WO2002005922A1 - Method for extracting proteins from plants in pure form - Google Patents

Abstract

The invention relates to a method for extracting a desired native or recombinant protein from a plant in pure form. After extracting a raw extract using an appropriate extraction agent, the desired protein is removed from the raw extract and purified by means of ion-exchange chromatography used in expanded bed technology. The desired protein can be, for example, napin, napin-like 2S albumin, cruciferin, legumin-like 11S globulin or a recombinant scFv antibody.

Description

 Process for obtaining proteins from plants in pure form

The present invention relates to a process for obtaining a desired native or recombinant protein from a plant in pure form, the desired protein being purified from the crude extract by means of expanded exchange technology using ion-exchange chromatography using expanded bed technology after obtaining a crude extract. In a preferred embodiment of the method according to the invention, the method according to the invention is used for the isolation and purification of napin, napin-like 2S albumin, cruciferin, legumin-like IIS globulin or a recombinant scFv antibody.

Vegetable proteins are a hitherto rarely used reservoir of renewable raw materials. Vegetable storage proteins are obtained in particularly large quantities, for example in the production of oil from rapeseed, sunflowers and other oil plants, or from lupins or also in other agricultural processes. They are usually added to animal feed as a waste product, since no pure and thus technically usable components are available with technically and economically usable methods. Other vegetable proteins occur in smaller quantities, but their functionality is extremely interesting. So far, however, only a few vegetable proteins have been put to commercial use. This is due, among other things, to complex and therefore uneconomical cleaning processes which are also unsuitable for a larger scale. This contrasts with the extremely interesting properties, such as isolated vegetable storage proteins for various areas of application. The two predominant storage proteins in rapeseed, napin and cruciferin, which could previously only be obtained technically as a crude protein fraction, are suitable, for example, due to their physicochemical properties as isolated components for use as foams or adhesives (napin) or for film production (cruciferin). Furthermore, they are due to these properties also suitable for use in the food industry, e.g. as foaming agents and stabilizers.

So far, only precipitation and extraction processes have been used to obtain crude protein fractions or enriched compounds on an industrial scale. In this connection, reference is made to US Pat. Nos. 4370267 and 4368151, in which the enrichment of a vegetable storage protein component after isoelectric precipitation is described by extraction under suitable conditions. All of these processes are characterized by low efficiency. In addition, no pure components, but only enriched fractions, are obtainable in this way, and in most cases such processes can only be optimized to obtain a single component from a protein mixture. So far, purer substances could only be obtained in complicated processes involving a combination of a large number of different cleaning steps, e.g. 12S globulins from rapeseed by a combination of precipitation, dialysis, gel chromatography and anion exchange chromatography.

Furthermore, "Molecular Farming" (foreign protein production in transgenic plants) is developing new areas of application for protein materials and active ingredients, for which technically and economically efficient cleaning processes, even on an industrial scale, are necessary. While the production of therapeutically valuable proteins usually allows high profit margins, this is for "mass proteins", e.g. Serum proteins, or diagnostics, and for technically usable proteins are not the case. For this reason, all the more robust, easily scalable and economically efficient processes for protein purification from plant tissue are required.

The invention is therefore essentially based on the technical problem of providing a purification method for proteins from plants which has the disadvantages of the prior art does not have the process described in the art, ie above all it is simple, efficient and can be carried out on an industrial scale and also ensures that the desired proteins are of high purity and therefore preferably no further purification steps are required.

This technical problem has been solved by providing the embodiments characterized in the patent claims.

Surprisingly, it has been found that both native and recombinant proteins from plant tissue are separated into pure substances with high efficiency using the process according to the invention, which comprises an extraction step and a subsequent selective adsorption on cation and anion exchangers in the "expanded bed" technology or can be isolated as pure substances. It has been shown here that the degree of purity of the proteins is so high that an economically competitive production of new protein materials is also possible in comparison to plastic production based on fossil raw materials. It has also been shown that the otherwise necessary pre-treatment steps, such as degreasing of oil-containing seeds, or pre-precipitation steps are not necessary in the process according to the invention, but can, if appropriate, be combined therewith. Furthermore, it has been shown that the process according to the invention can be carried out on an industrial scale. This has been shown by the separation of napin (albumin) and cruciferin (globulin) from rapeseed. Furthermore, it has been shown that the method according to the invention is also suitable for the use of foreign proteins, e.g. to isolate and purify scFv antibodies from transgenic plants. Reference is made to the examples below.

