NL1018211C2 - Winning a stress protein. - Google Patents

Description

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Title: Winning 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 its use.

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-sensitive genes are expressed. According to current insights, such proteins can contribute to a protection against harmful effects due to such environmental effects. For that reason, the use of stress proteins is attracting a great deal of interest from, among others, medicine, molecular biology, the cosmetic industry and the plant protection products producing industry.

It is known that stress proteins have a number of important natural therapeutic functions in plants and animals, including humans. These proteins involved as a regulator of 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 have a positive contribution to combating cancer.

However, the availability of stress proteins, such as HSP70, is a problem because they are often isolated from bovine brain. In addition to the small amounts that can be isolated from this and the associated extremely high cost price, the possible presence of BSE in the brain is a major obstacle to the use of these proteins. HSP 70 can also be extracted from microorganisms and cancer cells, but also these production methods are expensive and only give a low yield.

Alternatives for the production of the stress proteins, in particular for medical applications, lie in the field of the production of fusion proteins using expression systems. This relatively complicated way of production makes it possible to obtain the stress proteins without the risks associated with the use of bovine brain. Possible problems that may persist are the high cost price and the small quantities that can be produced.

Stress proteins of vegetable origin could offer an alternative. Since the production of the stress proteins is dependent on the condition of the cell, the synthesis of the stress proteins in the plant cell can be greatly increased by changing the conditions outside the cell.

WO 00/70931 describes a method for the preparation of stress proteins from vegetable material. By giving the plants a heat treatment, the amount of stress protein can be increased. A disadvantage of using plants as a source for stress proteins is the large amount of other proteins (bulk proteins) and other bulk compounds that are 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 hampered by the high concentrations of other proteins. The ratio of stress proteins to bulk proteins is generally 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 an excess of bulk proteins. For example, when using a crude stress protein product in a food or pharmaceutical application, the amount of product to be administered to administer a desired amount of stress protein would be so high that there is an excess of 1018211 '3 bulk proteins and other bulk compounds, such as polyphenols would be introduced into the food or pharmaceutical preparation. Moreover, bulk proteins can have a negative effect on the activity of a stress protein, for example because the stress protein becomes bound to a bulk protein. Bulk proteins (such as enzymes) can also lower the stability of a stress protein product.

It is an object of the present invention to provide an economically attractive way of increasing the stress protein content (as a percentage of the total protein content) in a protein-containing fluid.

It has now been found that it is possible to selectively separate disturbing bulk compounds, such as bulk proteins, from a liquid containing proteins by means of precipitation achieved by a pH reduction. The present invention therefore relates to a method for increasing the stress protein content (as a percentage of the total protein content) in a liquid containing proteins, wherein bulk compound from the liquid is precipitated by lowering the pH of the liquid, 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 interfering bulk connections, and in particular interfering bulk proteins, in a simple and industrially attractive manner. A method is thus extremely suitable for purifying 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.

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In particular, a method appears to be very suitable for separating chlorophyll (in the form of a chlorophyll-protein complex and / or as free chlorophyll) and / or Rubisco as a precipitate, from a liquid containing vegetable proteins. By removing these two components, it appears that a subsequent concentration or isolation of the stress protein is greatly facilitated. For example, after precipitation of chlorophyll-protein complexes and / or Rubisco, stress protein can be efficiently isolated from the supernatant or can be concentrated by conventional techniques, such as, for example, (preperative) column chromatography, which technique was unsuitable due to binding to the column material (chlorophyll) and the transfer due to the high concentration of protein (Rubisco).

The term stress protein is herein understood to mean a protein which, under the influence of an exposure to a stressful environment factor ("stress") not determined internally by the organism, is expressed by a "stress" inducible gene. Very suitable examples of such a "stress" are exposure to temperature change (exposure to a hot or cold environment), to radiation, for example 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 a stress protein inducing substance, for example a transition metal ion (for example silver or copper) or organic xenobiotic substance (for example paraquat).

Examples of stress proteins that are particularly suitable for recovery by means of 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 are those according to the invention

101821H

Cytochrome p450 proteins, metallothioneins and heme oxygenases can be recovered.

By the term bulk compound, it is meant compounds that are present in a large amount in the liquid from which the desired stress protein can be recovered, such as proteins and other peptidic compounds and other contaminants that are not stress proteins. Typical examples of bulk compounds are chlorophyll and proteins such as, chlorophyll complexes with chlorophyll binding proteins, rubisco, enzymes (for example proteases and polyphenol oxidases) and other proteins present in the fluid to such an extent that they efficiently extract stress protein. Interfering proteins, such as Rubisco, "chlorophyll binding protein" and chlorophyll protein complexes are referred to herein as bulk proteins.

In a preferred embodiment, a pH reduction and separation of the precipitate formed removes the bulk proteins Rubisco, "chlorophyll binding protein", chlorophyll protein complexes and any other interfering bulk compounds such as uncomplexed chlorophyll from the liquid. The stress protein can optionally be recovered from the resulting supernatant by a subsequent purification step, wherein a remainder of the said bulk proteins and / or other components such as polyphenols, water and / or organic solvents are removed from the stress protein fraction.

Very good results have been achieved in increasing the stress protein content in a liquid, the stress protein coming from an organism in which one or more stress proteins are 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 minutes-12 hours and even more preferably 1-4 hours.

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The invention is preferably applied to a fluid with stress proteins derived from a plant, such as, for example, alfalfa, a grain (e.g., barley), soy, grass, beet, potato, or a water plant (e.g., an alga). This technique can also be applied to stress proteins (such as HSPs) from yeasts, fungi and bacteria.

Particularly good results were achieved with stress proteins from lucerne and barley.

The leaf of the plant is preferably used as a source for one or more stress proteins. Beet leaves, alfalfa leaves, barley leaves and potato leaves are particularly suitable. In addition to the particular suitability of such leaf material as a source of stress proteins, its use also offers the possibility of using such leaf material, which is usually a waste product, in a useful manner.

The plant can be grown in a conventional manner. Very good results have also been achieved with a plant grown in the dark or under low-light conditions. By allowing the plant, for example barley, to grow in the dark or under low-light conditions, it appears possible not only to lower the chlorophyll content but also the Rubisco content, in some cases even below the detection limit. It has been found that a plant grown in this way is a very good source for stress proteins, which can easily be obtained in a very efficient manner with a high degree of purity by a pH reduction according to the invention and / or by isolation or concentration. in another manner, for example as described herein.

In principle, any liquid with vegetable proteins can be used as a source from which a stress protein according to the invention can be obtained. Particularly suitable is, for example, the sap of the plant obtained by pressing vegetable material and an extract from a vegetable material. Suitable methods of preparation for such a liquid are known, for example from WO 00/70931 and S. Lewis et al, 101821n 7

Ecotoxicology 8, 351-368 (1999). Extraction can for instance take place with water, a buffer (for example Tris, borate, phosphate, optionally with additives such as polyvinylpyrolidone or EDTA), a saline solution (for example NaCl, KCl) 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.

In a method according to the invention, the pH is preferably lowered to a target value ("set-point"). Very good results have been achieved with a process in which the pH is gradually or gradually lowered. The pH to which the pH is lowered and the speed at which the pH is preferably lowered depends, among other things, 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 lowered stepwise or gradually with an average speed of about 0.1-2 pH unit per hour, more preferably with an average speed of about 0.2-1 pH unit per hour, particularly preferably about 0 3-0.5 pH unit per hour, for example, stepwise every half hour with a stepwise 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 liquid is stirred during the addition of the acid and the stirring between two reductions is temporarily stopped.

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

101821) - 8

For the recovery of 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 that further contains chlorophyll and / or Rubisco proteins for example a method very suitable, wherein chlorophyll 5 and / or Rubisco is precipitated from the liquid by gradually or gradually reducing the pH of the liquid with the proteins by adding an acid from a pH of about 6.5 or higher to a pH of about 4.8-5.2, the precipitate formed is separated from the supernatant containing the stress protein and optionally the stress protein is isolated or concentrated in the supernatant. Particularly good results have been achieved with such a process in which HSP70 was recovered from alfalfa or barley.

In principle, any acid can be used for reducing the pH by means of 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, partly due to economic reasons. In addition to hydrochloric acid and acetic acid, lactic acid and citric acid are also preferred in human and animal applications.

In addition to the pH reduction, other parameters can be changed, such as the salt concentration, the concentration of organic solvents, the protein concentration (by dilution or concentration) and the temperature, in order to separate bulk compounds in a desired manner.

A method according to the invention can be carried out within a wide temperature range, which temperature can be kept constant or changed if desired. By changing the temperature during the pH reduction, the precipitation point (pH at which precipitation occurs) can be influenced. The temperature is preferably between approximately 1 and 50 ° C, more preferably between 5 and 30 ° C, even more preferably between 5 and 20 ° C.

The precipitate can be separated from the supernatant in any suitable manner, for example by centrifugation, filtration or decantation. The stress protein in the supernatant can then be isolated or concentrated in any suitable manner. Very suitable techniques for this include gel filtration, ultrafiltration, ultracentrifugation, cross-flow filtration, precipitation, drying (in whole or in part), membrane filtration, ion exchange, chromatography (for example gel filtration, gel permeation or ion exchange chromatography) and electrophoresis.

The equipment and other material (e.g. columns, filters) have been found to last longer than in conventional methods.

In a preferred embodiment, the stress protein is concentrated or isolated by chromatography or a filtration technique. In such an embodiment, it has been found that a stress protein with high nativity and / or activity is obtained on a large scale. Particularly good results for the purification of HSPs, such as HSP 70, have been achieved with affinity chromatography, preferably with ATP agarose as a column material. With affinity chromatography, after removal of Rubisco and chlorophyll-protein complex, other contaminants, such as polyphenols, can be removed very quickly and effectively without much column contamination, so that an end product with a high degree of purity can be obtained in an economically attractive manner. It has been found that by removing the bulk compounds the isolation or concentration can be achieved efficiently with simpler equipment and can be applied on an industrial scale. Fewer separation steps are required to prepare a product with a certain purity according to the invention. In addition to advantages for the equipment required and the shorter preparation time, the loss of stress protein as a result of the purification can also be greatly reduced.

Thanks to a method according to the invention, it also appears to be possible to isolate the stress protein by gel filtration, after separation of bulk compounds such as Rubisco. This has made it possible to win an HSP as one well-defined fraction, in contrast to separation methods where Rubisco was not first separated.

It is also possible to isolate a stress protein by precipitation, for example by ammonium sulphate precipitation, precipitation with an organic solvent (acetone, ethanol and the like) or acid. Isolation by precipitation has the simplicity of the process.

In a preferred embodiment, the stress protein is additionally purified from polyphenolic compounds and / or other contaminants, in particular if the stress protein is derived from leaf material. This maintains a higher activity of the stress protein. Suitable methods for removing polyphenols are known. A very suitable method for removing polyphenolic compound from a protein product is described in Dutch application NL 101 72 41.

Described herein is 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 around the isoelectric point (± 1 pH unit) and then the protein is purified . It has now been found that such a method is also suitable for removing polyphenols from a stress-protein product, while maintaining stress-protein activity.

The invention further relates to a stress-protein product that is available by a method according to the invention. Such a product can for instance be a solution, a dispersion, an emulsion or a dry product.

101821 11

A product according to the invention can be obtained without genetic modification and without the possible health risks that stress proteins, such as bovine brain HSPs, may have.

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 many applications. Therefore, the invention also relates to a food for human or animal consumption, a pharmaceutical or cosmetic preparation or a crop protection agent, which comprises a stress protein, which stress protein is obtainable according to a method according to the present invention.

In a human food according to the invention, a low content of chlorophyll is highly desirable. Although the Rubisco is an interesting protein for human nutrition, an excessive amount of stress protein will result in an excess of Rubisco. A human food according to the invention appears to have a very high stress protein content compared to the Rubisco and chlorophyll content.

In particular for a pharmaceutical, cosmetic and crop protection agent according to the invention, it has been found that a very low degree of complexation of stress proteins (such as HSPs) with Rubisco occurs. This keeps the activity of the stress protein high. part of the HSP's action can be canceled out because the complexation results in a reduced dosage of the free HSP. In addition, the other components (Rubisco and chlorophyll) can have a negative effect.

In particular, the invention also relates to its use in animal nutrition or human nutrition, in a pharmaceutical preparation, such as an immune system stimulating agent or a preparation for the treatment of cancer, or a cosmetic preparation or in a crop protection agent.

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

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Example 1: HSP 70 from lucerne 5

Induction of the stress protein was achieved by heating Lucerne immediately (within half an hour) after mowing for 2 hours at 40 ° C.

