CN1079226A - The novel method of preparation biological activity protein from people's urine - Google Patents
The novel method of preparation biological activity protein from people's urine Download PDFInfo
- Publication number
- CN1079226A CN1079226A CN92108938A CN92108938A CN1079226A CN 1079226 A CN1079226 A CN 1079226A CN 92108938 A CN92108938 A CN 92108938A CN 92108938 A CN92108938 A CN 92108938A CN 1079226 A CN1079226 A CN 1079226A
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- urine
- column
- people
- resin
- protein
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Links
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 64
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- 238000002360 preparation method Methods 0.000 title abstract description 7
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- 108090000435 Urokinase-type plasminogen activator Proteins 0.000 claims abstract description 34
- 229960005356 urokinase Drugs 0.000 claims abstract description 32
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
-
- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
-
- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- 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
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- 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
- 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
- C07K14/76—Albumins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
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Abstract
The present invention relates to from people's urine, prepare the method for biological activity protein, specifically, the present invention relates to use ultrafiltration, add acid or add metal ion, and a series of column chromatographys, the economy of some kinds of biological activity proteins of preparation (as Urogastron, urokinase and white protein) and high-efficiency method from people urine simultaneously.
Description
The present invention relates to the novel method of preparation biological activity protein from people urine, more particularly, relate to proteinic, the economy that from people's urine, prepares some kinds of pharmaceutically usefuls simultaneously and high-efficiency method.
Human body will be discharged about 1.5 liters of urine every day, contains salts such as muriate, phosphoric acid salt and vitriol in the urine; Mineral ions such as potassium, sodium and calcium; And a large amount of ureas and a small amount of protein.
In above-mentioned protein, found such as urokinase (Williams, J.R.B., Br.J.Exp.Pathol.32:530,1951), Urogastron (Jaspar, J.M. and Franchimant, P., Eur.J.Biochem.166:295,1987) and G CFS (Xu Tao etc., Biol.Chem.Hopper-Seyler, biological activity protein such as 368:187,1987).
Yet, have only from urine isolated urokinase be proved to be the related industries that can be applicable to pharmaceutical field, other proteic attentions are only concentrated on the research needs that satisfy some technical task in the purge process.
For isolated protein from people's urine, prior art has adopted use aluminum oxide, gac and diatomaceous purification process (United States Patent (USP) 3711377; Japanese Patent 82037316); The precipitator method of applied chemistry additive such as metal ion; Be used to separate the ultrafiltration process of metakentrin (United States Patent (USP) 4123510) and human chorionic gonadotrophin (United States Patent (USP) 4123509); And base exchange method (Japanese Patent 52061289; Japanese Patent 50069285) etc.Yet these methods are ineffective with regard to yield, and restricted to the proteinic kind of want purifying.
So, still need to study a kind of economy and purification process efficiently, make its various biological activity proteins that can be applied to derive from people's urine, can reduce the loss of urine sample simultaneously again.
So main purpose of the present invention is, use a series of efficient and simple purification process, fractional separation goes out various biological activity proteins simultaneously from people's urine, and these biological activity proteins also have Urogastron and white protein etc. except that urokinase.
Above-mentioned purpose of the present invention and other purposes and feature can be found out from the description of doing below in conjunction with accompanying drawing.In the accompanying drawing:
Fig. 1 to 8 illustrates the separation method of preparation biological activity protein from people's urine;
Fig. 9,10,14,15,16,18,20,21, the 23rd, various chromatography eluant curves of the present invention;
Figure 11,12 and 13 has illustrated the influence to urine protein content in the gained throw out of pH, zine ion and cupric ion respectively;
Figure 17,19 and 22 has provided Urogastron, urokinase and albuminised SDS-PAGE (SDS-PAGE) figure respectively.
Further specify the present invention with following separation and purifying procedure.
