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WO1996000239A1 - Procede permettant d'isoler des isolectines de gui - Google Patents

Procede permettant d'isoler des isolectines de gui Download PDF

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Publication number
WO1996000239A1
WO1996000239A1 PCT/EP1995/002445 EP9502445W WO9600239A1 WO 1996000239 A1 WO1996000239 A1 WO 1996000239A1 EP 9502445 W EP9502445 W EP 9502445W WO 9600239 A1 WO9600239 A1 WO 9600239A1
Authority
WO
WIPO (PCT)
Prior art keywords
mistletoe
lectins
lactosyl
type
isolectins
Prior art date
Application number
PCT/EP1995/002445
Other languages
German (de)
English (en)
Inventor
Rudolf Eifler
Uwe PFÜLLER
Original Assignee
Private Universität 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 Universität Witten/Herdecke Gmbh filed Critical Private Universität Witten/Herdecke Gmbh
Priority to AU28875/95A priority Critical patent/AU2887595A/en
Priority to JP8502798A priority patent/JPH10504287A/ja
Priority to EP95924317A priority patent/EP0766697A1/fr
Publication of WO1996000239A1 publication Critical patent/WO1996000239A1/fr

Links

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)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un procédé permettant d'isoler des isolectines de gui de type ML I, selon lequel: du gui est récolté sous forme de plante fraîche sur des plantes hôtes telles que le bouleau ou l'érable, il est broyé et mis en suspension dans un milieu aqueux; la suspension aqueuse est éventuellement filtrée et son pH est ajusté à 7; la suspension est éventuellement centrifugée, le surnageant obtenu est traité au moyen d'un échangeur de cations, la fraction d'élution qui contient des lectines de gui est recueillie; cette opération est suivie d'une chromatographie sur des matériaux supports modifiés par lactosyl; désorption des lectines de type MLI par traitement du matériau support modifié par lactosyl, séparation par échangeur de cations, suivie de la collecte des fractions de lectine de type ML I-1 et ML I-2 du gui. L'invention concerne des lectines de gui de type ML I-1 et ML I-2, obtenues selon un procédé décrit d'après une des revendications 1 à 5, ainsi que des fragments obtenus par dissociation réductive avec des réactifs dissociant les liaisons de bisulfures.
PCT/EP1995/002445 1994-06-23 1995-06-23 Procede permettant d'isoler des isolectines de gui WO1996000239A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU28875/95A AU2887595A (en) 1994-06-23 1995-06-23 Method of isolating isolectins from mistletoe
JP8502798A JPH10504287A (ja) 1994-06-23 1995-06-23 ヤドリギからイソレクチンを単離する方法
EP95924317A EP0766697A1 (fr) 1994-06-23 1995-06-23 Procede permettant d'isoler des isolectines de gui

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19944421895 DE4421895A1 (de) 1994-06-23 1994-06-23 Verfahren zur Isolierung von Isolektinen aus der Mistel
DEP4421895.8 1994-06-23

Publications (1)

Publication Number Publication Date
WO1996000239A1 true WO1996000239A1 (fr) 1996-01-04

Family

ID=6521266

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1995/002445 WO1996000239A1 (fr) 1994-06-23 1995-06-23 Procede permettant d'isoler des isolectines de gui

Country Status (5)

Country Link
EP (1) EP0766697A1 (fr)
JP (1) JPH10504287A (fr)
AU (1) AU2887595A (fr)
DE (1) DE4421895A1 (fr)
WO (1) WO1996000239A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19639375A1 (de) * 1996-09-25 1998-04-02 Aar Pharma Mistel-Trockenextrakte
US6792715B2 (en) 2001-07-09 2004-09-21 University Of Copenhagen Methods and cuttings for mass propagation of plant parasites

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ526202A (en) * 2000-11-14 2005-08-26 Ian Pryme Orally ingestible preparation of mistletoe lectins, ML-I, ML-II and ML-III and methods fro treating cancer and autoimmune diseases

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD296703A5 (de) * 1990-07-16 1991-12-12 Staatliches Institut Fuer Immunpraeparate Und Naehrmittel,De Verfahren zur gewinnung von a- und b-kette des mistellektins i
DE4229876A1 (de) * 1992-09-04 1994-03-10 Uwe Dr Pfueller Verfahren zur Gewinnung von Lektinen aus Mistelpflanzen

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD296703A5 (de) * 1990-07-16 1991-12-12 Staatliches Institut Fuer Immunpraeparate Und Naehrmittel,De Verfahren zur gewinnung von a- und b-kette des mistellektins i
DE4229876A1 (de) * 1992-09-04 1994-03-10 Uwe Dr Pfueller Verfahren zur Gewinnung von Lektinen aus Mistelpflanzen

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19639375A1 (de) * 1996-09-25 1998-04-02 Aar Pharma Mistel-Trockenextrakte
US6792715B2 (en) 2001-07-09 2004-09-21 University Of Copenhagen Methods and cuttings for mass propagation of plant parasites

Also Published As

Publication number Publication date
DE4421895A1 (de) 1996-01-04
AU2887595A (en) 1996-01-19
JPH10504287A (ja) 1998-04-28
EP0766697A1 (fr) 1997-04-09

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