. Suitable preparation methods for such a hquid are known, for instance from

WO 00/70931 and S. Lewis et al, Ecotoxicology 8, 351 - 368 (1999). Extraction can for instance take place with water, a buffer (for instance Tris, borate, phosphate, optionally with additives such as polyvinyl pyrrolidone or EDTA) a saline solution (for instance NaCl, KC1) or another solvent in which stress proteins dissolve.

For the pH reduction in a method according to the invention, the liquid with vegetable proteins preferably has a pH in the range of pH 6 - 9, more preferably pH 6.5 - 8.

Good results are further obtained with a liquid with vegetable proteins to which, prior to the pH reduction, in a method according to the invention bisulfite is added, preferably sodium metabisulfite. Preferably, the bisulfite is added in a concentration of 0.1 - 5 g 1.

In a method according to the invention, the pH is preferably reduced to a target value ("set point"). Very good results have been obtained with a method in which the pH is reduced gradually or step by step. The pH to which the reduction takes place and the speed with which the pH is preferably reduced is dependent, inter aha, on the bulk compounds present, the type of stress protein that is isolated and can be routinely determined by the skilled person. Preferably, the pH is reduced step by step or gradually with an average speed of approximately 0.1 - 2 pH unit per hour, more preferably with an average speed of approximately 0.2 - 1 pH unit per hour, most preferably approximately 0.3 - 0.5 pH unit per hour, for instance step by step each half hour with a step-by-step reduction of 0.2 pH unit. The precipitate that forms can optionally be separated from the supernatant during the pH reduction or at the end.

In a preferred embodiment, the hquid is stirred during addition of the acid, and the stirring is temporarily discontinued between two reductions.

Depending on the protein composition of the liquid, the skilled person will know which suitable pH conditions to choose. Very good results have been obtained with a method in which the pH is reduced to a value in the range of approximately pH 4 - 6, preferably to a value in the range of approximately 4.5 - 5.5.

Very suitable for the production of HSP 20 - 30, HSP 40, HSP 60 (Chaperonin), HSP 70, HSP 90, HSP 130 Cytoehrome p450, metallothionein, heme oxygenase and/or ubiquitin from a liquid further containing chlorophyll and/or rubisco proteins is, for instance, a method in which chlorophyll and/or rubisco is precipitated from the liquid by step-by-step or gradual reduction of the pH )f the hquid with the proteins by adding an acid of a pH of approximately 6.5 or higher to a pH of approximately 4.8 - 5.2, the precipitate formed is separated from the supernatant containing the stress protein, and optionally the stress protein in the supernatant is isolated or concentrated. Particularly good results are obtained with such a method in which HSP70 was produced from lucerne, clover, oat or barley.

In principle, any acid can be used for the pH reduction through a method according to the invention. Very suitable are (relatively) strong inorganic or organic acids such as hydrochloric acid, nitric acid, phosphoric acid, boric acid, sulfuric acid, citric acid, acetic acid, lactic acid and the like. Of these acids, hydrochloric acid and acetic acid are particularly preferred because of economic reasons. In addition to hydrochloric acid and acetic acid, lactic acid and citric acid are also preferred in human and animal uses.

In addition to the pH reduction, other parameters, such as the salt concentration, the concentration of organic solvents, the protein concentration (by dilution or concentration) and the temperature, can be changed for separating bulk compounds in a desired manner. A method according to the invention can be carried out within a broad temperature range, which temperature, if desired, can be kept constant or can be changed. By changing the temperature during the pH reduction, the precipitation point (pH at which precipitation takes place) can be influenced. Preferably, the temperature lies between approximately 1 and 50°C, more preferably between 5 and 30°C, and still more preferably between 5 and 20°C. The precipitate can be separated from the supernatant in any suitable manner, for instance by centrifugation, filtration or decantation. Then the stress protein in the supernatant can be isolated or concentrated in any suitable manner. Very suitable techniques are, inter alia, gel filtration, ultrafiltration, ultracentrifugation, cross-flow filtration, precipitation (complete or partial) drying, membrane filtration, ion exchange, chromatography (for instance gel filtration, gel permeation or ion exchange chromatography) and electrophoresis.

The equipment and other material (for instance columns, filters) prove to have a longer hfe span than in conventional methods. In a preferred embodiment, the stress protein is concentrated or isolated with chromatography or a filtration technique. In such an embodiment, it has been found that a stress protein with a high nativity and/or activity is obtained on a large scale. Particularly good results for the purification of HSPs, such as HSP 70, are achieved with affinity chromatography, for instance with ATP agarose as column material. With affinity chromatography, other contaminants such as polyphenols can be removed very rapidly and effectively after removal of rubisco and chlorophyll protein complex without much column contamination, so that in an economically attractive manner an end product with a high degree of purity can be obtained. It has been found that by removing the bulk compounds the isolation or concentration can be obtained with simpler equipment in an efficient manner and can be applied on an industrial scale. For the preparation of a product with a particular purity according to the invention fewer separating steps are necessary. In addition to advantages for the required equipment and the shorter preparation time, the loss of stress protein as a result of the purification can thus also be strongly reduced.

Due to a method according to the invention, it also proves to be possible to isolate the stress protein by means of gel filtration, after separation of bulk compounds such as rubisco. It has thus been found possible to produce an HSP as one well defined fraction, as opposed to separation methods in which rubisco is not separated first.

It is also possible to isolate a stress protein by precipitation, for instance by ammonium sulfate precipitation, precipitation with an organic solvent (acetone, ethanol etc.) or acid. The advantage of isolation by precipitation is- hat ^'the process is simple.

In ,& preferred embodiment, the stress protein is additionally purified from polyphenolic compounds and/or other contaminants, particularly when the stress protein comes from leaf material. As a result, a higher activity of the stress protein is preserved. Suitable methods for removing polyphenols are known. A very suitable method for removing polyphenohc compound from a protein product is described in Dutch patent application NL 1017241. This application describes a method in which protein is precipitated by mixing a solution of the protein in an aqueous medium with a water-miscible organic solvent at a pH about the isoelectric point (± 1 pH unit), and the protein is then purified. It has now been found that such a method is also suitable for removing polyphenols from a stress protein product while preserving the stress protein activity.

The invention further relates to a stress protein product obtainable through a method according to the invention. Such a product can for instance be a solution, a dispersion, an emulsion or a dry product.

A product according to the invention can be obtained without genetic modification and without the possible health risks that may be attached to stress protein such as HSPs from bovine brain. A product according to the invention is characterized by a high nativity of the stress protein and/or a high activity of the stress protein.

A product according to the invention is very suitable in numerous uses. Therefore, the invention also relates to a food product for human or animal consumption, a pharmaceutical or cosmetic preparation or a plant protection product comprising a stress protein, which stress protein is obtainable according to a method of the present invention.

In a human food product according to the invention, a low content of chlorophyll is highly desirable. Although rubisco is an interesting protein for human food, a high dose of stress protein will result in an excess of rubisco. A human food product according to the invention proves to have a very high stress protein content with regard to the rubisco and chlorophyll content.

In particular for a pharmaceutical, cosmetic and plant protection product_according to the invention, it has been found that a very low degree of complexing of stress protein (such as HSPs) with rubisco occurs. As a result, the activity of the stress protein remains high. This can prevent a part of the effect of the HSP from being undone as a result of the undesired complexing of HSP to rubisco, which would cause a reduced dose of the free HSP. Additionally, the other components (rubisco and chlorophyll) can have a negative effect. In particular, the invention also relates to its use in animal food or human food, in a pharmaceutical preparation such as an immune system stimulating agent or a preparation for the treatment of cancer, or cosmetic preparation or in a plant protection product.

The invention will now be further illustrated with reference to some examples.

Example 1: HSP 70 from lucerne

Induction of the stress protein took place by heating lucerne directly (within half an hour) after mowing for 2 hours at 40°C.

The heat-treated lucerne was frozen with liquid nitrogen and then reduced in a mortar. To 10 grams of leaf powder was added a 20 ml buffer (25 g 1 polyvinyl pyrrolidone; 50 mM Tris pH 8, prepared 24 hours in advance and kept at 4°C). To this were added 2mM EDTA and 5 mM DTT (dithiothreitol). After 30 minutes keeping at 4°C while stirring, the extract was centrifuged for 15 min at 10.000 g). The supernatant contains both the HSP 70 and the chlorophyll and rubisco.

pH precipitation: The liquid fraction was brought to pH 6.5, and at a temperature of

5°C or 20°C, 0.1 M hydrochloric acid was then added step by step while stirring (steps of approximately 0.2 pH, 30 min between two reductions). Between, two pH reductions the stirring was discontinued for 30 minutes. First the chlorophyll precipitated and then rubisco between pH 6 and 5. After centrifugation of the hquid at pH 5, the pellet contained the chlorophyll and the rubisco and the supernatant HSP 70 protein. (Determined by means of western blotting and gel electrophoresis.)

The experiment was repeated with the sap of pressed lucerne as liquid fraction

Example 2: HSP 70 from barley

Barley was germinated in the dark. Leaf material from germinated barley was harvested between 1 and 10 days. After cutting, leaf material from barley was heated for 2 hours at

40°C.

Then the leaf materials was frozen with liquid nitrogen and then reduced in a mortar. After this was added a two times larger buffer (with the composition as indicated in Example 1), and then the extract was centrifuged. The liquid fraction contained HSP 70 as well as some other proteins.

The liquid fraction could optionally be treated further as described in Example 1.

Example 3: Temperature sensitivity of HSP 70

Leaf material of lucerne was pressed with a sap press, and the sap samples obtained were exposed to a temperature in the range of from 0 to 60°C for 30 minutes. Denaturated proteins were removed by means of centrifugation. The supernatant with soluble proteins was separated on an SDS/page gel and then blotted. With antibodies it was determined whether HSP 70 was still present. The results are shown in Fig. 1. The HSP band is indicated with the arrow. From the results it is clear that HSP is not heat stable. Already at 30 minutes exposure to 40°C, no detectable amount of HSP 70 is present anymore. Example 4: HSP from clover or oat.

Clover leaves or oat leaves were heated directly (within half an hour) after mowing at 40°C for 2 hours and then pressed with a sap press. To the sap obtained were added 3.9 g/1 of sodium metabisulfite, and then the pH precipitation was carried out as described in Example 1. After centrifugation of the liquid at pH 5, the pellet contained chlorophyll and the rubisco and the supernatant HSP 70 protein. (Determined by means of western blotting and gel electrophoresis.)

Claims

1. A method for increasing the stress protein content (as percentage of the total sohd matter content) in a liquid containing at least one stress protein and at least one bulk compound, wherein

- bulk compound from the liquid is precipitated by reducing the pH of the liquid, and

2. A method according to claim 1, wherein the pH is reduced gradually or step by step.

3. A method according to claims 1 or 2, wherein the pH is reduced at a speed of approximately 0.1 to approximately 2 pH unit per hour.

4. A method according to any one of the preceding claims, wherein the pH is reduced to a value in the range of approximately 4 to 6.

5. A method according to any one of the preceding claims, wherein the stress protein is induced by exposure to a temperature change, to radiation, for instance UV radiation, to hypoxia or anoxia, to an osmolarity change or to a substance inducing stress protein, for instance a transition metal ion or organic xenobiotic substance.

6. A method according to any one of the preceding claims, wherein chlorophyll, chlorophyll protein complex and/or rubisco is separated as precipitate.

7. A method according to any one of the preceding claims, wherein said liquid comprises ah extraet,.a sap or another fraction of a vegetable material.

8. A method according to any one of the preceding claims, wherein said liquid, prior to the reduction of the pH, has a value in the range of approximately 6 to 9.

9. A method according to any one of the preceding claims, wherein after the separation of the precipitate the stress protein in the supernatant is concentrated or isolated.

10. A method according to claim 9, wherein the concentration or isolation of the stress protein in the supernatant takes place by means of gel filtration, ultrafiltration, ultracentrifugation, cross-flow filtration, precipitation, drying, membrane filtration, ion exchange, chromatography or electrophoresis.

11. A method according to any one of the preceding claims, wherein the stress protein comes from a plant grown in the dark or in light- deficient conditions.

12. A method for increasing the stress protein content (as percentage of the total protein content) in a composition containing vegetable proteins, wherein the stress protein comes from a plant grown in the dark or in light- deficient conditions, and wherein the stress protein content is increased by a pH reduction according to any one of claim 1 — 10, by an isolation step, a concentration step or a combination thereof.

13. A method according to claim 12, wherein the isolation step and/or the concentration step of the stress protein takes place by means of gel filtration, ultrafiltration, ultracentrifugation, cross-flow filtration, precipitation, drying, membrane filtration, ion exchange, chromatography or electrophoresis.

14. A method according to any one of the preceding claims, wherein the stress protein is a HSP 20-30, HSP 40, HSP 60 (Chaperonin), HSP 70, HSP 90, HSP 130 or ubiquitin, cytochrome p450, metallothionein, heme oxygenase.

15. A method for producing HSP 20-30, HSP 40, HSP 60 (Chaperonin), HSP 70, HSP 90, HSP 130, Cytochrome p450, metallothionein, heme oxygenase and/or ubiquitin from a liquid containing vegetable proteins, wherein - chlorophyll and/or rubisco is precipitated from the liquid by reducing the pH of the liquid with the proteins step by step or gradually, by adding an acid of a pH of approximately 6.5 or higher to a pH of approximately 4.8 - 5.2 - the precipitate formed is separated from the supernatant containing stress protein

- optionally the stress protein in the supernatant is isolated or concentrated.

16. A method according to any one of the preceding claims, wherein the proteins in the liquid come from lucerne, a cereal, soy, grass, beet, potato or a water plant.

17. A stress protein product, obtainable by means of a method according to any one of the preceding claims.

18. A food product for human or animal consumption, a pharmaceutical or cosmetic preparation or a plant protection product comprising a stress protein according to claim 17.

19. The use of a stress protein according to claim 17 in animal or human food, in a pharmaceutical or cosmetic preparation, in a plant protection product.