Concentrating of urine
Get the urine of picking up from healthy male, in 4-30 ℃ temperature range, concentrate.At filter membrane (aperture is 0.2-0.8 μ m) with have on the ultra-filtration equipment of 1,000 and 5,000 dalton molecules sieves, urine is concentrated 10-100 doubly.The concentrated solution of gained is dissolved in 1-50mM damping fluid (pH6-9) as in the damping fluids such as acetate, Citrate trianion and Tris, and concentrates once more and obtain denseer solution.Can obtain the powdery enriched material with vacuum vapor deposition method and freezing and drying lyophilization, be dissolved in the described damping fluid it standby.
The adding of acid/metal ion
Above-mentioned concentrated solution can directly carry out column chromatography, but does ineffective with regard to yield and/or purifying multiple like this.In this case, the inventor adopts a series of purification steps to obtain beat all result, adds acid or metal ion in the urine concentrated solution that is:, and supernatant liquor and throw out are carried out fractional separation; Then the two-phase of gained is carried out column chromatography respectively.
Fractional separation is to realize by the pH or the concentration of metal ions of control concentrated urine, with hydrochloric acid or sulphuric acid soln pH is controlled at 2-6 that is:; With zinc chloride or copper sulfate concentration of metal ions is controlled at 1-50mM.With gained solution stirring 5 minutes to 24 hours, centrifugation supernatant liquor and throw out; The gained throw out is dissolved in the described damping fluid.
PH to the principle that urine protein carries out fractional separation institute foundation is by control: protein is in the iso-electric point place precipitation of himself.Contain 15% total urinary protein in the supernatant liquor of gained, particularly under the situation of Urogastron, can obtain than prior art (Jaspar with isoelectric point precipitation, J.M. and Franchimant, P., Eur.J.Biochem.166:295,1987) high twice purification efficiency how.Urokinase and white protein major part all are present in the throw out, can carry out fractional separation with anionite by controlling acidity respectively; Adopt affinity column that urokinase (Holmberg, L. etc., Biochem.Biophys.Acta.445:215,1976) and white protein (Travis, J. etc., Biochem.J.157:301,1976) are further purified.
On the other hand, the principles of chemistry of carrying out protein precipitation with metal ion are: metal ion such as zinc or copper and proteinic histidine residues form title complex.This separation method is very high to specific protein efficient of the present invention.
The sample that aforesaid method is obtained be added to filter or centrifugal device on remove behind the insolubles standby.
Chromatogram
Resin used in the column chromatography is selected,, and reduced concentrated/solvent exchange step with satisfied economy/requirement efficiently.Used resin is as follows: ion-exchange chromatography: the hydrophobic polysaccharide that is combined with diethyllaminoethyl (DEAE), season aminoethyl (QAE), carboxymethyl (CM) or sulfopropyl (SP); Adsorption chromatography: silica gel or aluminum oxide; Gel filtration chromatography: Sephadex or Sephacryl; Affinity chromatography: benzenyl amidine-Sepharose 6B or blue Sepharose CL-6B.
The damping fluid that adopts in the column chromatography is as follows: anionite: 1-50mM phosphoric acid salt or Tris damping fluid (pH6-9); Cationite: 1-50mM acetate or citrate buffer (pH3-6).Concentration or controlling acidity (pH) by control salt such as Repone K or sodium-chlor are carried out wash-out.
In gel-filtration and adsorption chromatography, use 1-50mM damping fluid (pH6-9) and the 5-100mM damping fluid (pH3-6) that contains 10-200mM sodium-chlor respectively, carry out wash-out by adding alcoholic solvent or controlling acidity.
In affinity column chromatography, use 1-50mM phosphoric acid salt or Tris damping fluid (pH6-9) or 5-500mM glycine buffer (pH2-4); In reversed-phase column chromatography, use the silica gel of 18 carbon, carry out wash-out with the solvent that contains trifluoroacetic acid and acetonitrile.