The present invention thus relates to a method for obtaining a desired native or recombinant protein from a plant in pure form, the method comprising the following steps: (a) disintegrating the plant to obtain a crude extract using a suitable extracting agent; and

(b) Separation of the desired protein via ion exchange chromatography using "expanded bed" technology, the crude extract being bound at a defined pH.

Preferably the method according to the invention comprises only steps (a) and (b), i.e. there are no pretreatment and / or further purification steps, especially those that are based on specific physicochemical characteristics of the desired protein, e.g. Molecular weight, sedimentation coefficient, pl value, based, necessary. However, pretreatment and / or cleaning steps can be added if necessary.

Suitable methods for digestion of the plant are known to the person skilled in the art and the latter can select suitable digestion methods according to the desired protein and the plant used. These can e.g. homogenization, such as in a grinder or mixer, and / or lysis with suitable lysis agents. Reference is made to the examples below.

The person skilled in the art also knows suitable extraction agents and selects them, inter alia, according to the known properties of the desired protein. The extractant is preferably an aqueous solution, in particular phosphate buffer, TRIS buffer, MOPS buffer or an ethanolic extractant. In the process according to the invention, a denaturation and possibly renaturation step is optionally carried out before or after the extraction step if the desired protein should be in insoluble form. Suitable denaturation processes and renaturation processes are known to the person skilled in the art and the person skilled in the art also knows the conditions which are required in these processes for the desired protein not to lose its biological activity or to regain it. According to the invention, ion exchange chromatography is carried out using "expanded bed" technology in order to purify the desired protein. "Expanded bed" technology is a very robust process that does not require any organic solvents or other high amounts of salt. In addition, the "expanded bed" technology is easily transferable to the industrial scale and does not require any complex apparatus technology. The adsorbents show no signs of "fouling", so that long service lives of the chromatography columns are guaranteed. In addition, Straetkvern et al. , Bioseparation 7 (1999), 333-345. According to the invention, however, the "expanded bed" technology is not carried out in connection with affinity chromatography but, for the first time, with ion exchange chromatography. Depending on the known properties of the desired protein, for example with regard to the pI value, either anion exchange or cation exchange chromatography is carried out for the process according to the invention. The choice of the defined pH value for the binding of the protein to the chromatography material (loading) also depends on these properties. For example, the person skilled in the art selects the pH value on the basis of the pI value of the desired protein, the pI value either being calculated from the protein sequence or being determined by means of isoelectric focusing. It is expedient if the pH value is chosen so that the desired protein has a surface charge opposite to the main contamination and the person skilled in the art will then select the sorbent suitable for the surface charge of the desired protein, ie a cation or anion exchanger. In any case, in the method according to the invention the setting of a defined pH so that the protein components of a crude protein mixture contained in the extract can be selectively bound to the given chromatography material is of crucial importance. This makes it possible, for example, to purify two or more protein components, the pI values of which differ sufficiently, from a mixture. The term "in pure form" as used herein means that the protein is essentially free of contaminants, preferably a purity of at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99 % having.

The method according to the invention is suitable for the purification of a desired protein from a plant of any plant species, i.e. it can be both a monocot and a dicot. The term "plant" also includes gramineae, chenopodia, leguminous plants, Brasicaceae, Solanaceae, fungi, mosses and algae. They are preferably useful plants, e.g. plants such as wheat, barley, rice, corn, sugar beet, sugar cane, rapeseed, mustard, turnip, flax, pea, bean, lupine, tobacco and potato.

Any plant part or tissue of the plant can be used to obtain the protein, the selection being made according to the different concentration of the desired protein in the individual plant parts or tissues. The desired protein is preferably obtained from seeds, leaves, tubers, root pieces, seedlings, cuttings, etc., with potato tubers being particularly preferred.

In a further preferred embodiment of the method according to the invention, the desired protein is obtained from a storage protein mixture. Examples of storage proteins which can be isolated and purified using the method according to the invention are napin, cruciferin, patatin, legumin and vicillin.

In an even more preferred embodiment of the method according to the invention, the binding of the desired protein to the ion exchange material takes place at a pH of 7.5 to 9, most preferably the desired protein is eluted with a buffer containing NaCl of defined molarity or by means of a NaCl gradients. The A person skilled in the art can determine the optimal elution conditions on the basis of the known characteristics of the desired protein and / or by preliminary tests on a laboratory scale.

Depending on the pI value of the desired protein, the person skilled in the art chooses (a) anion exchange chromatography or (b) cation exchange chromatography, for (a) preferably a chromatography material with the properties of Streamline DEAE ™ or Streamline QXL (A ersham Pharmacia , Upsala, Sweden) and for (b) preferably Streamline SP XL ™ (Amersham Pharmacia).

The method according to the invention allows, among other things, the isolation and purification of native proteins, for example napin, napin-like 2S albumin proteins, cruciferin and legumin-like IIS globulin proteins from plant seeds. The napin-like proteins are proteins with an isoelectric point that is 8.0 or above, a molecular weight between 10 and 20 kDa and a composition of two heterodimeric subunits (α and β chains), which are linked via one or more disulfide bridges. Legumin-like IIS globulin proteins are characterized by an isoelectric point between 4.0 and 8.0, a molecular weight between 250 and 400 kD and a composition as hexamers from subunits, which are each composed of an α and β subunit, which in turn are linked by one or more disfuld bridges. In a further particularly preferred embodiment of the method according to the invention, the desired proteins to be isolated are napin, napin-like 2S albumin proteins, cruciferin and legumin-like IIS globulins which, for example, from non-defatted rapeseed under the conditions specified in Example 1 below can be isolated and cleaned. With the method according to the invention, for example, the proteins napin and cruciferin can be isolated and purified from the same crude extract, the surface charges of the proteins being adjusted by varying the pH so that only one of the two main components (napin and cruciferin) each aqueous rapeseed extract selectively interacts with anion and cation exchangers. An efficient adsorption chromatographic separation can thus be achieved.

Alternatively, the method according to the invention also allows the isolation and purification of recombinant proteins, preferably scFv antibodies, which e.g. can be isolated and purified, as indicated in Examples 2 to 4 below, in which an aqueous extract of potato leaves was prepared, which was then introduced into an "expanded bed" process without precipitation or other pre-cleaning steps.

Overall, the method according to the invention is distinguished in many respects from conventional methods. Above all, its simplicity, technical scalability without high equipment expenditure, the robustness of the column materials, the possible elimination of oil removal from plant tissues such as seeds, and extensive pre-treatment steps before applying the aqueous extract to the chromatography material and the achievable high Purity of the native or recombinant proteins and thus their availability at economically favorable conditions. Furthermore, the order of isolation of several proteins from the same protein mixture can be chosen arbitrarily, taking into account the physicochemical characteristics of the respective proteins.

Brief description of the figures

Figure 1: Results of SDS-PAGE of the napin purified according to Example 1 (A)

A 10% Novex gel from Invitrogen (Groningen, NL) was used; Running buffer: MES, color: Coomaassie Brilliant Blue. Lanes 1 and 2 show the washing functions with 150 mM NaCl from the Streamline SP XL ™ column. Lane 3 is the SDS-7 molecular weight marker from Sig a (Deisenhofen, Germany). Lanes 4-7 show purified napin in the Elution fractions (peak tip and peak shoulder).

Figure 2: Results of the SDS-PAGE of cruciferin purified according to Example 1 (B)

A 10% Novex gel from Invitrogen (Groningen, NL) was used; Running buffer: MES, color: Coomaassie Brillant Blue. Lane 1 shows the passage of the Streamline DEAE column, lane 2 is the SDS-7 molecular weight marker from Sigma (Deisenhofen). Lanes 3-5 show purified cruciferin in the elution fractions (peak tip and peak shoulder).

Figure 3: Results of SDS-PAGE of the scFv antibody purified according to Example 2

SDS page made from transgenic potato leaves. A 10% Novex gel from Invitrogen (Groningen, NL) was used; Running buffer: MES, color: silver. Lane 1 shows the leaf extract, lane 2 the passage of the Streamline QXL column, lane 3 the eluate from the Streamline Q XL column, lane 4 is the wash fraction with 0.5 M NaCl. Lane 5 is the SDS-7 molecular weight marker from Sigma (Deisenhofen).

The following examples illustrate the invention.

Example 1: Purification of Napin and Cruciferin from Non-Oiled Rapeseed

(A) extraction

Rapeseed (100 g) was ground in a grinder to medium grinding strength and extracted with five times the amount of buffer (20 mM phosphate buffer, pH 7.5). For this, the mixture was about 30 min. stirred and then the solid portions were centrifuged off. After decanting the supernatant into a collecting vessel, the precipitate was extracted a second time with the same amount of buffer as before and processed further as described above. The combined liquid phases were adjusted to a pH of 7.5 and then fed directly to the fluid bed adsorption ("expanded bed" technology). (B): Extraction of napin

Napin from the rapeseed extract was first adsorbed onto a strong cation exchanger (Streamline SP XL ™; Amersham Pharmacia, Uppsala, Sweden). For this purpose, the liquid phase with a flow of 200 cm / h. pumped through the equilibrated fluid bed. A Streamline 25 column with a bed of 2.5 x 11.5 cm = 56.5 ml was used. Subsequently, in the expanded state (due to the outflow, the bed expands, at 200 cm / h approximately twice) with 10 column volumes of charge buffer (20 mM phosphate buffer, pH 7.5). All other work steps were carried out in the sedimented state of the bed. The flow is no longer from below, but from above, so that it returns to the original bed height of 11.5 cm. First washing was carried out with 3 column volumes of washing buffer (20 mM phosphate buffer, pH 7.5, 0.15 M NaCl), followed by elution with elution buffer (20 mM phosphate buffer, pH 7.5, 0, 5 M NaCl). The napin thus purified was desalted and lyophilized using methods known to those skilled in the art. The column was then regenerated by washing with 3 column volumes of 0.2 M NaOH and 0.2 M HCl.

(C): Obtaining cruciferin

The cruciferin was obtained from the run of the napin adsorption from (B) by "expanded bed" adsorption. The continuous fraction from (B) was adjusted to a pH of 8.5 and adsorbed on an anion exchanger (Streamline DEAE ™; Amersham Pharmacia). For this purpose, the liquid phase with a flow of 200 cm / h. pumped through the equilibrated fluid bed (column volume: 2.5 x 11.5 cm = 56.5 ml). The expanded state was then washed with 10 column volumes of charge buffer (20 mM phosphate buffer, pH 8.5). All other work steps were carried out in the sedimented state of the bed. First with 3 column volumes of buffer La ^¬ dung was washed, and then elution was performed with elution tion buffer (20 mM phosphate buffer, pH 7.5, 0.2 M NaCl). The thus purified cruciferin was the most professional ^¬ th process and lyophilized. The pillar was then regenerated by washing with 3 column volumes of 0.5 M NaCl and 0.2 M HC1.

(D): Determination of the purity of the proteins obtained in (B) and (C)

The purified storage proteins napin and cruciferin were subjected to a purity analysis using SDS-PAGE (see FIG. 1 for napin and FIG. 2 for cruciferin). In both cases the purity of the proteins is 99%.

Example 2: Isolation of recombinant scFv antibodies from transgenic potato leaves

Transgenic potato leaves transformed with a vector encoding an scFv antibody were mixed in a ratio of 1: 1 (mass: volume) with 20 mM phosphate buffer (pH 7.0) and homogenized in a "Warring Blender" for 3 6 seconds , The green juice obtained was freed from particles by centrifugation (15 min, 10,000 rpm), adjusted to a pH of 7.0 and at a flow of 200 cm / h. pumped through the equilibrated (20 mM phosphate buffer, pH 7.0) fluid bed ("expanded bed") (column volume: 2.5 × 11.5 cm = 56.5 ml). Adsorption was carried out on Streamline Q XL ™ (Amersham Pharmacia). After loading, the expanded state was washed with 10 column volumes of loading buffer (20 mM phosphate buffer, pH 7.0) and 3 column volumes of loading buffer in the sedimented state of the bed, followed by elution of the scFv antibodies using a linear salt gradient from 0 to 0. 5 M NaCl in 20 mM phosphate buffer, pH 7.0. The purified antibodies were desalted and lyophilized using methods known to those skilled in the art. SDS-PAGE was able to demonstrate that the main protein component of the leaves, ribulose bisphosphate carboxylase, was completely separated in one step and antibodies with a purity of 90% could be obtained (FIG. 3).

