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EP0766697A1 - Method of isolating isolectins from mistletoe - Google Patents

Method of isolating isolectins from mistletoe

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Publication number
EP0766697A1
EP0766697A1 EP95924317A EP95924317A EP0766697A1 EP 0766697 A1 EP0766697 A1 EP 0766697A1 EP 95924317 A EP95924317 A EP 95924317A EP 95924317 A EP95924317 A EP 95924317A EP 0766697 A1 EP0766697 A1 EP 0766697A1
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EP
European Patent Office
Prior art keywords
mistletoe
lectins
lactosyl
type
isolectins
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95924317A
Other languages
German (de)
French (fr)
Inventor
Rudolf Private Universität Witten/Herdecke EIFLER
Uwe Private Universität Witten/Herdecke PFÜLLER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Private Universitat Witten/herdecke GmbH
Original Assignee
Private Universitat Witten/herdecke GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Private Universitat Witten/herdecke GmbH filed Critical Private Universitat Witten/herdecke GmbH
Publication of EP0766697A1 publication Critical patent/EP0766697A1/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C07K14/42—Lectins, e.g. concanavalin, phytohaemagglutinin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants

Definitions

  • 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.
  • Mistletoe lectin I consists of an A and B chain, which can be obtained by reductive cleavage of the mistletoe lectin.
  • the B chain fraction is optionally subsequently determined by affinity chromatography on monoclonal anti-ML IA - 2nd
  • DE 42 21 836 relates to a biochemically purified mistletoe lectin (ML-I) as a therapeutically applicable immunomodulator.
  • ML-I mistletoe lectin
  • 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. , -
  • 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.
  • 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.
  • the method ML I-1 and ML 1-2 fractions are to be delivered in preparatively manageable quantities.
  • 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.
  • 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.
  • the process according to the invention can also be carried out as a column chromatographic process.
  • the lectins are bound in a biospecific manner to suitable affinity carriers, preferably to lactosyl-Sepharose.
  • 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.
  • 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.
  • diagnostic agents are e.g. mono- or polyclonal antibodies or anti-idiotype antibodies, which can be obtained by immunizing rabbits.
  • Mistletoe plant (Viscum album L. or other species) from various host trees; as a fresh plant, dried powder or mistletoe tea.
  • 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
  • Lactosyl-Sepharose 4B Lactosyl-Sepharose 4CL, Lactosyl-
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Botany (AREA)
  • Analytical Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

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

Verfahren zur Isolierung von Isolektinen aus der MistelProcess for isolating isolectins from mistletoe
Gegenstand der vorliegenden Erfindung ist ein Verfahren zur Isolierung von Isolektinen aus der Mistel vom Typ ML I, Mi- stelisolektine des Typ ML I, deren biologisch aktiven Frag¬ mente erhältlich durch Peptidbindungen spaltende Mittel sowie ein Diagnostikmittel erhältlich durch Immunisierung von Säugern mit den erfindungsgemäßen Isolektinen.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 .
Mistellektine sind biologisch aktive Proteine, welche als immunstimulierend/modulierend und cytotoxisch wirkend be¬ schrieben sind.Mistletoe lectins are biologically active proteins which are described as immunostimulating / modulating and having a cytotoxic effect.
Man unterscheidet drei Gruppen von Isolektinen, die in der Mistel vorkommen, beispielsweise Mistellektin I, II, III. Das Mistellektin I besteht aus einer A- und B-Kette, die durch reduktive Spaltung des Mistellektins gewonnen werden können.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.
Zur Gewinnung einzelner Ketten des Mistellektins I wird dieses gemäß DD 296 703 in homogener Phase in Anwesenheit von 20 bis 40 Vol.-% Glycerol reduziert. Das entstandene Gemisch wird auf eine Immunglobulin-Sepharose- oder eine Asialofetuin-Säule aufgebracht und danach nacheinander mit einem Waschpuffer und einem Elutionspuffer, die jeweils 20 bis 40 Vol.-% Glycerol enthalten, eluiert.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.
Die B-Kettenfraktion wird gegebenenfalls nachfolgend durch Affinitats-Chromatographie an monoklonalen anti-ML I-A- - 2The B chain fraction is optionally subsequently determined by affinity chromatography on monoclonal anti-ML IA - 2nd
Ketten-Antikörpern getrennt.Chain antibodies separated.
Die DE 42 21 836 betrifft ein biochemisch aufgereinigtes Mistellektin (ML-I) als therapeutisch anwendbarer Immun¬ modulator. Diese Substanz wird aus wäßrigem Mistelextrakt gewonnen und gereinigt. Das ML I besteht aus je einer toxischen A-Kette und einer zuckerbindenden B-Kette. Es sind in der Offenlegungsschrift auch Strukturdaten genannt. Dabei existieren zwei unterschiedliche Modifikationen der A-Kette. Die Ketten wurden jeweils vom N-Terminus beginnend partiell sequenziert. , -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. , -
Das in der DE 42 21 836 beschriebene Verfahren ist nach¬ teilig, da es bei der Reinigung keine reinen Isolektin- Fraktionen ergibt, sondern ein komplexes Gemisch von Iso¬ lektinen isoliert wird, das weiteren chromatographischen Reinigungen widersteht. Dies ist überwiegend auf Bildung von Dimeren oder anderen Aggregaten zurückzuführen, die nur unter denaturierenden Bedingungen, beispielsweise SDS-Gelelek- trophorese, aufgebrochen werden können. Solche Strukturen stellen jedoch keine nativen Lektine dar, die pharmakolo- gisch-toxikologischen Untersuchung zugeführt werden können. Eine Renaturierung des mittels denaturierender Verfahren gewonnenen Mistellektins ist bislang nicht gelungen.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.
Die DE 42 29 876 beschreibt ein Verfahren zur Gewinnung von Lektinen aus Mistelpflanzen, wobei aus frischen oder getrock¬ neten Mistelpflanzen ein Extrakt gewonnen wird, der die Mistellektine I bis III enthält. Das Verfahren geht aus von einer Batch-Adsorption des wäßrigen Mistelpflanzenextrakts gefolgt von einer Affinitätsadsorption an Lactosyl-Sepharose. Nachfolgend werden die zwei erhaltenen Fraktionen durch Ionenaustauscher-Chromatographie in einzelne Lektinfraktionen getrennt.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.
Durch Chromatographie der ML I-Fraktion an einer Kationenaus¬ tauschersäule mit steigendem Kochsalzgradienten werden zwei - 3Chromatography of the ML I fraction on a cation exchange column with increasing saline gradient makes two - 3rd
Subtraktionen der Lektine ML I, nämlich ML I-l und ML 1-2 erhalten. Auch dieses Verfahren liefert die angesprochenen" Isolektine lediglich in analytischen Mengen, die eine prä- parative Gewinnung nicht zuläßt. Mit diesem Gewinnungsver¬ fahren lassen sich Isolektine nicht in hinreichender Menge herstellen, um zuverlässige biochemische, pharmakologische und toxikologische Daten über die Isolektine zu ermitteln. Das beschriebene Verfahren liefert auch Isolektine, die nicht mehr trennbar sind, möglicherweise aufgrund von Dimerisierun- gen, die unter nicht denaturierenden Bedingungen nicht aufgehoben werden können.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.
Das der Erfindung zugrundeliegende technische Problem besteht zunächst darin, ein Verfahren anzugeben, mit dem es gelingt, die oben genannten Nachteile zu vermeiden, insbesondere in reproduzierbarer Weise die Anzahl der Lektinverbindungen im Gemisch zu reduzieren. Das Verfahren soll insbesondere die Isolektine ML I-l und ML 1-2 aber nicht deren gemischte Ag¬ gregationsprodukte liefern. Weiterhin soll das Verfahren ML I-l sowie ML 1-2 Fraktionen in präparativ handhabbaren Mengen liefern.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.
Überraschenderweise wurde das der Erfindung zugrundeliegende technische Problem gelöst durch ein Verfahren mit den Merk¬ malen des Anspruchs 1. Die sich daran anschließenden Unter¬ ansprüche 2 bis 5 betreffen bevorzugte Ausführungsformen des erfindungsgemäßen Verfahrens.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.
Anspruch 6 betrifft Isolektine aus der Mistel erhältlich nach dem erfindungsgemäßen Verfahren. Anspruch 7 betrifft bio¬ logisch aktive Fragmente der erfindungsgemäßen Isolektine der Mistel. Anspruch 8 betrifft ein Diagnostikmittel erhält¬ lich durch Immunisierung von Säugetieren durch Verwendung der erfindungsgemäßen Isolektine.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.
Erfindungsgemäß wird Birke oder Ahorn als Wirtspflanze verwendet. Die darauf kultivierten Mistelpflanzen werden als Frischpflanzen geerntet und zerkleinert, danach mit de¬ stilliertem Wasser gerührt und der resultierende filtrierte Extrakt wird mit Essigsäure angesäuert. Nach Zentrifugation wird der Überstand mit einem geeigneten Kationenaustauscher, bevorzugt SP-Sephadex, gerührt. Der Kationenaustauscher adsorbiert die lektinhaltige Proteinfraktion, die nach Waschen mit einer Pufferlösung mit einem basischen Puffer höherer Ionenstärke eluiert wird. Neben dem Batch-Verfahren kann das erfindungsgemäße Verfahren auch als säulen-chromato- graphisches Verfahren durchgeführt werden. Aus der Rohlektin- lösung werden die Lektine biospezifisch an geeigneten Äffini- tätsträgern, vorzugsweise an Lactosyl-Sepharose, gebunden. Dabei können die Lektine der Typen II und III mit iso¬ tonischer phosphatgepufferter 0,8 bis 1 %iger Kochsalzlösung eluiert werden, wohingegen das ML I nachfolgend spezifisch mit galactosehaltigen Lösungen eluiert wird. Durch Rechroma- tographie am Kationenaustauscher, vorzugsweise einem Mono-S- Chromatographiematerial, wird das ML I in die Lektine ML I-l und ML 1-2 getrennt.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.
Die durch das erfindungsgemäße Verfahren erhältlichen Iso¬ lektine der Mistel ML I-l und ML 1-2 fallen in präparativ handhabbaren Mengen an, so daß pharmakologische und toxiko¬ logische Untersuchungen durchgeführt werden können.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.
Es ist dem Fachmann bekannt, daß Proteinstrukturen durch pep- tidspaltende Agenzien, wie Chemikalien oder Enzyme in Unter¬ abschnitte zerlegt werden können. Diese Bruchstücke können eine ähnliche biologische Aktivität wie die der Stammver¬ bindung aufweisen.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.
Gegenstand der vorliegenden Erfindung sind auch Bruchstücke der Mistelisolektine, die sich aus der Aminosäuresequenz, wie sie oben angegeben ist, ableiten lassen z.B. durch spezifische, peptidbindung-spaltende Reagenzien wie Cyanogen- bromid oder durch Behandlung mit spezifischen Proteasen wie Endoproteasen. Erfindungsgemäß beansprucht werden solche Peptide, die eine biologische Aktivität z. B. als Immunmodulator oder eine antigene Struktur aufweisen.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.
Durch Immunisierung von Säugern mit den Isolektinen aus der Mistel oder deren Bruchstücken lassen sich wertvolle diagno¬ stische Mittel erhalten. Diese Diagnostikmittel sind z.B. mono- oder polyklonale Antikörper oder antiidiotype Anti¬ körper, die durch Immunisierung von Kaninchen erhalten werden können.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.
Die Erfindung wird anhand des folgenden Ausführungsbeispiels näher erläutert.The invention is explained in more detail using the following exemplary embodiment.
1. Reagenzien und Ausgangsstoffe1. Reagents and starting materials
Mistelpflanze (Viscum album L. oder andere Spezies) von ver¬ schiedenen Wirtsbäumen; als Frischpflanze, getrocknetes Pulver oder Misteltee.Mistletoe plant (Viscum album L. or other species) from various host trees; as a fresh plant, dried powder or mistletoe tea.
Essigsäure: 2 MAcetic acid: 2 M
Acetatpuffer: 0,1 M, pH 4,0Acetate buffer: 0.1 M, pH 4.0
Acetatpuffer: 0,015 M, pH 3, 8Acetate buffer: 0.015 M, pH 3.8
Citratpuffer: 0,015 M, pH 3,8, NaCl-Gradient von 0 bis 0,6 MCitrate buffer: 0.015 M, pH 3.8, NaCl gradient from 0 to 0.6 M
Trispuffer: 0,1 M, 0,5 M NaCl, pH 8,0Tris buffer: 0.1 M, 0.5 M NaCl, pH 8.0
Kochsalzlösung: 0,9 % in dest. WasserSaline: 0.9% in dist. water
Galaktose-Lösung: 0,1 M, 0,9 % NaClGalactose solution: 0.1 M, 0.9% NaCl
Ammoniumsulfatlösung: 3 MAmmonium sulfate solution: 3 M
AmmoniumsulfatAmmonium sulfate
SP-Sephadex C-50®, PharmaciaSP-Sephadex C- 50® , Pharmacia
Lactosyl-Sepharose 4B, Lactosyl-Sepharose 4CL, Lactosyl-Lactosyl-Sepharose 4B, Lactosyl-Sepharose 4CL, Lactosyl-
Sephacryl 400Sephacryl 400
Mono-S-Säule®, Pharmacia 2. Isolierung und Trennung der Lektine ML I, ML II, ML III, ML I-l und ML 1-2Mono S column ® , Pharmacia 2. Isolation and separation of lectins ML I, ML II, ML III, ML Il and ML 1-2
Frischmistel von Birke oder Ahorn werden geerntet. Frisch¬ pflanze: 250 g grob zerkleinert mit 500 ml. dest. Wasser homogenisiertes Material 1 Stunde gerührt. Die entstandene Suspension wird über ein grobmaschiges Tuch filtriert. Die stark getrübte gefärbte Lösung wird mit 2 M Essigsäure auf pH 4, 0 eingestellt. Der resultierende Niederschlag wird durch lOminütiges Zentrifugieren bei 5000 x g abgetrennt. Dem klaren Zentrifugat werden 4 g festes, gequollenes SP-Sephadex C-50® zugesetzt, und es wird 1 Stunde gerührt. Der Ionenaus¬ tauscher wird über eine Glasfilternutsche abgetrennt und mit 0,1 M Acetatpuffer, pH 4, 0 solange gewaschen, bis der Wasch¬ puffer farblos die Nutsche verläßt. Die adsorbierten Proteine werden anschließend mit 0,1 M Trispuffer (0,5 M NaCl, pH 8,0) nach dem Säulenverfahren eluiert. Die resultierende Roh- lektinlösung wird über eine Lactosyl-Sepharose-Säule mit 100 ml Bettvolumen gegeben. Nachgewaschen wird die Säule mit dem dreifachen Säulenvolumen 0,9 %iger Kochsalzlösung. Die letzten 200 ml enthalten die Lektine II und III. Das ML I wird anschließend mit 1 Säulenvolumen 0,1 M Galaktoselösung eluiert.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.
Die ML II/III-Fraktion wird auf 1/10 eingeengt und darauf über einer Sephadex-G 25-Säule in 0,015 M Citratpuffer, pH 3,8 umgepuffert. Diese Lösung wird an einer Mono-S-Säule (10/10 Mono-S, Pharmacia) chromatographiert. Mit einem Kochsalzgradienten von 0 bis 0,5 M in einem 0,015 M Citrat¬ puffer, pH 3, 8 und einem Gesamtvolumen von 160 ml werden die Lektine II und III nacheinander eluiert. Die beiden so erhaltenen Fraktionen werden unter diesen Bedingungen re- chromatographiert. Das ML II wird bei 0,15 M und das ML III bei 0,21 M NaCl eluiert.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.
Die ReChromatographie der isolierten ML I-Fraktion, umge¬ puffert in 0,015 M Citratpuffer, pH 3, 8 and Mono-S (10/10) mit einem NaCL-Gradienten von 0,2 bis 0,6 M in Citratpuffer (0,015 M, pH 3,8) ergibt die Lektine ML I-l bei einer NaCl-' Konzentration von 0,36 M und das ML 1-2 bei 0,42 M.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
Alle drei Lektine sind suspendiert in 3 M Ammoniumsulfat- lösung mindestens 5 Jahre ohne Aktivitätsverlust und Beein¬ trächtigung der Löslichkeiten haltbar. 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

P a t e n t a n s p r ü c h e Patent claims
1. Verfahren zur Isolierung von Isolektinen aus der Mistel vom Typ ML I, wobei1. Method for the isolation of isolectins from the mistletoe of type ML I, wherein
Mistelpflanzen von Birke oder Ahorn als Wirts¬ pflanze als Frischpflanzen geerntet, zerkleinert, in wäßrigem Milieu suspendiert werden, die wäßrige Suspension wird gegebenenfalls filtriert und auf einen pH-Wert < 7 eingestellt, gegebenenfalls zentrifugiert, der erhaltene Über¬ stand mit einem Kationenaustauscher behandelt, die eluierende Fraktion, enthaltend die Lektine aus der Mistel, aufgefangen, gefolgt von einer Chromatographie an Lactosyl- modifizierten Trägermaterialien und Desorption der Lektine des Typs ML I durch Be¬ handlung des Lactosyl-modifizierten Träger¬ materials, Trennung an Kationenaustauscher mit an¬ schließendem Auffangen der Mistellektin-Fraktionen vom Typ ML I-l und ML 1-2.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. Verfahren nach Anspruch 1, wobei der Kationenaus¬ tauscher SP-Sephadex und das Lactosyl-modifizierte Trägermaterial Lactosyl-Sepharose ist.2. The method according to claim 1, wherein the cation exchanger SP-Sephadex and the lactosyl-modified carrier material is lactosyl-Sepharose.
3. Verfahren nach einem der Ansprüche 1 und/oder 2, wobei die Behandlung der wäßrigen AufSchlußlösung mit Kationenaustauscher im Batch-Verfahren und/oder Säulen¬ chromatographie erfolgt.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. Verfahren nach mindestens einem der Ansprüche 1 bis 3, wobei die Behandlung mit Lactosyl-modifizierten Träger¬ materialien im Batch-Verfahren und/oder durch Säulen¬ chromatographie erfolgt. Verfahren nach mindestens einem der Ansprüche 1 bis 4, wobei die ML I-Fraktionen vom Kationenaustauscher mit einer 0,2 bis 0,6 M Kochsalzgradientenlösung eluiert werden, wobei bei niedriger Ionenstärke ML I-l und bei höheren Ionenstärke ML 1-2 eluiert.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.
Mistellektine vom Typ ML I-l und ML 1-2 erhältlich durch ein Verfahren gemäß einem der Ansprüche 1 bis 5 sowie Fragmente erhältlich durch reduktive Spaltung mit Disulfidbindungen spaltenden Reagenzien.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.
Mistellektine, wobei die Lektine erhältlich sind durch Behandlung der Mistellektine nach Anspruch 6 mit spe¬ zifischen Peptidbindungen spaltenden chemischen Reagenzien oder Peptidbindungen spaltenden Enzymen und Isolierung der Bruchstücke mit immunmodulatorischer Wirkung.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.
Diagnostikmittel mit mono- oder polyklonalen Anti¬ körpern oder Antiidiotypen, erhältlich durch Immuni¬ sierung von Säugern mit den Mistellektinen gemäß mindestens einem der Ansprüche 6 und/oder 7. 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.
EP95924317A 1994-06-23 1995-06-23 Method of isolating isolectins from mistletoe Withdrawn EP0766697A1 (en)

Applications Claiming Priority (3)

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DE4421895 1994-06-23
DE19944421895 DE4421895A1 (en) 1994-06-23 1994-06-23 Process for isolating isolectins from mistletoe
PCT/EP1995/002445 WO1996000239A1 (en) 1994-06-23 1995-06-23 Method of isolating isolectins from mistletoe

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DE19639375A1 (en) * 1996-09-25 1998-04-02 Aar Pharma Mistletoe dry extracts
WO2002040041A2 (en) * 2000-11-14 2002-05-23 Ian Pryme Orally ingestible preparation of mistletoe lectins and method
ATE361664T1 (en) 2001-07-09 2007-06-15 Univ Copenhagen METHOD AND CUT-OFFS FOR THE MASS PROPAGATION OF PLANT PARASITES

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DD296703A5 (en) * 1990-07-16 1991-12-12 Staatliches Institut Fuer Immunpraeparate Und Naehrmittel,De METHOD FOR OBTAINING THE A AND B CHAIN OF THE MISTELECTURE I
DE4229876A1 (en) * 1992-09-04 1994-03-10 Uwe Dr Pfueller Mistletoe lectin extn. - using a cationic ion exchanger for the isolation of MTLI-1 and MTL-1-2, MTLII and MTL-III, useful in bio-technology and diagnostics

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DE4421895A1 (en) 1996-01-04
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