The analysis of purifying urine protein
Urokinase is a kind of serine protease that contains 411 amino-acid residues, and it is that molecular weight is 54,000 daltonian plasminogen activators.Downcut 135 amino-acid residues from its N-terminal, obtaining molecular weight is 34,000 daltonian modification urokinases, and it is the same with 54,000 daltonian complete urokinases, also can hydrolytically condensable hemase (White, W.F. etc., Biochemistry 5:2160,1966).The measuring method of urokinase comprises: measure fibrinolysis (Astrup.T. and Mullertz, S., Arch.Biochem.Biophys.40:346,1952); The burnt Glu-Gly-Arg-p-Nitroaniline of the mensuration S-2444(N-of chromophore) dissociate (Pannell, R. and Gurewich, V., Blood 69:22,1987).
Urogastron is a kind of about 5,000 daltonian protein, and it is a kind of factor,mitogenic that contains 53 amino-acid residues.The present invention adopts radioimmunoassay (Jaspar, J.M. and Franchimant, P., Eur.J.Cancer Clin.Oncol.20:1343,1985) to measure its biological activity.
White protein is a kind of 66,000 daltonian protein of keeping the colloidal osmotic pressure effect that play, its relevant with cytotropic transhipments such as lipid acid, amino acid (Theodore, P.Jr., The Plasma Proteins I:133,1975); The measuring method that the present invention adopts is the standard white protein peak of measuring in the HPLC elution curve.
Method quantitative assay total protein (Lowry, O.H. etc., J.Biol.Chem.193:265,1951) with Lowry etc.
Further specify the present invention with the following example, these embodiment should not think to limit the scope of the invention.
Gather 100 liters of man's urine, on 0.45 μ m membrane filter, filter, be limited to 2000 daltonian molecular sieves with exclusion and be concentrated into 2 liters.(20mM Tris-HCl pH7.6), obtains 2 liters of urinary concentrations with method same as described above to add 20 liters of damping fluids in this solution.From urinary concentration, get 0.5 liter of urine concentrated solution and carry out frost drying, obtain 4.3g urine powder (see figure 1).2g is urinated powder to be dissolved in the 5ml damping fluid (40mM Tris-HCl, pH7.6), gained solution carries out the fractional separation (see figure 4) by the Sephacryl S-300(16 * 190mm) with the damping fluid pre-equilibration that contains 50mM sodium-chlor.With 5ml is that a fraction is collected each fraction that wash-out goes out, to measure the photoabsorption at 280nm place.Measure urokinase, Urogastron and albuminised activity respectively.The results are shown in Fig. 9 and table 1.
Table 1.
| Sample | Total protein (mg) | Activity and amount | Than * alive (mg/mg) (unit/mg) (ug/mg) | Yield (%) | Purity (multiple) | ||
| White protein (mg) | Urokinase (unit) | h-EGF (ug) | |||||
| The urine concentrated solution | 1,048 | 440 | 7.830 | 333 | 0.42/7.5 /0.32 | 100 | 1 |
| The white protein fraction | 461 | 295 | 0.64 | 67 | 1.5 | ||
| The urokinase fraction | 264 | 6,573 | 24.9 | 84 | 3.3 | ||
| The h- |
38 | 147 | 3.8 | 44 | 11.9 | ||
H-EGF: human epidermal growth factor
*: than living with mg/mg or μ g/mg is that unit represents, the amount of urine protein in promptly every mg total protein.
By mode similar to Example 15 liters of urine are concentrated into 0.2 liter, the urinary concentration of gained is being used damping fluid (20mM Tris-HCl, pH7.6) the DEAE-Sepharose post crossed of balance (carries out fractional separation on 50 * 200mm), with the damping fluid that contains 0-1M NaCl linear gradient (40mM sodium acetate, pH4.6) wash-out (see figure 4).With 40ml is that a fraction is collected each fraction that wash-out goes out, and measures the photoabsorption and the activity at 280nm place by mode similar to Example 1.The results are shown in Figure 10 and table 2.
Table 2.
| Sample | Total protein (mg) | Activity and amount | Than * alive (mg/mg) (unit/mg) (ug/mg) | Yield (%) | Purity (multiple) | ||
| White protein (mg) | Urokinase (unit) | h-EGF (ug) | |||||
| The urine concentrated solution | 235 | 99 | 1,559 | 65 | 0.42/6.6 /0.27 | 100 | 1 |
| The white protein fraction | 94 | 64 | 0.68 | 65 | 1.6 | ||
| The urokinase fraction | 14 | 727 | 48 | 51.9 | 47 | 7.9 | |
| The h-EGF fraction | 74 | 48 | 0.65 | 74 | 2.4 | ||
H-EGF: human epidermal growth factor
*: than living with mg/mg or μ g/mg is that unit represents, the amount of urine protein in promptly every mg total protein.
By mode similar to Example 13 liters of urine being concentrated into 0.2 liter, is that a urine concentrated solution with gained is divided into some equal portions with 10ml.Add 5N HCl in each sample aliquot its pH is transferred to 3.0 to 5.6 scope, the pH fixed interval of each sample is 0.2.Behind the stir about 1 hour, gained solution is with centrifugal 10 minutes (see figure 2)s of 12,000 * g.In the throw out of gained, add damping fluid (40mM sodium acetate, pH7.2), measure the activity of total protein content and urokinase, white protein and Urogastron, confirm as Figure 11, when pH3.4 to 4.4, carry out supernatant liquor and sedimentary fractional separation, very effective concerning described proteic purifying.
In the 10ml that tells by mode similar to Example 3 urine concentrated solution sample aliquot, add zinc chloride to final concentration and be 1,2,4,6,8,10 and 20mM, gained solution carries out centrifugal (see figure 3) by mode similar to Example 3.Each throw out is dissolved in 10ml damping fluid (20mM sodium phosphate, pH7.2 contain 5mM EDTA).The activity of the total protein content of each solution and white protein, urokinase, Urogastron is shown in Figure 12.
Experimentize by mode similar to Example 4, the different copper sulfate that are to use replace the zinc chloride (see figure 3).The results are shown in Figure 13.
By mode similar to Example 1 20 liters of urine are concentrated into 0.5 liter.By mode similar to Example 3, above-mentioned urinary concentration fractional separation under pH4.0 is become supernatant liquor and throw out.The gained supernatant liquor carries out following processing (see figure 6): the 485ml supernatant liquor is added to silicagel column (on 50 * 200mm), with containing 20%(v/v) the alcoholic acid 40mM sodium phosphate buffer of crossing with 40mM sodium acetate buffer (pH4.2) pre-equilibration (pH8.0) carries out chromatographic separation.The active fraction that will contain Urogastron merges, and uses Amicon YMO2 membrane concentration to 20ml.
Gained solution is using the Sephadex G-50 post of the 20mM sodium acetate buffer pre-equilibration that contains 50mM NaCl (to carry out fractional separation on 26 * 1500mm).The active fraction that will contain Urogastron is added to uses 0.1%(v/v) the PepRPC(trade(brand)name crossed of trifluoroacetic acid pre-equilibration) on the reversed-phase HPLC post, use 50%(v/v) acetonitrile carries out fractional separation as eluent.The active fraction that will contain activities of epidermal growth factor with vacuum-evaporator and freeze dryer is dried to 46 μ g.A series of preparation process are shown in table 3 and Figure 14,15,16 and 17.
Figure 14,15 and 16 provides the urine concentrated solution respectively when the enterprising circumstances in which people get things ready for a trip spectrum of silica gel, Sephadex G-50 and reversed-phase HPLC post is separated, human epidermal growth factor's (h-EGF) elution curve.
