CN1108309C - Process for purifying nerve growth factor from venin - Google Patents
Process for purifying nerve growth factor from venin Download PDFInfo
- Publication number
- CN1108309C CN1108309C CN 99115898 CN99115898A CN1108309C CN 1108309 C CN1108309 C CN 1108309C CN 99115898 CN99115898 CN 99115898 CN 99115898 A CN99115898 A CN 99115898A CN 1108309 C CN1108309 C CN 1108309C
- Authority
- CN
- China
- Prior art keywords
- chromatography
- elutriant
- balance
- sample
- hydrophobic
- Prior art date
- Legal status (The legal status 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 status listed.)
- Expired - Fee Related
Links
Landscapes
- Peptides Or Proteins (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a new method for purifying a nerve growth factor from snake venom by using the combination of hydrophobic materials, ion exchange materials and gel row materials. The method causes the purity of the obtained nerve growth factor to be over 97% and the minimum specific activity to be 5 ul/ml; the used time is 1/10 of the traditional method; the activity ratio of the obtained nerve growth factor is two times of the traditional method. The method can be linearly used for laboratory production to medium-scale production until scale production.
Description
The present invention relates to a kind of three-step approach purifying nerve growth factor (Nerve Growth Factor, method NGF) from snake venom with hydrophobic material, ion-exchange material and gel exclusion combination of materials.
Nerve growth factor is present unique somatomedin that can promote nerve growth that obtains identifying, is the necessary a kind of protein that grows of sympathetic neuron and some Sensory neurone.Clinically, the main curative effect of nerve growth factor shows the following aspects: (1) promotes repairing nerve damage.The main clinical disease that is suitable for has: neuritis, idiopathic epilepsy, neurasthenia, angioneurotic headache, meningitis, cerebral surgery operation sequela, pesticide intoxication encephalopathy (HIE) sequela, cerebral apoplexy sequela, neural heariing loss, diseases such as poliomyelitis, encephalatrophy, the hard rope sclerosis of muscle; (2) senile degeneration illnesss such as treatment and alleviation Alzheimer type senile dementia, senile chorea, Parkinsonism, Guillain Barre syndrome; (3) can suppress the mitotic division of some tumour effectively, impel it to optimum differentiation, to melanoma, tumours such as colorectal carcinoma have certain curative effect as NGF; (4) healing of promotion skin histology, particularly chronicity being festered has better curative effect; (5) promote early embryo development and adjusting antibody mediated immunity function; (6) has the function that prevents the learning and memory functional defect.
At present, although many chromatography method reports that extract NGF from snake venom are arranged on document, as document 1[Siigur, G., etal., Comp.Biochem.Physiol., B:Comp.Biochem, 1987,87B (2), 329-34] using monoclonal antibody immunoaffinity chromatography method purifying the NGF in 10 kinds of different sorts snake venom; Document 2[Kilmon, J., Am.Biotechnol.Lab., 1992,110 (11), 18-20] reported a kind of that constitute by silica gel and poly-ammonia propylene, can enough different ions exchanges and affinity ligands activatory, be called the method that the microporous fibre bed of Acti-Disk Cartridge comes NGF in the purifying snake venom; Document 3[Stoeckel, K., et al., J.Neurochem, 1976,26 (6) 1207-11] reported and a kind of 2.5S NGF has been connected on the Sepharose 4B by CNBr, utilize the affinity chromatography method from sheep blood serum, to extract the method for NGF antibody; Document 4[Khamidor, D.K., Khim.Prir.Soedim, 1985, (4) 546-51] and document 5[Siigur, J., et al., Comp.Biochem.Physiol., B:comp.Biochem., 1986,83B (3), 621-5] reported the method for from snake venom, extracting NGF with the method for gel exclusion+ion-exchange+isoelectrofocusing/chromatofocusing respectively; Document 6[Koyama, J., et al., Biophys.Acta, 1992,1160 (3) 287-92] adopted gel to cross to coagulate+method of ion-exchange+affinity chromatography (Blue-Sepharose CL-6B) extracted NGF from adder (Vipera russelli resselli); And recently, document 7[Kostiza, T, et al., Toxico, 1995,33 (10), 1249-61] reported the method for from Chinese cobra-venom, extracting NGF with the method for weak cation exchange+anti-phase liquid chromatography (LC), or the like.But, chemical stability and commodity availability from separating medium, consider so that reach from the reliability and stability aspect of purifying process, the method of extracting NGF from snake venom that really has practical use then is the comparison classics and gel-filtration+ion-exchange+gel-filtration pattern [document 8:ogue-Angeleui that use in a large number on document at present, R.A., et al., Biochemistry, 15 (1976), 26; Document 9: Liu's one equality, Chinese biochemical drug magazine, 1994:15 (1)].But there are following two deadly defects in this method: (1) generally all needs more than 100 hour in the gel exclusion process of the first step, not only the operating time is oversize, and the Long contact time of albumen and medium, make the activity of required target protein to reduce.(2) adopt the gel exclusion method in the first step,, make that quantity of sample handling is very little, therefore, can only be used for the preparation of laboratory small amount of sample, be difficult to be amplified in pilot scale and the scale production because the gel media loading capacity is less.