Example 3: Isolation of recombinant scFv antibodies from transgenic rapeseed

Rapeseed (100 g) was homogenized as described in Example 1 nized and extracted with five times the amount of buffer (20 mM phosphate buffer, pH 7.5). For this, the suspension was stirred for 15 minutes. After decanting the liquid phase, the extraction was repeated once in the manner described. In the combined liquid phases the pH was adjusted to 7.5 and these were then at a flow of 200 cm / h. pumped through the equilibrated (20 mM phosphate buffer, pH 7.5) fluid bed ("Expanded bed") (column volume: 2.5 × 11.5 cm = 56.5 ml). Adsorption was carried out on Streamline Q XL ™ (Amersham Pharmacia). After loading, the expanded state was washed with 10 column volumes of loading buffer (20 mM phosphate buffer, pM 7.0) and 3 column volumes of loading buffer in the sedimented state of the bed, followed by elution of the scFv antibodies with 6 column volumes of washing buffer (phosphate buffer, pH 7, 5) with 0.5 M NaCl. The highly purified scFv antibodies were desalted and lyophilized using methods known to those skilled in the art.

Example 4: Isolation of recombinant scFv antibodies from defatted transgenic rapeseed

Rapeseed (100 g) was comminuted using methods known to those skilled in the art and gently degreased with hexane. After the hexane treatment, the seeds were pulverized. The seed powder was extracted with five times the mass of buffer (20 mM phosphate buffer, pH 7.5). For this, the suspension was stirred for 15 minutes. After decanting the liquid phase, the extraction was repeated once in the manner described. In the combined liquid phases, the pH was adjusted to 7.5 and then with a flow of 200 cm / h. pumped through the equilibrated (20 mM phosphate buffer, pH 7.5) expanded bed (column volume: 2.5 × 11.5 cm = 56.5 ml). Adsorption was carried out on Streamline Q XL ™ (Amersham Pharmacia). After loading, the expanded state was washed with 10 column volumes of loading buffer (20 M phosphate buffer, pH 7.5) and 3 column volumes of loading buffer in the sedimented state of the bed, followed by elution of the scFv antibodies with 6 column volumes of loading buffer with 0.5 M NaCl. The highly purified scFv antibodies were with the expert known processes desalted and lyophilized.

Claims

claims 1. A method for obtaining a desired native or recombinant protein from a plant in pure form, the method comprising the following steps: (a) disintegrating the plant to obtain a crude extract using a suitable extracting agent; and (b) Separation of the desired protein via ion exchange chromatography in "expanded bed" technology, the binding of the crude extract taking place at a defined pH. 2. The method according to claim 1, wherein the plant is a gramineae, a chenopodia, a legume, a brassica, a solanacee, a fungus, a moss or an algae. 3. The method according to claim 2 ,. where the plant is wheat, barley, rice, corn, sugar beet, sugar cane, rapeseed, mustard, turnip, flax, pea, bean, lupine, tobacco or potato. 4. The method according to any one of claims 1-3, wherein the desired protein is obtained from seeds, leaves, tubers, rhizomes, seedlings or cuttings. 5. The method according to any one of claims 1 to 4, wherein the desired protein is obtained from a storage protein mixture. 6. The method according to any one of claims 1 to 5, wherein the extractant is phosphate buffer, TRIS buffer, pug buffer or an ethanolic extractant. 7. The method according to any one of claims 1 to 6, wherein in step (b) the pH is in the range of 7.5 to 9.0. 8. The method according to any one of claims 1 to 7, wherein in step (b) the desired protein is eluted with a NaCl-containing buffer of defined molarity, by means of a NaCl gradient or by changing the pH. 9. The method according to any one of claims 1 to 8, wherein the ion exchange chromatography is an anion exchange chromatography. 10. The method of claim 9, wherein the anion exchange chromatography material is Streamline DEAE ™ or Streamline QXL. 11. The method according to any one of claims 1 to 8, wherein the ion exchange chromatography is a cation exchange chromatography. 12. The method of claim 11, wherein the cation exchange chromatography material is Streamline SP XL ™. 13. The method according to any one of claims 2 to 12, wherein the desired protein is napin, napin-like 2S albumin, cruciferin or legumin-like IIS globulin. 14. The method according to any one of claims 2 to 12, wherein the desired protein is an scFv antibody.