Figure 17 has provided the human epidermal growth factor's who is purified into SDS-polyacrylamide gel (20%(v/v) from urinary concentration) electrophoretic patten: swimming lane 1 is the high molecular marker; Swimming lane 2 is urine concentrated solutions; Swimming lane 3 is supernatant liquors of pH4.0; Swimming lane 4 is the isolated human epidermal growth factor of silica gel chromatography (h-EGF) fractions; Swimming lane 5 is isolated h-EGF fractions on the Sephadex G-50; Swimming lane 6 is that PepRPC goes up isolated h-EGF fraction; Swimming lane 7 is mouse EGFs; Swimming lane 8 is lower molecular weight markers.
Table 3.
| Sample | White protein (mg) | h-EGF (ug) | Than * alive (ug/mg) | Yield (%) | Purity (multiple) |
| The urine concentrated solution | 950 | 272 | 0.29 | 100 | 1 |
| Supernatant liquor | 148 | 244 | 1.65 | 90 | 6 |
| The silicagel |
26 | 126 | 4.58 | 46 | 17 |
| Sephadex G-50 post effluent liquid | 3.3 | 98 | 29.7 | 36 | 102 |
| The reversed-phase column effluent liquid | 0.05 | 46 | 920 | 17 | 3,172 |
H-EGF: human epidermal growth factor
*: than living with μ g/mg is that unit represents, the amount of h-EGF in promptly every mg total protein.
For preparation urokinase and white protein, the centrifugal throw out that obtains under the pH4.0 is further purified (see figure 5).In throw out, add 200ml 20mM Tris-HCl damping fluid (pH7.8), the gained solution stirring was obtained consoluet solution in 2 hours, and (50 * 200mm) enterprising circumstances in which people get things ready for a trip spectrums are separated at the DEAE-Sepharose post of crossing with damping fluid pre-equilibration same as described above.Carry out wash-out with initial buffer liquid and 40mM sodium acetate buffer (pH4.6), collection contains urokinase and albuminised active fraction (seeing Figure 24) respectively.
It is 200ml that the urokinase fraction that wash-out is gone out is concentrated into volume, be added to again and used damping fluid (20mM Tris-HCl, pH7.6) benzenyl amidine of pre-equilibration-Sepharose 6B post is (on 26 * 150mm), (50mM glycine-HCl pH3.69) carries out fractional separation (seeing Figure 18) with damping fluid.Merge the fraction and the frost drying that contain urokinase activity, obtain the urokinase of 29,800 units.The results are shown in table 4 and Figure 18,19.
Figure 18 has provided the urokinase elution curve of urine concentrated solution when the enterprising circumstances in which people get things ready for a trip spectrum of benzenyl amidine-Sepharose 6B post is separated.Figure 19 has provided the SDS-polyacrylamide gel (12.5%(v/v) of the urokinase that is purified into from urinary concentration) electrophoretic patten: swimming lane 1 is the intermediate molecular weight marker; Swimming lane 2 is the urine concentrated solution; Swimming lane 3 is the throw out of pH4.0; Swimming lane 4 is the first half of isolated urokinase fraction on DEAE-Sepharose; Swimming lane 5 is the latter half of described fraction; Swimming lane 6 is an isolated urokinase fraction on benzenyl amidine-Sepharose 6B; Swimming lane 7 is the standard urinary kinases; Swimming lane 8 is the lower molecular weight marker.
Table 4.
| Sample | White protein (mg) | Urokinase activity (unit) | Ratio work (unit/mg) | Yield (%) | Purity (multiple) |
| The urine concentrated solution | 950 | 62，000 | 65.2 | 100 | 1 |
| Throw out | 807 | 59,100 | 73.2 | 95 | 1.1 |
| DEAE-Sepharose post effluent liquid | 48.5 | 33,100 | 682 | 53 | 10.5 |
| Benzenyl amidine-Sepharose 6B post effluent liquid | 0.34 | 29,800 | 87,647 | 48 | 1,344 |
The white protein fraction that will obtain from the DEAE-Sepharose post, blue Sepharose CL-6B post (20 * 100mm) with Sephacryl S-300 post (16 * 900mm) enterprising circumstances in which people get things ready for a trip spectrums are separated.Carry out wash-out with the 20mM Tris-HCl damping fluid (pH7.6) that contains 2M NaCl.With gained fraction frost drying, obtain the 188mg white protein, the results are shown in table 5 and Figure 20,21,22.
Figure 20 and 21 has provided the white protein elution curve of urine concentrated solution when blue Sepharose CL-6B separates with the enterprising circumstances in which people get things ready for a trip spectrum of Sephacryl S-300 gel-filtration column respectively.
Figure 22 has provided the albuminised SDS-polyacrylamide gel (12.5%(v/v) that is purified into from urinary concentration) electrophoretic patten: swimming lane 1 is the standard bovine serum albumin(BSA); Swimming lane 2 is the urine concentrated solution; Swimming lane 3 is the throw out of pH4.0; Swimming lane 4 is an isolated white protein fraction on DEAE-Sepharose; Swimming lane 5 is an isolated white protein fraction on blue Sepharose CL-6B; Swimming lane 6 is an isolated white protein fraction on Sephacryl S-300; Swimming lane 7 is the standard serum white protein; Swimming lane 8 is the high molecular marker.
Table 5.
| Sample | White protein (mg) | White protein (mg) | Than * alive (mg/mg) | Yield (%) | Purity (multiple) |
| The urine concentrated solution | 950 | 410 | 0.43 | 100 | 1 |
| Throw out | 807 | 372 | 0.46 | 91 | 1.1 |
| DEAE-Sepharose post effluent liquid | 404 | 275 | 0.68 | 67 | 1.6 |
| Blue Sepharose CL-6B post effluent liquid | 210 | 198 | 0.94 | 48 | 2.2 |
| Sephacry1 s-300 post effluent liquid | 190 | 188 | 0.99 | 46 | 2.3 |
*: than living with mg/mg is that unit represents, albuminised amount in promptly every mg total protein.
Getting 20 times of urinary concentrations that 250ml is purified into by mode similar to Example 4, is 8mM to wherein adding zinc chloride to concentration, and with about 1 hour of gained solution stirring, centrifugal (12,000 * g, 20 minutes) obtained throw out.In the gained throw out, add 4ml 500mM EDTA and 20ml damping fluid (20mM Tris-HCl, pH7.6), obtain complete lysate, (20mM Tris-HCl, pH7.6) the Sephadex LH-20 post of pre-equilibration (carries out fractional separation (seeing Figure 23) to gained solution on 26 * 900mm) using damping fluid.Merge the protein fractions in the effluent liquid and be added to (see figure 7) on the DEAE-Sepharose post.Result and table 2 and result shown in Figure 10 are similar.
Carry out purification step by mode similar to Example 7, different is to replace zinc chloride with copper sulfate.Result and table 2 and result shown in Figure 10 are similar.
Get the protein fractions that makes with Sephadex LH-20 column chromatography among the embodiment 7, on Sephacryl S-300 post, carry out fractional separation, obtain white protein, urokinase and Urogastron.Result and table 1 and result shown in Figure 9 are similar.
Carry out purification step by the mode identical with embodiment 9, different is to replace zinc chloride with copper sulfate.The result is similar to Example 9.
As embodiment 6 is illustrated, white protein, urokinase and the Urogastron that is obtained by embodiment 7,8,9 and 10 carried out further purifying.Urokinase is fractional separation on benzenyl amidine-Sepharose 6B post is enterprising, white protein carries out fractional separation on blue Sepharose CL-6B and Sephacryl S-300, Urogastron carries out fractional separation on Sephadex G-50 and PepRPC reversed-phase column.
Confirm that as above method of the present invention can be applicable to separate simultaneously such as biological activity proteins such as urokinase, white protein, Urogastrons.The present invention provides specially with distinctive fractional separation and prepurification step and comes the economy of purifying urine protein and high-efficiency method.
Claims (11)
1, a kind of method that from people's urine, prepares white protein, urokinase and Urogastron, this method comprises:
Concentrate people's urine with ultra-filtration equipment;
In described urinary concentration, add acid or metal ion;
To by described add acid or add supernatant liquor and the throw out that the metal ion step obtains carry out fractional separation respectively.
2, the process of claim 1 wherein that described ultra-filtration equipment uses exclusion to be limited to 1,000 to 5,000 daltonian filter membrane.
3, the process of claim 1 wherein add described acid with the acidity control of concentrated solution between pH2 and 6.
4, the process of claim 1 wherein that described metal ion is zinc chloride or copper sulfate, the final concentration after the adding is 1 to 50mM.
5, the process of claim 1 wherein and add the supernatant liquor that sour step obtains and separate, isolate Urogastron in the enterprising circumstances in which people get things ready for a trip spectrum of adsorption column, gel-filtration column and reversed-phase column by described; Gained throw out fraction is separated in the enterprising circumstances in which people get things ready for a trip spectrum of ion exchange column, gel-filtration column and affinity column, isolates white protein and urokinase respectively.
6, the process of claim 1 wherein and add the throw out that the metal ion step obtains and separate, isolate urine protein at anion-exchange column or the enterprising circumstances in which people get things ready for a trip spectrum of gel-filtration column by described.
7, the method for claim 5, wherein said adsorption column uses the resin that is selected from silica gel and aluminum oxide.
8, the method for claim 5, wherein said gel-filtration column uses the resin that is selected from Sephadex and Sephacryl.
9, the method for claim 5, wherein said reversed-phase column are the C18-PepRPC post.
10, the method for claim 5, wherein ion exchange column uses the resin be selected from diethyllaminoethyl resin, season aminoethyl resin, carboxymethyl resin and sulfopropyl resin.
11, the method for claim 5, wherein affinity column uses the resin that is selected from benzenyl amidine-Sepharose 6B and blue Sepharose CL-6B.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR8884/92 | 1992-05-26 | ||
| KR1019920008884A KR960009051B1 (en) | 1992-05-26 | 1992-05-26 | Simultaneous preparation process of useful proteins from human urine |
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| Publication Number | Publication Date |
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| CN1079226A true CN1079226A (en) | 1993-12-08 |
| CN1037103C CN1037103C (en) | 1998-01-21 |
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| CN92108938A Expired - Fee Related CN1037103C (en) | 1992-05-26 | 1992-08-28 | A novel process for preparing biologically active proteins from human urine |
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| CN (1) | CN1037103C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064049C (en) * | 1995-10-05 | 2001-04-04 | 南京大学 | Thrombopoietin and its producing process |
| CN1113896C (en) * | 1998-01-04 | 2003-07-09 | 刘荣秀 | Preparation of epidermal growth factor with marine products and relevant discard |
| CN103509104A (en) * | 2013-08-23 | 2014-01-15 | 扬州艾迪生物科技有限公司 | LLarge scale human urine protein collection method |
| CN103694334A (en) * | 2013-12-23 | 2014-04-02 | 扬州艾迪生物科技有限公司 | Method for preparing hEGF (human Epidermal Growth Factor) crude product |
| WO2018133778A1 (en) * | 2017-01-20 | 2018-07-26 | 北京蛋白质组研究中心 | Preparation method for urine protein and detection method for urine proteome |
| CN112250753A (en) * | 2020-10-28 | 2021-01-22 | 宁波博睿瀚达生物科技有限公司 | Method for dissociative adsorption concentration of recombinant epidermal growth factor |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1088597C (en) * | 1999-10-26 | 2002-08-07 | 合肥永生制药有限公司 | Uropoly aic-peptide composition and its usage |
| CN1090971C (en) * | 1999-10-26 | 2002-09-18 | 合肥永生制药有限公司 | Uropoly acid-peptide composition |
| CN1090970C (en) * | 1999-10-26 | 2002-09-18 | 合肥永生制药有限公司 | