CN1263107A - Ribosome deactivated protein-pumpkin protein - Google Patents
Ribosome deactivated protein-pumpkin protein Download PDFInfo
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- CN1263107A CN1263107A CN 99102290 CN99102290A CN1263107A CN 1263107 A CN1263107 A CN 1263107A CN 99102290 CN99102290 CN 99102290 CN 99102290 A CN99102290 A CN 99102290A CN 1263107 A CN1263107 A CN 1263107A
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- cucurmosin
- protein
- ribosome inactivating
- inactivating protein
- albumen
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Abstract
The ribosome deactivated protein-pumpkin protein (Cucurmosin) is separated from common vegetable cucurbitaceous plant pumpkin seed, its molecular weight is 27KD (using SDS-PAGE to make detection), and its isoelectric point is greater than 9. It adopts moderate extraction method, including DEAE-cellulose filtration and twice SP-agarose fast-velocity gel ion-exchange chromatography, to obtain high-purity protein. Said protein is cultured into crystal body for x-ray diffraction. It can be used as medicine for induction of labor and resisting tumor, can be used as N-glycosidase in the field of molecular biology and immunology research, also can be used as toxin material of immune toxin.
Description
The present invention relates to organic chemistry filed, particularly relate to a kind of ribosome inactivating protein-Cucurmosin and preparation and application in the biological chemistry.
In this article, ribosome inactivating protein can be defined as having at least the protein of one of following activity: RNA-N glycosidase activity, induced labor protein-active, antitumor or antiviral activity.This albumen is the simple albumen or the glycoprotein of the single chain polypeptide of oligomerization.
From higher plant, extract ribosome inactivating protein, can be divided into two classes according to its molecular structure, type i is a single chain polypeptide albumen, for example, Trichosanthin (Trichosanthin) from cucurbitaceous plant piece root and seed, sponge gourd seed albumen (Luffin), octagon towel gourd seed albumen (Luffaculin) and from saponaretin (Saporin) of pinkwort seed etc.Type II is two peptide catenins, for example, and from the Ricin (Ricin) of euphorbia plant seed with from the abrin (Abrin) of leguminous plants seed.The proteic A chain of type i albumen and Type II all is that the N-glycosidic link by 4324 VITAMIN B4 of 28S rRNA in the cracking rrna makes eukaryotic cell rrna 60S subunit inactivation (Endo and Tsaragi 1987, J.Biol.Chem.262:8128-8130, Stirpe, F.et al., 1988, Nacl.Acid Res.16:1349-1357).Ribosome inactivating protein about type i and Type II has had a lot of patents and bibliographical information.
Pumpkin (Cucurbita moschata) is the kind of Curcurbitaceae Cucurbita, and pumpkin and seed thereof all are common vegetables and dry fruit.(1994, journal of biological chemistry 10:397-401) is separated to a kind of ribosome inactivating protein Maximin to people such as Zhong Xuemei from winter squash seed (Cucurbita maxima), and its molecular weight is 28 KD, IC
50Be 4.0 * 10
-10Mol/L, similar to the Trichosanthin activity.Yoshinari, S; Et al isolates a kind of ribosome inactivating protein Pepocin from summer squash (Cucurbita pepo), its molecular weight is 26 KD, IC
50Be 15.4 * 10
-12Mol/L.Other ribosome inactivating protein does not appear in the newspapers in this platymiscium.
At present, isolated two kinds of ribosome inactivating proteins from the melon and fruit of cucurbitaceous plant, a kind of is Loshinari, S et al isolating pepocin from summer squash, a kind of is Lee-Huang, the MAP that S et al is separated to from balsam pear (Momordica charantia).
The objective of the invention is from the cucurbita plants pumpkin, to isolate a kind of new ribosome inactivating protein-Cucurmosin, and disclose its Preparation method and use.The molecular weight that shows it through SDS-PAGE is 27 KD, iso-electric point pI＞9.
The present invention adopts a kind of separation purification method of gentleness, be that the pumpkin chopping is after acetate buffer solution, or phosphoric acid buffer, or water, or salt solution extracting, extract is after weak anionic gel medias such as DEAE-Mierocrystalline cellulose or DEAE-agarose filter, strong or the weak cation gel media secondary ions displacement chromatography separation through SP-agarose, S-agarose, CM-Mierocrystalline cellulose, CM-agarose etc. again, just can obtain the pure protein of electrophoresis one band, we are its called after Cucurmosin (Cucurmosin), SDS-PAGE shows that its molecular weight is 27 KD, iso-electric point pI＞9.
Cucurmosin of the present invention and Trichosanthin are similar, can use as drug inducing labor or anti-tumor virus formulation.
Cucurmosin pair cell internal protein of the present invention biosynthesizing has the strongly inhibited effect, is suitable for doing the toxin material of immunotoxin.
Cucurmosin of the present invention can also be used for molecular biology and immunology research as RNA N-Glycosylase.
Use separation purification method of the present invention, can from the sophisticated melon and fruit (as: Snakegourd Fruit, colocynth, wax gourd etc.) of other kinds of Curcurbitaceae platymiscium, isolate ribosome inactivating protein, its molecular weight is about 27 KD, the iso-electric point meta-alkalescence, and have strongly inhibited intracellular nucleic sugar body inactivating protein synthetic activity.
Use separation purification method of the present invention, can from the sporophore of Basidiomycetes fungi (as mushroom, mushroom), isolate ribosome inactivating protein, its molecular weight about 27 KD, iso-electric point meta-alkalescence, and have strongly inhibited intracellular nucleic sugar body protein synthetic activity.
Cucurmosin of the present invention, can use vapor diffusion technique, free interface method or dialysis process, in the phosphoric acid buffer of 0.05～0.2 Mol/L, citrate buffer solution, acetate buffer solution, imidazole buffer, polyoxyethylene glycol with 10%～40% is under the precipitation agent condition, grows the monocrystalline that can supply X-ray diffraction to use.
The present invention compared with prior art has following positively effect:
(1). pumpkin is vegetables commonly used among the people, adopts gentle extracting method can obtain a kind of new ribosome inactivating protein-Cucurmosin.
(2). isolating winter squash seed albumen and Pepocin are similar in the present invention and this platymiscium, and they all belong to the type i ribosome inactivating protein, the about 26～28KD of molecular weight.
(3). Cucurmosin crystal provided by the invention, can carry out X-ray diffraction analysis and molecular structure work in a deep going way.
Now accompanying drawing is made drawing explanation: Fig. 1. show that ribosome inactivating protein-Cucurmosin carries out the cation-exchange chromatography figure of purifying; Fig. 2. show the cation-exchange chromatography figure that Cucurmosin carries out repurity; Fig. 3. show the 12%SDS-PAGE figure of (Fig. 1); Fig. 4. show the 12%SDS-PAGE figure of (Fig. 2).
Further describe the present invention below in conjunction with accompanying drawing by embodiment:
Get the flavous sophisticated pumpkin wooden dipper of 3 Kg, be cut into small pieces, add an amount of 20mM acetate buffer solution (pH4.5) homogenate, with the centrifugal collection crude extract of water extracter, Glacial acetic acid with 50% is transferred pH4.0, through frozen centrifugation (15,000rpm, 15 ') collect clear liquid, the 0.2 M phosphoric acid buffer (pH6.0) that adds 1/10 volume is transferred pH neutrality, adds DEAE-Mierocrystalline cellulose-52 (swelling in 20mM pH6.0 phosphoric acid buffer in advance) and stirs the fast flow velocity gel of SP-agarose ion exchange column on the clear liquid after the filtration, wash post with 20mM pH6.0 phosphoric acid buffer, use 0～1M NaCl (in 20mM pH6.0 phosphoric acid buffer) gradient elution then.Component 17-19 is exactly the Cucurmosin of preliminary purification.Merge component 17-19, transfer pH4.0,, wash post, use 0～0.5M NaCl (in 20mM pH6.0 phosphoric acid buffer) gradient elution then with the 20mMpH6.0 phosphoric acid buffer through the repurity of the fast flow velocity gel of SP-agarose ion exchange column with hydrochloric acid.The component 50 that is obtained is exactly a Cucurmosin.SDS-PAGE shows that its molecular weight is 27 KD.
Embodiment 2.SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Ribosome inactivating protein-Cucurmosin, on the micro-slab-electrophoresis instrument of Bio-Rad company, carry out SDS-PAGE, SDS-PAGE is undertaken by the method for Leammli, resolving gel concentration is 12%, concentrated gum concentration is 4%, press monarch's Guo Yao method (1991 behind the electrophoresis, biological chemistry and biophysics progress, 18:32-37), dye with Coomassie brilliant blue, (((MW～14KD) is the molecular weight standard contrast, shows that the molecular weight of Cucurmosin is 27 KD for MW～27KD) and N,O-Diacetylmuramidase for MW～67KD), Trichosanthin with bovine serum albumin.SDS-PAGE the results are shown in accompanying drawing 3 and accompanying drawing 4.
Claims (8)
1. a ribosome inactivating protein-Cucurmosin (Cucurmosin) is characterized in that, this albumen is separated from pumpkin, and its molecular weight (SDS-PAGE detection) is 27 KD, iso-electric point＞9.
2. ribosome inactivating protein-Cucurmosin as claimed in claim 1 is characterized in that, it has strongly inhibited intracellular nucleic sugar body protein synthetic activity.
3. the separation purification method of the ribosome inactivating protein-Cucurmosin of a claim 1, comprise that buffered soln extracting, DEAE-Mierocrystalline cellulose filter, the fast flow velocity gel of secondary SP-agarose ion exchange chromatography, it is characterized in that, filter and the fast flow velocity gel of secondary SP-agarose ion exchange chromatography by the DEAE-Mierocrystalline cellulose.
4. the separation purification method of the ribosome inactivating protein-Cucurmosin of a claim 3, it is characterized in that, can from the sophisticated melon and fruit (as: Snakegourd Fruit, colocynth, wax gourd etc.) of other kinds of Curcurbitaceae platymiscium, isolate ribosome inactivating protein with similar methods, its molecular weight is about 27 KD, the iso-electric point meta-alkalescence, and have strongly inhibited intracellular nucleic sugar body protein synthetic activity.
5. the separation purification method of the ribosome inactivating protein-Cucurmosin of a claim 3, it is characterized in that, can from the sporophore of Basidiomycetes fungi (as mushroom, mushroom), isolate ribosome inactivating protein with similar methods, its molecular weight is about 27 KD, the iso-electric point meta-alkalescence, and have strongly inhibited intracellular nucleic sugar body protein synthetic activity.
6. the application of the ribosome inactivating protein-Cucurmosin of a claim 1 is characterized in that, this albumen can be used as induced labor albumen and is applied to induced labor, and the sick and viral infection disease of treatment related neoplasms.
7. the application of a kind of ribosome inactivating protein-Cucurmosin as claimed in claim 6 is characterized in that, this albumen also can be developed the toxin material as the preparation immunotoxin.
8. the crystal growth of the ribosome inactivating protein-Cucurmosin of a claim 1, it is characterized in that, can use vapor diffusion technique, free interface method or dialysis process, in the phosphoric acid buffer of 0.05～0.2 Mol/L, citrate buffer solution, acetate buffer solution, imidazole buffer, polyoxyethylene glycol with 10%～40% is under the precipitation agent condition, can success grow the monocrystalline that can use for X-ray diffraction.
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|---|---|---|---|
| CN 99102290 CN1263107A (en) | 1999-02-11 | 1999-02-11 | Ribosome deactivated protein-pumpkin protein |
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|---|---|---|---|
| CN 99102290 CN1263107A (en) | 1999-02-11 | 1999-02-11 | Ribosome deactivated protein-pumpkin protein |
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| CN1263107A true CN1263107A (en) | 2000-08-16 |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100370011C (en) * | 2005-11-14 | 2008-02-20 | 中国农业大学 | Method for extracting pumpkin seed oil and pumpkin seed protein |
| CN100429224C (en) * | 2003-09-29 | 2008-10-29 | 中国科学院福建物质结构研究所 | Antitumor activity of squash protein and use thereof |
| CN1830486B (en) * | 2004-11-05 | 2010-08-25 | 福建医科大学 | Application of pumpkin protein in preparation of medicine |
| CN101822819B (en) * | 2004-11-05 | 2011-10-26 | 中国科学院福建物质结构研究所 | Application of pumpkin protein to preparation of medicament for treating gastric cancer |
| CN101843894B (en) * | 2005-11-03 | 2012-02-15 | 中国科学院福建物质结构研究所 | Application of pumpkin protein to preparation of medicament for treating lung cancer |
| CN102038934B (en) * | 2005-11-03 | 2012-08-29 | 福建医科大学 | Application of cucurmosin in preparation of drug for treating breast cancer |
| CN102038935B (en) * | 2005-11-03 | 2012-08-29 | 福建医科大学 | Application of Cucurmosin in preparation of medicaments for treating cervical cancer |
| CN101386644B (en) * | 2007-01-04 | 2012-11-28 | 中国科学院福建物质结构研究所 | Pumpkin protein 2 and preparation method and application thereof |
| CN104498494A (en) * | 2015-01-06 | 2015-04-08 | 山西大学 | Plasmid for detecting activity of ribosome inactivating protein as well as construction method and application of plasmid |
-
1999
- 1999-02-11 CN CN 99102290 patent/CN1263107A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100429224C (en) * | 2003-09-29 | 2008-10-29 | 中国科学院福建物质结构研究所 | Antitumor activity of squash protein and use thereof |
| CN1830486B (en) * | 2004-11-05 | 2010-08-25 | 福建医科大学 | Application of pumpkin protein in preparation of medicine |
| CN101822819B (en) * | 2004-11-05 | 2011-10-26 | 中国科学院福建物质结构研究所 | Application of pumpkin protein to preparation of medicament for treating gastric cancer |
| CN101843894B (en) * | 2005-11-03 | 2012-02-15 | 中国科学院福建物质结构研究所 | Application of pumpkin protein to preparation of medicament for treating lung cancer |
| CN102038934B (en) * | 2005-11-03 | 2012-08-29 | 福建医科大学 | Application of cucurmosin in preparation of drug for treating breast cancer |
| CN102038935B (en) * | 2005-11-03 | 2012-08-29 | 福建医科大学 | Application of Cucurmosin in preparation of medicaments for treating cervical cancer |
| CN100370011C (en) * | 2005-11-14 | 2008-02-20 | 中国农业大学 | Method for extracting pumpkin seed oil and pumpkin seed protein |
| CN101386644B (en) * | 2007-01-04 | 2012-11-28 | 中国科学院福建物质结构研究所 | Pumpkin protein 2 and preparation method and application thereof |
| CN104498494A (en) * | 2015-01-06 | 2015-04-08 | 山西大学 | Plasmid for detecting activity of ribosome inactivating protein as well as construction method and application of plasmid |
| CN104498494B (en) * | 2015-01-06 | 2017-11-03 | 山西大学 | Detect plasmid and its construction method and the application of ribosome inactivating protein activity |
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