WO1996000239A1 - Method of isolating isolectins from mistletoe - Google Patents

Abstract

The invention concerns a method of isolating type ML I isolectins from mistletoe, the method calling for the following operations: mistletoe plants freshly harvested from birch or sycamore host plants are chopped and an aqueous suspension made; the aqueous suspension is optionally filtered and its pH adjusted to < 7; the suspension is optionally centrifuged, the supernatant liquid passed through a cation-exchange column and the eluting fraction containing the lectins from the mistletoe collected; this fraction is subjected to chromatography using a lactosyl-modified column support material; and the type ML I lectins are desorbed by treating the lactosyl-modified support material and separated on a cation-exchange column and from which the type ML I-1 and ML I-2 fractions are collected. The invention also concerns type ML I-1 and ML I-2 mistletoe lectins obtained by a method as described in one of claims 1 to 5, plus fragments obtained by reductive splitting with disulphide-bond-splitting reagents.

Description

Process for isolating isolectins from mistletoe

The present invention relates to a method for isolating isolates from mistletoe of type ML I, mistletoe isolectins of type ML I, their biologically active fragments obtainable by agents which break peptide bonds and a diagnostic agent which can be obtained by immunizing mammals with the isolectins according to the invention .

Mistletoe lectins are biologically active proteins which are described as immunostimulating / modulating and having a cytotoxic effect.

A distinction is made between three groups of isolectins that occur in mistletoe, for example mistletoe lectin I, II, III. Mistletoe lectin I consists of an A and B chain, which can be obtained by reductive cleavage of the mistletoe lectin.

To obtain individual chains of mistletoe lectin I, this is reduced in accordance with DD 296 703 in a homogeneous phase in the presence of 20 to 40% by volume glycerol. The resulting mixture is applied to an immunoglobulin-Sepharose or an Asialofetuin column and then eluted successively with a washing buffer and an elution buffer, each containing 20 to 40% by volume glycerol.

The B chain fraction is optionally subsequently determined by affinity chromatography on monoclonal anti-ML IA - 2nd

Chain antibodies separated.

DE 42 21 836 relates to a biochemically purified mistletoe lectin (ML-I) as a therapeutically applicable immunomodulator. This substance is obtained from aqueous mistletoe extract and purified. The ML I consists of a toxic A chain and a sugar-binding B chain. Structural data are also mentioned in the published specification. There are two different modifications to the A chain. The chains were partially sequenced starting from the N-terminus. , -

The process described in DE 42 21 836 is disadvantageous because it does not result in pure isolectin fractions during purification, but rather a complex mixture of isolectins is isolated which resists further chromatographic purifications. This is mainly due to the formation of dimers or other aggregates that can only be broken up under denaturing conditions, for example SDS gel electrophoresis. However, such structures are not native lectins that can be subjected to pharmacological-toxicological studies. So far, the mistletoe lectin obtained by means of denaturing processes has not been renatured.

DE 42 29 876 describes a process for obtaining lectins from mistletoe plants, an extract being obtained from fresh or dried mistletoe plants which contains the mistletoe lectins I to III. The method is based on a batch adsorption of the aqueous mistletoe extract followed by an affinity adsorption on lactosyl-Sepharose. The two fractions obtained are subsequently separated into individual lectin fractions by ion exchange chromatography.

Chromatography of the ML I fraction on a cation exchange column with increasing saline gradient makes two - 3rd

Subtractions of the lectins ML I, namely ML Il and ML 1-2 obtained. This method also supplies the ^" isolectins ^" only in analytical amounts which do not permit preparative extraction. With this extraction method, isolectins cannot be produced in sufficient quantities to determine reliable biochemical, pharmacological and toxicological data on the isolectins. The method described also provides isolectins that are no longer separable, possibly due to dimerizations that cannot be eliminated under non-denaturing conditions.

The technical problem on which the invention is based consists first of all in specifying a method with which the disadvantages mentioned above can be avoided, in particular in a reproducible manner reducing the number of lectin compounds in the mixture. The method is intended in particular to provide the isolectins ML I-1 and ML 1-2 but not their mixed aggregation products. Furthermore, the method ML I-1 and ML 1-2 fractions are to be delivered in preparatively manageable quantities.

Surprisingly, the technical problem on which the invention is based was solved by a method having the features of claim 1. The subsequent subclaims 2 to 5 relate to preferred embodiments of the method according to the invention.

Claim 6 relates to isolectins from the mistletoe obtainable by the process according to the invention. Claim 7 relates to biologically active fragments of the isolates of mistletoe according to the invention. Claim 8 relates to a diagnostic agent obtainable by immunizing mammals by using the isolectins according to the invention.

According to the invention, birch or maple is used as the host plant. The mistletoe plants cultivated on it are called Fresh plants are harvested and ground, then stirred with distilled water and the resulting filtered extract is acidified with acetic acid. After centrifugation, the supernatant is stirred with a suitable cation exchanger, preferably SP-Sephadex. The cation exchanger adsorbs the lectin-containing protein fraction, which after washing with a buffer solution is eluted with a basic buffer of higher ionic strength. In addition to the batch process, the process according to the invention can also be carried out as a column chromatographic process. From the raw lectin solution, the lectins are bound in a biospecific manner to suitable affinity carriers, preferably to lactosyl-Sepharose. The lectins of types II and III can be eluted with isotonic phosphate-buffered 0.8 to 1% saline solution, whereas the ML I is subsequently eluted specifically with solutions containing galactose. The ML I is separated into the lectins ML II and ML 1-2 by rechromatography on the cation exchanger, preferably a mono-S chromatography material.

The isolectins of the mistletoe ML I-1 and ML 1-2 obtainable by the process according to the invention are obtained in preparatively manageable amounts, so that pharmacological and toxicological studies can be carried out.

It is known to the person skilled in the art that protein structures can be broken down into subsections by peptide-cleaving agents, such as chemicals or enzymes. These fragments can have a biological activity similar to that of the parent compound.

The present invention also relates to fragments of the mistletoe isolectins which can be derived from the amino acid sequence as stated above, for example by means of specific peptide bond-cleaving reagents such as cyanogen bromide or by treatment with specific proteases such as endoproteases. According to the invention, such peptides are claimed which have a biological activity z. B. as an immunomodulator or have an antigenic structure.

By immunizing mammals with the isolectins from the mistletoe or their fragments, valuable diagnostic means can be obtained. These diagnostic agents are e.g. mono- or polyclonal antibodies or anti-idiotype antibodies, which can be obtained by immunizing rabbits.

The invention is explained in more detail using the following exemplary embodiment.

1. Reagents and starting materials

Mistletoe plant (Viscum album L. or other species) from various host trees; as a fresh plant, dried powder or mistletoe tea.

Acetic acid: 2 M

Acetate buffer: 0.1 M, pH 4.0

Acetate buffer: 0.015 M, pH 3.8

Citrate buffer: 0.015 M, pH 3.8, NaCl gradient from 0 to 0.6 M

Tris buffer: 0.1 M, 0.5 M NaCl, pH 8.0

Saline: 0.9% in dist. water

Galactose solution: 0.1 M, 0.9% NaCl

Ammonium sulfate solution: 3 M

Ammonium sulfate

SP-Sephadex C- ^50® , Pharmacia

Lactosyl-Sepharose 4B, Lactosyl-Sepharose 4CL, Lactosyl-

Sephacryl 400

Mono S column ^® , Pharmacia 2. Isolation and separation of lectins ML I, ML II, ML III, ML Il and ML 1-2

Fresh mistletoe from birch or maple is harvested. Fresh plant: 250 g roughly chopped with 500 ml. Dest. Water homogenized material stirred for 1 hour. The resulting suspension is filtered through a coarse-meshed cloth. The strongly cloudy colored solution is adjusted to pH 4.0 with 2 M acetic acid. The resulting precipitate is separated off by centrifugation at 5000 xg for 10 minutes. 4 g of solid, swollen SP-Sephadex C-50 ^{® are} added to the clear centrifugate and the mixture is stirred for 1 hour. The ion exchanger is separated off via a glass suction filter and washed with 0.1 M acetate buffer, pH 4.0, until the washing buffer leaves the Nutsche colorless. The adsorbed proteins are then eluted with 0.1 M Tris buffer (0.5 M NaCl, pH 8.0) using the column method. The resulting crude lectin solution is passed through a lactosyl-Sepharose column with a bed volume of 100 ml. The column is washed with three times the column volume of 0.9% saline. The last 200 ml contain lectins II and III. The ML I is then eluted with 1 column volume of 0.1 M galactose solution.

The ML II / III fraction is concentrated to 1/10 and then buffered over a Sephadex G 25 column in 0.015 M citrate buffer, pH 3.8. This solution is chromatographed on a Mono-S column (10/10 Mono-S, Pharmacia). The lectins II and III are eluted in succession with a saline gradient of 0 to 0.5 M in a 0.015 M citrate buffer, pH 3, 8 and a total volume of 160 ml. The two fractions thus obtained are re-chromatographed under these conditions. The ML II is eluted at 0.15 M and the ML III at 0.21 M NaCl.

The re-chromatography of the isolated ML I fraction, buffered in 0.015 M citrate buffer, pH 3, 8 and Mono-S (10/10) with a NaCl gradient of 0.2 to 0.6 M in citrate buffer (0.015 M, pH 3.8) yields the lectins ML Il NaCl at a ^{'concentration} of 0.36 M and the ML 1-2 at 0, 42 sts

Suspended in 3 M ammonium sulfate solution, all three lectins can be kept for at least 5 years without loss of activity and impairment of the solubilities.

Claims

Patent claims 1. Method for the isolation of isolectins from the mistletoe of type ML I, wherein Mistletoe plants from birch or maple as a host plant are harvested as fresh plants, crushed, suspended in an aqueous medium, the aqueous suspension is optionally filtered and adjusted to a pH <7, optionally centrifuged, the supernatant obtained is treated with a cation exchanger, the eluting fraction containing the lectins from the mistletoe is collected, followed by chromatography on lactosyl-modified carrier materials and desorption of the lectins of type ML I by treatment of the lactosyl-modified carrier material, separation on cation exchangers with subsequent collection the mistletoe lectin fractions of type ML II and ML 1-2. 2. The method according to claim 1, wherein the cation exchanger SP-Sephadex and the lactosyl-modified carrier material is lactosyl-Sepharose. 3. The method according to any one of claims 1 and / or 2, wherein the treatment of the aqueous digestion solution with cation exchanger is carried out in a batch process and / or Säulen¬ chromatography. 4. The method according to at least one of claims 1 to 3, wherein the treatment with lactosyl-modified support materials is carried out in a batch process and / or by column chromatography. Method according to at least one of claims 1 to 4, wherein the ML I fractions are eluted from the cation exchanger with a 0.2 to 0.6 M saline gradient solution, eluting at low ionic strength ML II and higher ionic strength ML 1-2. Mistletoe lectins of the types ML I-1 and ML 1-2 obtainable by a process according to one of claims 1 to 5 and fragments obtainable by reductive cleavage with reagents cleaving disulfide bonds. Mistletoe lectins, the lectins being obtainable by treating the mistletoe lectins according to claim 6 with specific chemical reagents or peptide bond-cleaving enzymes and isolating the fragments with immunomodulatory activity. Diagnostic agents with mono- or polyclonal antibodies or anti-idiotypes, obtainable by immunizing mammals with the mistletoe lectins according to at least one of claims 6 and / or 7.