CN1120174C - Process for preparing marine conidae analgesic polypeptide - Google Patents
Process for preparing marine conidae analgesic polypeptide Download PDFInfo
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- CN1120174C CN1120174C CN 00128525 CN00128525A CN1120174C CN 1120174 C CN1120174 C CN 1120174C CN 00128525 CN00128525 CN 00128525 CN 00128525 A CN00128525 A CN 00128525A CN 1120174 C CN1120174 C CN 1120174C
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Abstract
The present invention discloses a method for preparing active conidae polypeptide. The present invention has the steps of synthesizing linear peptide, folding the linear peptide and purifying peptide. The present invention characterized in that the step of folding the linear peptide is carried out in an oxidation reduction system under the existence of 0.1 to 2 M of sodium chloride or sodium sulfate. The conidae polypeptide with high efficiency and highly activity can be obtained by the method of the present invention, and the method of the present invention is suitable for scale production.
Description
Conus gasteropod, the whole world have 500 kinds approximately, spread all over each warm sea area, the world, and there is cone shell 60-70 kind in China, mainly is distributed in the Xisha Islands, Hainan Island and marine site, Taiwan.Worm and other mollusks and fish in the cone shell happiness food seawater can be divided into ichthyophagy, carnivorism, three kinds of cone shells of food spiral shell according to its habit.Conotoxin mostly is the polypeptide of genes encoding by the poison gland secretion of venom duct and malicious capsule inner wall, and by 12～40 amino acid, 2～3 pairs of disulfide linkage are formed.This class active polypeptide has the high selectivity effect to different ionic channels and passage hypotype, can be divided into types such as α, ω, μ, δ, κ and μ/o by its target site.Found that at present conotoxin has broad application prospects at aspects such as disease treatment and diagnosis; wherein omega-conotoxin M VII A is a N type ockers; has the intensive analgesic activity, big thousand times in specific activity morphine, and unlikely addiction; be used for analgesia and neuroprotective by the exploitation of Neurex company; enter the II-III clinical trial phase, seen Scott Bowersox etc., Drug of Future; 1998,23 (2): 152-160.
The SO3 conotoxin peptide is by strain line cone shell (Conus Striatus) the poison pipe gene clone (Lu Baisong etc. to China South Sea, Science Bulletin, 44 (16): 1737-1739,1999) and be synthesized into through peptide, its amino acid whose sequence is: CKAAGKPCSRIAYNCCTGSCRSGKC (C holds amidation) contains three pairs of disulfide linkage.It is 0.75 μ g/kg (ED that application hot plate method, optical heat radiation method and chemical stimulation method have been measured its median effective dose to the analgesic activities of mouse
50), ED
50Than mld LD
50Little 18000 times, the specific activity morphine is more than big thousand times.The chronic sleeve pipe administration 0.5 μ k/kg threshold of pain in the rat spinal cord sheath is improved 50%, administration 1.2 μ g/kg, the threshold of pain improves 100%, and action time more than 4 hours, has the excellent drug development prospect.
Chinese patent application 99106070.9 (CN1237584A) discloses 14 kinds of conotoxin peptides and gene clone method thereof, wherein also specifically discloses a kind of escherichia coli expression of peptide and the chemical synthesis process of another kind of peptide.The disclosed chemical synthesis process of this application directly prolongs the routine techniques of having used the synthetic field of peptide, is unsuitable for obtaining fairly large peptide with high yield high reactivity.
Purpose of the present invention is exactly to attempt to provide a kind of yield height to be suitable for the method for preparing active conotoxin peptide of industrial application, makes the clinical application of this class polypeptide become possibility.
The present inventor has carried out extensive studies to the preparation technology of conotoxin peptide, has found that some are particularly suitable for the processing condition of such polypeptide, thereby has finished the present invention.
The invention provides a kind of method for preparing active conotoxin peptide, the folding and peptide purification step that it comprises synthetic, the linear peptides of linear peptides, it is characterized in that described peptide is folding carries out in redox system in the presence of 0.1-2M sodium-chlor or sodium sulfate.
Synthesizing of linear peptides of the present invention, can adopt any method well known by persons skilled in the art to finish.These methods are (but being not limited to) clonal expression method, chemical synthesis for example, and the latter comprises the synthetic and solid phase synthesis of liquid phase.About solid phase synthesis, it is synthetic automatically and manual synthetic to be divided into instrument again.About another patent application 99106070.9 (CN1237584A) of the cloning expression method of conotoxin peptide and instrument solid phase synthesis referring to the applicant.This paper has also provided SO3 linear peptides instrument synthetic embodiment.In brief, the instrument of linear peptides is synthetic adopts commercial Rink resin and the Fmoc amino acid of buying to carry out on peptide synthesizer.Ile for SO3
15, Alg
16And Lys
20Three amino acid adopt double coupling method to connect, and all the other amino acid all adopt single coupling process to connect.Gained resin-peptide conjugate lytic reagent cracking and deprotection that contains phenol, dimercapto acetate, thioanisole, water and trifluoroacetic acid.The gained free peptide is through ether sedimentation, filtration, purifying obtains pure substantially linear peptides on dextrane gel again.
Because instrument synthetic output is limited, the applicant has studied and has been suitable for industrial manual synthetic method.This method also is based on the principle of solid phase synthesis, from Rink resin and Fmoc amino acid, with DCC-HOBt as activator and with piperidines as the deprotection agent synthetic.Wherein the protecting group of each seed amino acid employing is respectively Trt (Cys), Boc (Lys), Pmc (Arg), tBu (Ser, Tyr, Thr), OtBu (Asp).Adopt the manual synthetic not only a collection of a large amount of peptides (number restrains hundreds of grams or more) that synthesize, save a large amount of solvents, and it is cheap and easy to get mainly to consume solvent.
Linear peptides folding is the important step of peptide in synthetic, because peptide just has its biological activity when only being correct folding conformation form, and is all to be the forms such as inclusion body, linear peptides of non-activity through the expression of prokaryotic cell prokaryocyte or through the peptide of chemosynthesis.The folding of linear peptides is that one of peptide synthetic must be through step, and its efficient directly influences peptide synthetic yield.The applicant now makes us finding uncannily, carries out the folding of linear peptides in the presence of the sodium-chlor of 0.1-2M or sodium sulfate in redox system, and compared with prior art, the folding efficiency of conotoxin peptide is significantly increased.
In the method for the invention, the concentration of sodium-chlor or sodium sulfate is 0.1-2M, and preferred 0.2-1M most preferably is about 0.5M.
In one embodiment, the used redox system of the inventive method is the damping fluid that contains cysteine plus cystine.In this embodiment, the mol ratio of halfcystine and Gelucystine is preferably 20: 1 to 2: 1, and preferred 15: 1 to 5: 1, most preferably from about 10: 1.
In another embodiment, the used redox system of the inventive method is the damping fluid that contains dithiothreitol (DTT).
In a preferred embodiment, the used redox system of the inventive method is the damping fluid that contains reduced form and Sleep-promoting factor B.In this embodiment, the mol ratio of reduced glutathion and Sleep-promoting factor B is preferably 20: 1 to 2: 1, is more preferably 15: 1 to 5: 1, most preferably from about 10: 1.
In the above-described embodiment, described damping fluid is preferably ammonium acetate buffer or phosphate buffered saline buffer or TrisHCl damping fluid.The pH value of damping fluid is about 7.0-8.2.
The mensuration of folding efficiency is measured by HPLC, by the ratio between the total area of measuring correct folding main peak area and all folding peak, can determine folding efficiency.
Conversion further concentrated, that purifying reaches salt form in case of necessity that the conotoxin peptide solution that folds needs just has to form to be suitable for medicinal product.Certainly, the present invention includes the various forms of conotoxin peptide products after adopting the inventive method folding, no matter its whether through concentrating, the conversion of purifying or salt form.Describedly concentrate, the conversion of purifying and salt can adopt any method well known by persons skilled in the art to carry out.The concentrated and purified of the conotoxin peptide of reporting in the prior art is to adopt freeze-drying-HPLC purifying process.Be freeze-drying after the acetate acidifying in the folding solution of polypeptide, use the HPLC purifying then.This technology is unsuitable for scale operation.Therefore, the applicant researchs and develops out that HPLC concentrates, ion-exchange concentrates, normal pressure is anti-phase concentrates or ultrafiltration and concentration adds the method for HPLC purifying.Preferred HPLC and ion-exchange concentrate method of enrichment.
At last, in order to be prepared into the preparation of clinical application,, then also need carry out the conversion of salt form if product is the trifluoroacetic acid salt form.Typically, the peptide that will contain trifluoroacetic acid is dissolved in the acetate, by sephadex column, uses the acetate wash-out again, collects the purified peptide that target component and freeze-drying can get the acetate form then.Certainly according to the needs of clinical application, those skilled in the art can be transformed into this peptide species various suitable salt forms.
The active polypeptide that the inventive method obtains is suitable for the conventionally form administration with peptide, preferably with injection administration.For the preparation of polypeptide aseptic parenteral solution, purified peptide can be dissolved in stroke-physiological saline solution, degerming after filtration is diluted to proper concn with physiological saline then, is divided in the ampoule of different size and sealing.
The invention described above method is specially adapted to be called the conotoxin peptide of SO3, and its aminoacid sequence is as follows: CKAAGKPCSRIAYNCCTGSCRSGKC (C holds amidation).
Being example with the SO3 polypeptide below further specifies the present invention in the mode of embodiment.The synthetic method A of embodiment 1 SO3 linear peptides: instrument is synthetic
Adopt the Fmoc/HoBt/DCC method, utilize Rink resin and Fmoc amino acid, the synthetic handbook of synthesis step reference instrument carries out.For reacting completely, distinguish proper extension at the piperidines deprotection and on the coupling time, first deprotection of piperidines, 3min is extended for 5min, and deprotection changes 30min into by 15min for the second time, and the coupling time goes up increases 20min.Ile
15, Arg
16, Lys
20Three amino acid are difficult to connect, and adopt two couling process, and all the other adopt single coupling.
Resultant quantity is 0.25mol, gets peptide-resin 1.50g, and total coupling rate is 95%.1.5g peptide resin is sloughed protecting group in following lytic reagent: 1.5g phenol, 0.5mL 1, the 2-3-mercaptoethanol; 1mL phenmethyl thioether, 1mL distilled water, 20mL trifluoroacetic acid; stir 2.5h, filter, with the washing of 2mL trifluoroacetic acid; with anhydrous ethylene dichloride washing, filtrate is steamed on rotatory evaporator to 8mL, adds a large amount of ice-cold ether again; produce precipitation immediately, filter, with ether washing four times; dissolve thick peptide with 20% acetate then, lyophilize, thick peptide 610mg.
The thick peptide of above-mentioned 610mg is dissolved in 10% the acetate, the thick peptide after the lyophilize is dissolved in 10% the acetate, crosses Sephadex G-25 (26 * 400mm), 10% acetate wash-out, main peak and freeze-drying collected in the 254nm record, must purer peptide 500mg. method B: manual synthetic
Select for use Rink resin and Fmoc protection amino acid (protecting group be respectively (Trt (Cys), Boc (Lys), Pmc (Arg), t-Bu (Ser, Tyr, Thr), OtBu (Asp)), DCC-HOBt is an activator, the piperidines deprotection.Connect the peptide method:
NMP 1×1min
30% piperidines/DMF 1 * 5
30% piperidines/DMF 1 * 40
NMP 2×1
CH
2Cl
2 1×1 MeOH(i-PrOH) 2×1
CH
2Cl
2 3×1
NMP 1×1Fmoc-AA/HBTU，DIEA 1～2h
NMP 2×1min
MeOH 2×1
CH
2Cl
2 2×1
NMP 1×1
Detect
(heavily condensation, sealing, next circulation)
Resultant quantity is 1.88mmol, gets peptide-resin 10g, and the coupling rate is 79.4%.
The peptide resin cracking: 6g peptide-resin places lytic reagent (3g phenol, 1mL 1,2-3-mercaptoethanol, 2mL phenmethyl thioether, 2mL distilled water, 40mL trifluoroacetic acid), and all the other steps are with method A.Get thick peptide 4.2g.
3.2g thick peptide is dissolved in 10% the acetate, the thick peptide after the lyophilize is dissolved in 10% the acetate, (main peak and freeze-drying collected in 36 * 700mm), 10% acetate wash-out, 254nm record, must purer peptide 2.8g to cross Sephadex G-25.
Embodiment 2 oxidations are folding
At 4～8 ℃ linear peptides is dissolved in the damping fluid that contains redox agent and complexing agent (EDTA) (about pH7.9), detects folding process with HPLC.After folding the finishing, be certain value, carry out purifying with various purification process then with acetate acidifying pH of buffer.Method A: oxidation in the presence of halfcystine
0.1 add the halfcystine of 0.8～7.2mmol/L and 0.08～0.72 Gelucystine, sodium ethylene diamine tetracetate, 2～0.1mol/L sodium-chlor or 2～0.1mol/L sodium sulfate and the 0.2～2mg/mlSO3 of 1mmol/L in the phosphoric acid salt of～2.0mol/L ammonium acetate or 0.05～0.1mol/L or the Tris.HCl damping fluid (pH is 7.0-8.2), 4-8 ℃ was stirred 1-5 days down, and folding efficiency is 10-50%.
Add 1.0mmol/L halfcystine and the Gelucystine of 0.1mmol/L, sodium ethylene diamine tetracetate, 0.5mol/L sodium-chlor or 2mol/L sodium sulfate and the 0.2mg/ml SO3 of 1mmol/L in A1.0.5mol/L ammonium acetate (pH the is 7.9) damping fluid, 4 ℃ were stirred 2 days down, and folding efficiency is 35%.
Add the halfcystine of 1.0mmol/L and the Gelucystine of 0.2mmol/L, sodium ethylene diamine tetracetate, 0.2mol/L sodium-chlor or 0.5mol/L sodium sulfate and the 0.2mg/mlSO3 of 1mmol/L in the phosphoric acid salt of A2.0.05mol/L (pH the is 7.9) damping fluid, 4 ℃ were stirred 2 days down, and folding efficiency is 30%.
Add the halfcystine of 1.0mmol/L and the Gelucystine of 0.1mmol/L, sodium ethylene diamine tetracetate, 1mol/L sodium-chlor or 2mol/L sodium sulfate and the 0.2mg/ml SO3 of 1mmol/L in the A3.0.1mmol/LTris.HCl damping fluid (pH7.9), 4 ℃ were stirred 3 days down, and folding efficiency is 30%.Method B: oxidation in the presence of reductive glutathione and Sleep-promoting factor B
0.1 add the reductive glutathione of 0.8～7.2mmol/L and 0.08～0.72 Sleep-promoting factor B, sodium ethylene diamine tetracetate, 2～0.1mol/L sodium-chlor or 2～0.1mol/L sodium sulfate and 0.2～2mg/ml SO3,4-8 ℃ of following stirring 1-5 days of 1mmol/L in the phosphoric acid salt of～2.0mol/L ammonium acetate or 0.05～0.1mol/L or the Tris.HCl damping fluid (pH is 7.0-8.2), folding efficiency is 10-60%.
Add the reductive glutathione of 1.0mmol/L and the Sleep-promoting factor B of 0.1mmol/L, sodium ethylene diamine tetracetate, 0.5mol/L sodium-chlor or 2mol/L sodium sulfate and the 0.2mg/mlSO3 of 1mmol/L in B1.0.5mol/L ammonium acetate (pH the is 7.9) damping fluid, 4 ℃ were stirred 3 days down, and folding efficiency is 40%.
Add the reductive glutathione of 1.0mmol/L and the Sleep-promoting factor B of 0.1mmol/L, sodium ethylene diamine tetracetate, 0.5mol/L sodium-chlor or 1mol/L sodium sulfate and the 0.2mg/mlSO3 of 1mmol/L in B2.0.05mol/L phosphoric acid salt (pH the is 7.9) damping fluid, 4 ℃ were stirred 3 days down, and folding efficiency is 30%.
Add the reductive glutathione of 1.0mmol/L and the Sleep-promoting factor B of 0.1mmol/L, sodium ethylene diamine tetracetate, 0.5mol/L sodium-chlor or 2mol/L sodium sulfate and the 0.2mg/mlSO3 of 1mmol/L in B3.0.1mol/L Tris.HCl (pH the is 7.9) damping fluid, 4 ℃ were stirred 3 days down, and folding efficiency is 30% method C: oxidation in the presence of dithiothreitol (DTT)
0.1 add the dithiothreitol (DTT) of 0.8～2.2mmol/L, sodium ethylene diamine tetracetate, 2～0.1mol/L sodium-chlor or 2～0.1mol/L sodium sulfate and the 0.2～2mg/mlSO3 of 1mmol/L in the phosphoric acid salt of～2.0mol/L ammonium acetate or 0.05～0.1mol/L or the Tris.HCl damping fluid (pH is 7.0-8.2), 4-8 ℃ was stirred 3-5 days down, and folding efficiency is 10-30%.
Add the dithiothreitol (DTT) of 1.2mmol/L, sodium ethylene diamine tetracetate, 0.5mol/L sodium-chlor or 1mol/L sodium sulfate and the 0.2mg/mlSO3 of 1mmol/L in the C1.0.5mol/L ammonium acetate buffer (pH is 7.9), 4 ℃ were stirred 5 days down, and folding efficiency is 20%.
Add the dithiothreitol (DTT) of 1.2mmol/L, sodium ethylene diamine tetracetate, 0.5mol/L sodium-chlor or 1mol/L sodium sulfate and the 0.2mg/mlSO3 of 1mmol/L in the phosphate buffered saline buffer of C2.0.05mol/L (pH is 7.9), 4 ℃ were stirred 5 days down, and folding efficiency is 25%.
Add the dithiothreitol (DTT) of 1.2mmol/L, sodium ethylene diamine tetracetate, 1.0mol/L sodium-chlor or 1mol/L sodium sulfate and the 0.2mg/mlSO3 of 1mmol/L in the C3.0.1mol/LTris.HCl damping fluid (pH is 7.9), 4 ℃ were stirred 5 days down, and folding efficiency is 25%.The comparative example
Do not add under sodium-chlor or the sodium sulfate situation folding:
0.1 the halfcystine of the middle adding of the phosphoric acid salt of～0.5mol/L ammonium acetate or 0.05～0.1mol/L or Tris.HCl damping fluid (pH is 7.0-8.2) 0.8～7.2mmol/L and 0.08～0.72 Gelucystine or the reductive glutathione of 0.8～7.2mmol/L and 0.08～0.72 Sleep-promoting factor B, sodium ethylene diamine tetracetate and the 0.2～2mg/mlSO3 of 1mmol/L, 4-8 ℃ was stirred 1-5 days down, and folding efficiency is 5-20%.
Increase phosphoric acid salt or Tris.HCl concentration in addition and will cause folding solution to produce precipitation, cause productive rate to descend.
Increase ammonium acetate concentration and also can reach higher yields, but neutralization buffer need consume a large amount of acetic acid, a large amount of in addition acetic acid makes troubles for the purifying of back, and needs dilution.
At the same terms of embodiment 2 method A, B and C but do not add under the situation of sodium-chlor or ammonium sulfate foldingly, folding efficiency sees the following form:
The purification process A of embodiment 3 SO3; Freeze-drying-HPLC purifying process
| Corresponding conditions | A1 | A2 | B1 | B3 | C1 | C2 |
| Folding efficiency | 20％ | 15％ | 20％ | 16％ | 10％ | 8％ |
SO3 adds the acetate acidifying, freeze-drying, HPLC purifying (C then after folding and finishing
18, 21.1 * 250mm, elutriant A are 0.1% trifluoroacetic acid, and elutriant B is 100% acetonitrile, and gradient is 1-40min, 10-30%B, flow velocity are 8ml/min).Yield 15%.Method B:HPLC concentrates-the HPLC purifying process
After SO3 folds and finishes, add acetate (the 2000ml damping fluid adds acetate 100ml) and transfer pH＜4.5, filter, last reversed-phase column absorption (C
18Post, 30 * 250mm), column flow rate 3-5ml/min, absorption finishes back water flushing, and (elutriant A is 0.1% trifluoroacetic acid, and elutriant B is 100% acetonitrile to carry out gradient elution then, gradient is 1-40min, 10-40%B, flow velocity are 8ml/min), collect main peak and other peaks, freeze-drying, polymer can be with pure acetonitrile wash-out, from collecting liquid pressure reducing and steaming acetonitrile, freeze-drying.
With freeze dried main peak, carry out reversed-phase HPLC once more and separate (C
18Post, 21.1 * 250mm, elutriant A are 0.1% trifluoroacetic acid, and elutriant B is 100% acetonitrile, and gradient is 1-40min, 10-30%B, flow velocity are 8ml/min).Through high pressure anti-phase concentrate and preliminary purification after, this step purifying gained main peak, purity is between 50-90%, through the anti-phase purifying of a step high pressure, product purity can reach more than 99% again, yield reaches 15～20%.Method C: ion-exchange concentrates-the HPLC purifying process
SO3 adds acetate (the 2000ml damping fluid adds acetate 40ml) after folding and finishing, and transfers pH5.0-5.2, filters, and is diluted to NH
4Ac concentration is 0.22M, last BiO-Rex70 ion column (H
+), flow velocity 5ml/min, after absorption finished, 50% acetate wash-out was used in the water flushing then, and rotary evaporation is removed acetate, freeze-drying.The freeze dried main peak anti-phase preparative column (C of high pressure
18Post, 21.1 * 250mm, elutriant A are 0.1% trifluoroacetic acid, and elutriant B is 100% acetonitrile, and gradient is 1-40min, 10-30%B, flow velocity are 8ml/min) separate.SO3 behind ion exchange chromatography, purifying gained main peak, purity is 50%-70%, the main peak yield is 20-30%, further anti-phase purifying can get 99% product.Method D: normal pressure is anti-phase to be concentrated-the HPLC purifying process
After SO3 folds and finishes, add acetate (the 2000ml damping fluid adds acetate 100ml) and transfer pH＜4.5, filter, last Source RPC30 or Poros reversed-phase column absorption (1.5 * 50), column flow rate 3-5ml/min, absorption finishes back water flushing, carry out gradient elution then, collect main peak and other peaks, freeze-drying, polymer can be with pure acetonitrile wash-out, from collecting liquid pressure reducing and steaming acetonitrile, freeze-drying.
Freeze dried main peak is carried out reversed-phase HPLC separate (C
18Post 21.1 * 250mm, elutriant A are 0.1% trifluoroacetic acid, and elutriant B is 100% acetonitrile, and gradient is 1-40min, and 10-30%B, flow velocity are 8ml/min).The SO3 productive rate of preparation is 15-25% (linear peptides relatively).Method E: ultrafiltration and concentration-HPLC purifying process
SO3 is folding finish after, transferring pH is 3.5, dilute one times, with YM1 (molecular weight cut-off is 1000Da) ultrafiltration membrance filter, pressure 0.35Mpa, dilute with water then, ultrafiltration again, collection mother liquor, freeze-drying.Dry powder HPLC purifying (C
18Post, 21.1 * 250mm, elutriant A are 0.1% trifluoroacetic acid, and elutriant B is 100% acetonitrile, and gradient is 1-40min, 10-30%B, flow velocity are 8ml/min).The SO3 productive rate of preparation is 15-20% (linear peptides relatively).The buffer-exchanged and the preparation of the pure product of embodiment 4 SO3
The SO3 peptide that 500mg is contained trifluoroacetic acid is dissolved in 20% the acetate, and (26 * 400mm), with 20% acetate wash-out, in the 254nm record, collection main peak and freeze-drying must contain the pure peptide 420mg of acetic acid to cross Sephadex G-25 post.With 0.2 μ m polyethersulfone membrane filtration degerming of sterilization, dilute with a small amount of autoclaving physiological saline solution at aseptic then, be distributed into the different size preparation with physiological saline.
Claims (11)
1. method for preparing active cone shell peptide, wherein said conotoxin peptide are the polypeptide that is called SO3 that the C end is amidated, have following sequence: CKAAGKPCSRIAYNCCTGSCRSGKC; This method comprises the automatic synthesis method of utilizing peptide synthesizer or manual synthesis method synthesizing linear peptide, linear peptides is folding and peptide purification, it is characterized in that described linear peptides is folding to carry out in redox system in the presence of 0.1-2M sodium-chlor or sodium sulfate.
2. the process of claim 1 wherein that the concentration of sodium-chlor or sodium sulfate is 0.2-1M.
3. the method for claim 2, wherein the concentration of sodium-chlor or sodium sulfate is about 0.5M.
4. the process of claim 1 wherein that described redox system is the damping fluid that contains cysteine plus cystine.
5. the process of claim 1 wherein that described redox system is the damping fluid that contains reduced form and Sleep-promoting factor B.
6. the process of claim 1 wherein that described redox system is the damping fluid that contains dithiothreitol (DTT).
7. the process of claim 1 wherein that the synthetic of linear peptides is to adopt the manual synthesis method of solid phase.
8. the process of claim 1 wherein that the purifying of peptide comprises that peptide folds the conversion of concentrating of solution and HPLC purifying and salt in case of necessity.
9. the method for claim 8, wherein enrichment step adopts lyophilization, HPLC method of enrichment, ion-exchange method of enrichment, the anti-phase method of enrichment of normal pressure or ultrafiltration and concentration method.
10. the method for claim 9, wherein enrichment step adopts the ion-exchange method of enrichment.
11. the method for claim 8, wherein the trifluoroacetate that is converted to peptide of salt is converted to acetate.
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| CN102875654B (en) * | 2012-09-25 | 2014-07-09 | 中国人民解放军军事医学科学院生物工程研究所 | Method for preparing conotoxin polypeptide Eb1.6 |
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