The heat-treated alfalfa was frozen with liquid nitrogen and then comminuted in a mortar. To 10 grams of leaf powder was added a 20 ml buffer (25 g / l polyvinylpyrolidone; 50 mM Tris pH 8, prepared 24 hours in advance and stored at 4 ° C). To this was added 2 mM EDTA and 5 mM DTT (dithiothreitol). After storage for 30 minutes at 4 ° C with shaking, the extract was centrifuged at 10,000 g for 15 minutes. The supernatant contains both the HSP 70 and chlorophyll and Rubisco.

pH precipitation:

The liquid fraction was adjusted to pH 6.5 and at a temperature of 5 ° C or 20 ° C, 0.1 M hydrochloric acid was then added stepwise with stirring (steps of approximately 0.2 pH, 30 minutes between two reductions). Stirring was stopped for 30 minutes between two pH reductions. Initially chlorophyll and then Rubisco precipitated between pH 6 and 5. After centrifugation of the liquid at pH 5, the pellet contained the chlorophyll and the Rubisco and the supernatant HSP 70 protein. (Determined by western blotting and gel electrophoresis)

The experiment was repeated with the juice of pressed alfalfa as the liquid fraction.

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Example 2: HSP 70 from barley

Barley was germinated in the dark. Leaf material from germinated barley was harvested between 1 and 10 days.

Barley leaf material was heated at 40 ° C after cutting

for 2 hours.

The sheet material was then frozen with liquid nitrogen and then comminuted in a mortar. A volume of buffer twice as large was then added, after which the extract was centrifuged.

The liquid fraction contained HSP 70 as well as some other proteins.

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

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Claims (19)

A method for increasing the stress protein content (as a percentage of the total protein content) in a liquid containing proteins, wherein - bulk compound is precipitated from the liquid by lowering the pH of the liquid, 5. the precipitate formed is separated from the supernatant containing the stress protein. The method of claim 1, wherein the pH is lowered gradually or stepwise. 3. A method according to claim 1 or 2, wherein the pH is lowered at a rate of about 0.1 to about 2 pH unit per hour. The method of any one of the preceding claims, wherein the pH is lowered to a value in the range of about 4 to 6. 5. Method according to any of the preceding claims, wherein the stress protein is induced by exposure to a temperature change, to radiation, for example UV radiation, to hypoxia or anoxia, to an osmolarity change or to a stress protein-inducing substance, for example a transition metal ion or organic xenobiotic substance. A method according to any one of the preceding claims wherein, chlorophyll, chlorophyll-protein complex and / or Rubisco is separated as a precipitate. A method according to any one of the preceding claims, wherein said liquid comprises an extract, a juice or another fraction of a vegetable material. 8. A method according to any one of the preceding claims, wherein said liquid has a value in the range of approximately 6 to 9 prior to lowering the pH. The method of any one of the preceding claims, wherein after separating the precipitate, the stress protein is concentrated or isolated in the supernatant. 10. A method according to claim 9, wherein concentrating or isolating 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 originates from a plant that has been grown in the dark or under low-light conditions. 12. Method for increasing the stress protein content (as a 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 under low-light conditions and wherein the stress protein content is increased by a pH reduction according to any of claims 1-10, by an isolation step, a concentration step or a combination thereof. 13. Method as claimed in claim 12, wherein the isolation step and / or 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 an HSP 20-30, HSP 40, HSP 60 (Chaperonin), HSP 70, HSP 90, HSP 130 or ubiquitin, Cytochrome p450, metallothionein, heme oxygenase . 15. Method for recovering HSP 20-30, HSP 40, HSP 60 (Chaperonin), HSP 70, HSP 90, HSP 130, Cytochrome p450, metallothioneine, heme oxygenase and / or ubiquitin from a liquid containing 1018211 µ vegetable proteins wherein - chlorophyll and / or Rubisco is precipitated from the liquid by gradually or gradually reducing the pH of the liquid with the proteins by adding an acid from a pH of about 6.5 or 5 higher to a pH of about 4, 8-5.2 - the precipitate formed is separated from the supernatant containing the stress protein - optionally the stress protein in the supernatant is isolated or concentrated. A method according to any one of the preceding claims wherein the proteins in the liquid come from alfalfa, a grain, soy, grass, beet, potato or a water plant. A stress protein product obtainable by a method according to any one of the preceding claims. A foodstuff for human or animal consumption, a pharmaceutical or cosmetic preparation or a crop protection agent, comprising a stress protein according to claim 17. 19. The use of a stress protein according to claim 17 in animal feed or human feed, in a pharmaceutical or cosmetic preparation, in a crop protection agent. J 0 1 82 1 1