WO1996000239A1 - Method of isolating isolectins from mistletoe - Google Patents
Method of isolating isolectins from mistletoe Download PDFInfo
- 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
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.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Botany (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Description
Claims
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 |
EP95924317A EP0766697A1 (en) | 1994-06-23 | 1995-06-23 | Method of isolating isolectins from mistletoe |
JP8502798A JPH10504287A (en) | 1994-06-23 | 1995-06-23 | Method for isolating lectin from mistletoe |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DEP4421895.8 | 1994-06-23 | ||
DE19944421895 DE4421895A1 (en) | 1994-06-23 | 1994-06-23 | Process for isolating isolectins from mistletoe |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996000239A1 true WO1996000239A1 (en) | 1996-01-04 |
Family
ID=6521266
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/EP1995/002445 WO1996000239A1 (en) | 1994-06-23 | 1995-06-23 | Method of isolating isolectins from mistletoe |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0766697A1 (en) |
JP (1) | JPH10504287A (en) |
AU (1) | AU2887595A (en) |
DE (1) | DE4421895A1 (en) |
WO (1) | WO1996000239A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19639375A1 (en) * | 1996-09-25 | 1998-04-02 | Aar Pharma | Mistletoe dry extracts |
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)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100832614B1 (en) * | 2000-11-14 | 2008-05-27 | 아이언 프라임 | Orally ingestible preparation of mistletoe lectins and method |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
-
1994
- 1994-06-23 DE DE19944421895 patent/DE4421895A1/en not_active Withdrawn
-
1995
- 1995-06-23 AU AU28875/95A patent/AU2887595A/en not_active Abandoned
- 1995-06-23 JP JP8502798A patent/JPH10504287A/en active Pending
- 1995-06-23 WO PCT/EP1995/002445 patent/WO1996000239A1/en not_active Application Discontinuation
- 1995-06-23 EP EP95924317A patent/EP0766697A1/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE19639375A1 (en) * | 1996-09-25 | 1998-04-02 | Aar Pharma | Mistletoe dry extracts |
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 |
---|---|
JPH10504287A (en) | 1998-04-28 |
AU2887595A (en) | 1996-01-19 |
EP0766697A1 (en) | 1997-04-09 |
DE4421895A1 (en) | 1996-01-04 |
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