The objective of the invention is to overcome the shortcoming of above-mentioned prior art, a kind of method of using three-step approach purifying nerve growth factor from snake venom of hydrophobic material, ion-exchange material and gel exclusion combination of materials is proposed, the used time is 1/10 of traditional method, and almost can be amplified to pilot scale until scale production from the laboratory linearly.
Fig. 1 realizes device required for the present invention.
As shown in Figure 1, this device comprises the infusion pump 1 of two parallel connections, and an end of the outlet of infusion pump 1 and mixing tank 2 is connected, and the other end of mixing tank is connected with chromatography column 3, chromatography column 3 a back Ultraviolet Detector 4 of configuration and registering instruments 5.During actually operating, two infusion pumps 1 are used for two kinds of solution are pumped into chromatography column 3, mixing tank 2 is used for two kinds of solution are mixed, and chromatography column 3 is used for target protein and other proteic separation, and Ultraviolet Detector 4 and registering instrument 5 are used for the detection and the record of sample separation.
1, the selection of hydrophobic material, elute soln and type of elution
Because NGF has stronger hydrophobicity, therefore selected hydrophobic medium should have certain hydrophobicity, but can not be too strong, so that NGF can not elute, and all can be used as one of selection as positive ethyl, polyethers of bonding, normal-butyl or phenyl medium.Selected elutriant should make the NGF in the sample dissolve fully, does not destroy the activity of NGF again because of precipitation, as pH at the buffered soln of 6.0-9.0 (as the Na that contains of 20-50mmol/L
+, K
+, Mg
++, Ca
++, NH
4 +, H
2PO
4 -, HPO
4 2-And SO
4 2-The ionic salts solution).Selected type of elution is advisable in the mode that lowers salt concn.
2, the selection of ion-exchange material, elute soln and type of elution
Because the iso-electric point of NGF is near 7.0, therefore selected Ion Exchange Medium both can be undertaken by the form of cationic exchange, also can be undertaken by the form of anionresin.All can be used as one of selection as phenmethyl, sulfonic group, phosphate and oxalic acid diethylin, quaternary amine base.Selected elutriant should make the NGF in the sample dissolve fully, does not destroy the activity of NGF again because of precipitation, as pH at the buffered soln of 5.0-7.0 and 7.0-9.0 (as the Na that contains of 20-50mmol/L
+, K
+, Mg
++, Ca
++, H
2PO
4 -, HPO
4 2-And Cl
-The ionic salts solution).Selected type of elution is advisable in the mode that increases salt concn.
3, the selection of gel exclusion material, elute soln and type of elution
Because the molecular weight of NGF is about 30,000, therefore the linearity range of selected gel exclusion medium should be in this scope, and has good non-specific adsorption performance.All can be used as one of selection as agarose, dextran or silica gel material.Selected elutriant should make the NGF in the sample dissolve fully, does not destroy the activity of NGF again because of precipitation, as pH at the buffered soln of 5.0-9.0 (as the Na that contains of 20-50mmol/L
+, K
+, Mg
++, Ca
++, H
2PO
4 -, HPO
4 2-And Cl
-The ionic salts solution).Selected type of elution is advisable in mode such as degree such as grade.Operational instances
Get 30.0 gram meal shape snake venom, be dissolved in the Na that contains of 120ml 10mM
+, K
+, Mg
++, Ca
++, H
2PO
4 -And HPO
4 2-In the damping fluid of ionic pH=7.0,4, centrifugal 30 minutes of 000rpm gets supernatant purifying in the following order.
(1), hydrophobic chromatography
Chromatography column: 2.6 * 50cm.
Filler: a kind of hydrophobic material that contains normal-butyl, 265ml glue
Balance liquid A:20mM PBS+1.5M (NH)
2SO
4, pH=7.0
Elutriant B:20mM PBS, pH=7.0
Gradient: elutriant B is from 0～100%, linear gradient
Flow velocity: 290cm/h
Operate: earlier with balance liquid A balance chromatography column 30min, will use the sample upper prop again, behind balance liquid A flushing 20min, beginning 100min gradient is kept 20min with elutriant B again, prepares against with balance liquid A balance chromatography column at last and uses next time again.Collection contains the active peak of NGF and is about 180ml.
(2), ion exchange chromatography
Chromatography column: 2.6 * 5.0cm
Filler: a kind of ion-exchange material that contains carboxymethyl, 265ml
Balance liquid C:20mM NaAc-HAc, pH=5.0
Elutriant D:20mM NaAc-HAc+1M NaCl, pH=5.0
Gradient: elutriant D is from 0-100%, linear mode.
Flow velocity: 290cm/h (26ml/min)
Operation: chromatography column is earlier with balance liquid C balance 30min, again previous step is collected and on the sample of desalting column desalination sample (having changed into NaAc-HAc solution), with balance liquid C towards liquid 20min, after the 100min gradient, continue to keep elutriant D 20min, again with 30min balance liquid C balance chromatography column.Collection contains the about 290ml in active peak of NGF.
(3), gel exclusion chromatography:
Chromatography column: 2.6 * 125cm
Filler: a kind of sepharose material, 660ml
Elutriant: 50mM PBS, pH=7.0
Flow velocity: 124cm/h
Operation: the sample behind the ion exchange chromatography is divided into three parts, goes up the about 100ml of sample at every turn.Collect the about 200ml of sample
Extract NGF with above-mentioned technology from raw venin, its purification result is summarised in the table 1.
Table 1 purification result is summed up
Sample size (g) rate of recovery
123 average substep rate of recovery total yields
Thick poison 30.0 30.0 30.0 30.0 100% 100%
Hydrophobic material 1.52 1.38 1.42 1.44 4.8% 4.80%
Ion-exchange 0.42 0.32 0.34 0.36 25.0% 1.20%
Gelatinous material 0.130 0.120 0.128 0.126 35.0% 0.42%
Each pacing is decided active and is really seen Table 2 than slip-knot.
Each pacing of table 2 is fixed active and than the slip-knot fruit
Productive rate gained amount gross activity is than living
(mg) (mg/ml) (B.U.) (B.U./mg) thick poison 30,000 3.50 1.98 * 10
76.60 * 10
2Hydrophobic material 1,420 0.45 3.69 * 10
62.60 * 10
3Ion-exchange 340 0.08 4.76 * 10
61.40 * 10
4Gelatinous material 128 0.01 2.94 * 10
72.30 * 10
5
Claims (5)
1. the method for a purifying nerve growth factor from snake venom, it is characterized in that, get a certain amount of meal snake venom, be dissolved in the damping fluid of PH=7.0, get supernatant after centrifugal and carry out hydrophobic chromatography successively, ion exchange chromatography and gel exclusion chromatography, the selected hydrophobic medium of hydrophobic chromatography is positive ethyl, the polyethers of bonding, normal-butyl or phenyl, selected elutriant is the buffered soln of PH at 6.0-9.0, selected type of elution is for lowering the mode of salt concn, the selected Ion Exchange Medium of ion exchange chromatography is a phenmethyl, sulfonic group, phosphate and oxalic acid diethylin or quaternary amine base, selected elutriant is the buffered soln of pH at 5.0-7.0 and 7.0-9.0, selected type of elution is to increase the mode of salt concn, the selected gel exclusion medium of gel exclusion chromatography is an agarose, dextran or silica gel material, selected elutriant is the buffered soln of pH at 5.0-9.0, and selected type of elution is a mode such as degree such as grade.
2. method according to claim 1 is characterized in that, said hydrophobic chromatography elutriant is the Na that contains of 20-50mmol/L
+, K
+, Mg
++, Ca
++, NH
4 +, H
2PO
4 -, HPO
4 2-And SO
4 2-The ionic salts solution.
3. method according to claim 1 is characterized in that, said ion-exchanging eluent is the Na that contains of 20-50mmol/L
+, K
+, Mg
++, Ca
++, H
2PO
4 -, HPO
4 2-And Cl
-The ionic salts solution.
4. method according to claim 1 is characterized in that, said ion-exchanging eluent is the Na that contains of 20-50mmol/L
+, K
+, Mg
++, Ca
++, H
2PO
4 -, HPO
4 2-And Cl
-The ionic salts solution.
5. method according to claim 1 is characterized in that, gets 30.0 gram meal shape snake venom, is dissolved in the Na that contains of 120ml 10mM
+, K
+, Mg
++, Ca
++, H
2PO
4 -And HPO
4 2-In the damping fluid of ionic pH=7.0,4, centrifugal 30 minutes of 000rpm, get supernatant purifying in the following order:
(1) hydrophobic chromatography
With balance liquid A balance chromatography column 30min, will use the sample upper prop more earlier, behind balance liquid A flushing 20min, beginning 100min gradient, keep 20min with elutriant B again, at last with balance liquid A balance chromatography column in order to the next time of usefulness again, collect and contain the active peak of NGF and be about 180ml; Wherein: chromatography column: 2.6 * 50cm, filler are: a kind of hydrophobic material that contains normal-butyl, 265ml glue, balance liquid A are: 20mM PBS+1.5M (NH)
2SO
4, pH=7.0, elutriant B are: 20mM PBS, and pH=7.0, gradient: elutriant B is from 0～100%, linear gradient, flow velocity: 290cm/h;
(2) ion exchange chromatography
Chromatography column is earlier with balance liquid C balance 30min, again previous step is collected and sample on the sample of desalting column desalination, with balance liquid C towards liquid 20min, after the 100min gradient, continue to keep elutriant D 20min, again with 30min balance liquid C balance chromatography column, wherein: chromatography column: 2.6 * 5.0cm, filler: a kind of ion-exchange material that contains carboxymethyl, 265ml glue, balance liquid C:20mM NaAc-HAc, pH=5.0, elutriant D:20mM NaAc-HAc+1MNaCl, pH=5.0, gradient: elutriant D is from 0-100%, linear mode, flow velocity: 290cm/h
(3) gel exclusion chromatography:
Sample behind the ion exchange chromatography is divided into three parts, goes up the about 100ml of sample at every turn, wherein: chromatography column: 2.6 * 125cm, filler: sepharose material, 660ml, elutriant: 50mM PBS, pH=7.0, flow velocity: 124cm/h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99115898 CN1108309C (en) | 1999-11-09 | 1999-11-09 | Process for purifying nerve growth factor from venin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 99115898 CN1108309C (en) | 1999-11-09 | 1999-11-09 | Process for purifying nerve growth factor from venin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1255503A CN1255503A (en) | 2000-06-07 |
| CN1108309C true CN1108309C (en) | 2003-05-14 |
Family
ID=5278787
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 99115898 Expired - Fee Related CN1108309C (en) | 1999-11-09 | 1999-11-09 | Process for purifying nerve growth factor from venin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1108309C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102485752A (en) * | 2010-12-01 | 2012-06-06 | 上海丽珠制药有限公司 | Purification method for human chorionic gonadotrophin (HCG) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105732796A (en) * | 2016-04-27 | 2016-07-06 | 广西医科大学 | Cobra venom nerve growth factor purification and preparation method |
| CN109762056A (en) * | 2017-11-10 | 2019-05-17 | 舒泰神（北京）生物制药股份有限公司 | A kind of extracting method of nerve growth factor |
-
1999
- 1999-11-09 CN CN 99115898 patent/CN1108309C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102485752A (en) * | 2010-12-01 | 2012-06-06 | 上海丽珠制药有限公司 | Purification method for human chorionic gonadotrophin (HCG) |
| CN102485752B (en) * | 2010-12-01 | 2014-07-23 | 上海丽珠制药有限公司 | Purification method for human chorionic gonadotrophin (HCG) |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1255503A (en) | 2000-06-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4519646B2 (en) | Purification method of preproinsulin | |
| US6627737B1 (en) | Factor IX purification methods | |
| Morse et al. | A new approach to the study of urinary macromolecules as a participant in calcium oxalate crystallization | |
| EP1127063B1 (en) | Methods of purifying recombinant human erythropoietin from cell culture supernatants | |
| EP0796865A2 (en) | Adsorbent and method for removing chemokines | |
| Ramos-de-la-Peña et al. | Progress and challenges in PEGylated proteins downstream processing: a review of the last 8 years | |
| CN1031943C (en) | Method of purifying protein | |
| US5466377A (en) | Chromatography media and their uses | |
| CN1108309C (en) | Process for purifying nerve growth factor from venin | |
| EP3280508B1 (en) | Method for chromatography | |
| Sparro et al. | Isolation and N-terminal sequence of multiple forms of granulins in human urine | |
| CN1175105C (en) | Purification of vitamin-K dependent proteins by membrane chromatography | |
| US7214319B2 (en) | Method of separating protein from animal milk | |
| Cooney | Chromatographic gel media for large scale protein purification | |
| CN105153294B (en) | A kind of Recombulin and insulin analog precursor purification process | |
| CN104447977A (en) | Purification production method for recombinant human bone morphogenetic protein-2 | |
| NZ208241A (en) | Purification of blood plasma or serum: selective removal of (very) low density lipoproteins (ldl and vldl) | |
| Flodin | The sephadex story | |
| Chataway et al. | Development of a two‐dimensional gel electrophoresis database of human lysosomal proteins | |
| JP3386498B2 (en) | Purification method of human TGF-β | |
| AU650532B2 (en) | Yeast derived mitogen | |
| CA1334390C (en) | Glycoprotein growth modulation materials | |
| CN109627324B (en) | Low electronegativity gelatin and preparation method and application thereof | |
| Murase et al. | Nerve growth factor-like immunoreactive substance in Panax ginseng extract | |
| RU2060274C1 (en) | Process for preparing purified ribonuclease dimer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |