CN107163118A - A kind of natural biological polypeptide SPNP 27 preparation method - Google Patents
A kind of natural biological polypeptide SPNP 27 preparation method Download PDFInfo
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- CN107163118A CN107163118A CN201710467845.5A CN201710467845A CN107163118A CN 107163118 A CN107163118 A CN 107163118A CN 201710467845 A CN201710467845 A CN 201710467845A CN 107163118 A CN107163118 A CN 107163118A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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Abstract
The invention discloses a kind of method for preparing novel bioactive polypeptide, obtained this novel bioactive polypeptide, entitled spider suppression sodium peptide 27（SPNP‑27）It is that the separation of totally three steps is obtained by cation exchange HPLC and two step reversed-phase HPLCs, is that a kind of new type nerve toxin identified is separated from Jingzhao chilobrachys spider venom, its primary structure is made up of 34 amino acid residues, wherein containing three pairs of disulfide bond of 6 cysteines and formation, the amidatioon of C ends.Molecular weight is 4086.4 Da, and the physicochemical character of the peptide purification freeze-dried powder is white or off-white color loosening body, odorlessness, and very soluble in water, the aqueous solution is bordering on water white transparency.
Description
Technical field
The present invention relates to a kind of novel bioactive polypeptide and preparation method thereof, and in particular to one kind passes through cation exchange
The method that HPLC and the separation of two step reversed-phase HPLCs obtain novel bioactive polypeptide.
Background technology
Biotoxin is a kind of important biological phenomena, is that living nature was formed in 1 years in evolutionary process in order to survive
, it is the valuable source of the new bioactive molecule of a huge potential discovery.One is successfully screened in the world at present
A little biotoxin molecules are used to treat some relevant diseases or the design reference as newtype drug.Spider produces various act on
The neurotoxin of nervous system, its neurotoxin is divided into protein and peptide neurotoxin and many amines according to their chemical constitution
Neurotoxin.Wherein spider polypeptide neurotoxins are most important, and spider polypeptide neurotoxin is special according to their function and molecule
Two types can be divided into by levying:The first is low molecular weight polypeptide class, and they can be with the cationic channel phase on excitable cells film
Interaction；Second is high molecular weight polypeptide class, and they make with the receptor components of presynaptic membrane and the neurohumoral secretion of enhancing
With closely related.Spider venom turn into neurotoxin an important new sources, and in spider venom specific drug and
Lps molecule is also that we find the preferable model molecule of agricultural fungicides.
The content of the invention
The purpose of the present invention is intended to provide the side for preparing novel bioactive polypeptide in a kind of thick poison from Guangxi tassel hair spider
Method, described biologically active polypeptide is spider suppression sodium peptide -27（SPNP-27）, its amino acid sequence is：
NH2- Asp Cys Leu Gly Leu Phe Trp Ile Cys Gln Tyr Met Asp Asp Lys Cys Cys
Pro Gly Tyr Lys Cys Glu Arg Ser Ser Pro Trp Cys Lys Ile Asp Ile Trp-NH2,
Between the 2nd cysteine of N-terminal and the 17th cysteine of described spider suppression sodium peptide -27, the 9th half Guang ammonia of N-terminal
Between acid and the 22nd cysteine, disulfide bond is formed respectively between the 16th cysteine of N-terminal and the 29th cysteine.
Methods described includes：（1）Thick poison dry powder distilled water dissolving is made into 5mg/ml toxin soiutions, 12 000rpm from
The min of the heart 5, supernatant is placed in 4 °C of preservations with after (0.22 μm) filtering of Millipore companies disposable filter.Thick poison loading
Cation exchange HPLC (ion-exchange HPLC) carries out first step separation.Using the type HPLC of Waters 650, the U.S.
The Hiprep of Pharmacia companiesTM16/10 CM FF prepacked columns are separated.486 detectors detect that Detection wavelength is
215nm.Mobile phase is four phase elution systems, and flow velocity is 1.5mL/min.Eluent is respectively A phase 0.1M NaH2PO4, B phases
0.1M Na2HPO4, C phase 1M NaCl, D phases ddH2O.Each elution collected after the above-mentioned separation through ion-exchange HPLC
Loading reversed-phase HPLC is further purified respectively at peak.Using analytic type Vydac C18 RP-HPLC reversed-phase columns (218TP54,
4.6 × 250 mm) in carrying out gradient elution on the high performance liquid chromatographs of analytic type Waters 2690,996 detectors are detected,
Detection wavelength is 215nm, and mobile phase is the water containing 0.1%TFA（A）And acetonitrile（B）, elution speed is 1mL/min, and column temperature is 40
℃。
（2）By above-mentioned cation exchange HPLC and two step the reversed-phase HPLCs SPNP-27 that the separation of totally three steps is obtained, use
Mass Spectrometric Identification purity reaches more than 98%, and its molecular weight is about 4086.4 Da, and through sequencing, the primary structure of the polypeptide is by 34
Individual amino acid residue composition, wherein containing 6 cysteines and forming three pairs of disulfide bond, the amidatioon of C- ends.Polypeptide SPNP-27 is purified
The physicochemical character of freeze-dried powder is white or off-white color loosening body, odorlessness, and very soluble in water, the aqueous solution is bordering on water white transparency.
Brief description of the drawings
Fig. 1 is the thick malicious cation exchange HPLC collection of illustrative plates of Guangxi tassel hair spider." * " represents purpose peak, and ordinate represents eluting peak
In 280 nm absorption value, abscissa represents elution time.
Fig. 2 is the thick malicious purpose cation exchange peak reversed-phase HPLC collection of illustrative plates of Guangxi tassel hair spider.Arrow represents purpose peak, ordinate
Absorption value of each eluting peak under 280 nm is represented, abscissa represents elution time.
Embodiment
It is of the invention with more abundant disclosure in order to be better understood from, below will be by extracellular method of administration, using the primary heart
Myocyte model illustrates spider suppression potassium peptide-XI myocardium sodium channel inhibitory action.
1st, experiment material and method
1st, experiment material and method
The acquisition of 1.1 thick poison
Guangxi tassel hair spider is collected in the mountain area in Hainan Province's fine jade in border of the county, and thick poison venom is adopted in female spider kind.It is summarized as follows：Make spider
Spider opens chela pawl and stretched into plastic cup, then with 3~5 s on the outside of 5~10 V chelicera base portion of pulse current stimulating two.Spider sense
After electro photoluminescence, i.e., wall of cup is tightly embraced with pedipalps, while chela pawl is effectively stabbed into glass inwall and venom is projected.Venom is collected
Afterwards, be put into -40 DEG C of freeze driers through vacuum freeze drying into light yellow or white powder be thick poison.
1.2 SPNP-27's isolates and purifies and mass spectral analysis
Thick poison dry powder distilled water dissolving is made into 5mg/ml toxin soiutions, and 12 000rpm centrifuge 5 min, and supernatant is used
After (0.22 μm) filtering of Millipore companies disposable filter, 4 °C of preservations are placed in.Thick poison loading cation exchange HPLC
(ion-exchange HPLC) carries out first step separation.Using Waters 650 type HPLC, Pharmacia companies of the U.S.
HiprepTM16/10 CM FF prepacked columns are separated.486 detectors detect that Detection wavelength is 215nm.Mobile phase is four phases
Elution system, flow velocity is 1.5mL/min.Eluent is respectively A phase 0.1M NaH2PO4, B phase 0.1M Na2HPO4, C phases 1M
NaCl, D phase ddH2O.Loading reversed-phase HPLC enters each eluting peak collected after the above-mentioned separation through ion-exchange HPLC respectively
Row is further purified.Using analytic type Vydac C18 RP-HPLC reversed-phase columns (218TP54,4.6 × 250 mm) in analysis
Gradient elution is carried out on the high performance liquid chromatographs of type Waters 2690,996 detectors detect that Detection wavelength is 215nm, flowing
It is mutually the water containing 0.1%TFA（A）And acetonitrile（B）, elution speed is 1mL/min, and column temperature is 40 DEG C.
Utilize MALDI-TOF mass spectrums（Voyager-DEÔSTR BiospectromitryÔ workstation）Determine and divide
Son amount.Linear cationic pattern；N2The nm of light source 337；Ion accelerating voltage is 20000 V.Matrix is alpha-cyano -4- hydroxyls-meat
Cinnamic acid（CCA）, sample is prepared in the following manner：Take 1 mL（About 0.5 mg）Sample liquid is added to 9 mL CCA 0.1%TFA/
Take and determined after 1 mL point samples, drying at room temperature after 50% acetonitrile saturated solution, mixing, and using internal standard method correction.
1.3 SPNP-27 determined amino acid sequence
SPNP-27 determined amino acid sequences are using N- ends Edman edman degradation Edmans in Applied Biosystems company 491A type gas
Completed on phase sequenator.36 circulations are surveyed, are sequenced using the standardization program of apparatus preparation, online HPLC detections are accurate to read
SPNP-27 amino acid sequence.
2nd, experimental result and analysis
2.1 SPNP-27's isolates and purifies
Because tassel hair spider composition in Guangxi is extremely complex, toxin polypeptide is generally isolated and purified using the method for Two way chromatograms：I.e. first
After the thick poison of Cation exchange separation, elution composition is further isolated and purified using reversed-phase HPLC.Two way chromatograms are a kind of non-
The method for often efficiently separating purifying, is separated by cation exchange HPLC and the step of reversed-phase HPLC two, and we are from Guangxi tassel hair
SPNP-27 is successfully separated in the thick poison of spider.Fig. 1 is the cation exchange HPLC collection of illustrative plates of the thick poison of Guangxi tassel hair spider, in 280 nm ripples
Long lower detection, can be observed 9 obviously eluting peaks, wherein the 9th peak is purpose peak.Collect behind this peak, it is pure in AKTA
Collection of illustrative plates obtained by carrying out desalting processing in change system is shown in Fig. 2.After collecting purpose peak and being freeze-dried, carried out in Alliance systems
Reversed-phase HPLC again.Eluting peak containing SPNP-27 is simple spike, and by Mass Spectrometric Identification, their purity reaches more than 98%.
2.2 mass spectral analysis
Eluting peak MALDI-TOF mass spectral analyses show that contained component is single.Identified SPNP-27 molecular weight is respectively
4086.4 Da.From figure, sample purity reaches more than 98%, and isolating and purifying for illustration purpose peptide is very successful.
2.3 amino acid sequence analysis
SPNP-27 sequencing is carried out on 491-A sequenators.Iodoacetamide subsequently is carried out to SPNP-27 before sequencing
Modification, as a result shows, SPNP-27 is single-chain polypeptide molecules, is made up of 34 amino acid residues, including 6 Cys.SPNP-
27 containing 6 cysteines, form three pairs of disulfide bond.The 2nd cysteine of N-terminal and the 17th half Guang of spider suppression sodium peptide -27
Between propylhomoserin, between the 9th cysteine of N-terminal and the 22nd cysteine, the 16th cysteine of N-terminal and the 29th half Guang ammonia
Disulfide bond is formed between acid respectively.SPNP-27 is the unique spider toxin molecule of a newfound sequence, SPNP-27 and its
Its ICK die body toxin cysteine residues position is guarded very much, therefore SPNP-27 is also a kind of ICK die bodys polypeptide toxin.
SEQUENCE LISTING
<110>Changsha Qin Cai bio tech ltd
<120>Spider suppression sodium peptide -27
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 34
<212> PRT
<213>Guangxi tassel hair spider
<400> 1
Asp Cys Leu Gly Leu Phe Trp Ile Cys Gln Tyr Met Asp Asp Lys
1 5 10 15
Cys Cys Pro Gly Tyr Lys Cys Glu Arg Ser Ser Pro Trp Cys Lys
16 20 25 30
Ile Asp Ile Trp
31
Claims (2)
1. preparing the method for novel bioactive polypeptide in a kind of thick poison from Guangxi tassel hair spider, described biologically active polypeptide is spider
Spider suppression sodium peptide -27（SPNP-27）, its amino acid sequence is：
NH2- Asp Cys Leu Gly Leu Phe Trp Ile Cys Gln Tyr Met Asp Asp Lys Cys Cys
Pro Gly Tyr Lys Cys Glu Arg Ser Ser Pro Trp Cys Lys Ile Asp Ile Trp-NH2。
2. preparation method according to claim 1, it is characterised in that methods described includes：（1）Thick poison dry powder distilled water
Dissolving is made into 5mg/ml toxin soiutions, and 12 000rpm centrifuge 5 min, supernatant Millipore companies disposable filter
After (0.22 μm) filtering, 4 °C of preservations are placed in；Thick poison loading cation exchange HPLC (ion-exchange HPLC) carries out first
Step separation；Using the type HPLC of Waters 650, the Hiprep of Pharmacia companies of the U.S.TM16/10 CM FF prepacked columns are carried out
Separation；486 detectors detect that Detection wavelength is 215nm；Mobile phase is four phase elution systems, and flow velocity is 1.5mL/min；Elution
Liquid is respectively A phase 0.1M NaH2PO4, B phase 0.1M Na2HPO4, C phase 1M NaCl, D phases ddH2O；It is above-mentioned through ion-
Loading reversed-phase HPLC is further purified each eluting peak collected after exchange HPLC separation respectively；Using analytic type
Vydac C18 RP-HPLC reversed-phase columns (218TP54,4.6 × 250 mm) are in the high-efficient liquid phase colors of analytic type Waters 2690
Gradient elution, the detection of 996 detectors are carried out on spectrometer, Detection wavelength is 215nm, and mobile phase is the water containing 0.1%TFA（A）And second
Nitrile（B）, elution speed is 1mL/min, and column temperature is 40 DEG C；
（2）By above-mentioned cation exchange HPLC and two step the reversed-phase HPLCs SPNP-27 that the separation of totally three steps is obtained, with mass spectrum
Identification purity reaches more than 98%, and its molecular weight is about 4086.4 Da, and through sequencing, the primary structure of the polypeptide is by 34 ammonia
Base acid residue composition, wherein containing 6 cysteines and forming three pairs of disulfide bond, the amidatioon of C- ends；Polypeptide SPNP-27 purifying is lyophilized
The physicochemical character of powder is white or off-white color loosening body, odorlessness, and very soluble in water, the aqueous solution is bordering on water white transparency.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111138516A (en) * | 2020-01-10 | 2020-05-12 | 大理大学 | Vespa mandarinia peptide and preparation method and application thereof |
| CN113214376A (en) * | 2021-03-30 | 2021-08-06 | 青岛大学 | Novel method for synthesizing centipede toxin RhTX and spider toxin GsMTx4 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104987372A (en) * | 2015-07-08 | 2015-10-21 | 长沙沁才生物科技有限公司 | Novel bioactive peptide and preparation method thereof |
-
2017
- 2017-06-20 CN CN201710467845.5A patent/CN107163118A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104987372A (en) * | 2015-07-08 | 2015-10-21 | 长沙沁才生物科技有限公司 | Novel bioactive peptide and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| CHENG TANG等: "A novel tarantula toxin stabilizes the deactivated voltage sensor of bacterial sodium channel", 《FASEB J.》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111138516A (en) * | 2020-01-10 | 2020-05-12 | 大理大学 | Vespa mandarinia peptide and preparation method and application thereof |
| CN111138516B (en) * | 2020-01-10 | 2023-04-25 | 大理大学 | Royal jelly peptide and preparation method and application thereof |
| CN113214376A (en) * | 2021-03-30 | 2021-08-06 | 青岛大学 | Novel method for synthesizing centipede toxin RhTX and spider toxin GsMTx4 |
| CN113214376B (en) * | 2021-03-30 | 2023-09-19 | 青岛大学 | New method for synthesizing centipede toxin RhTx and spider toxin GsMTx4 |
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Application publication date: 20170915 |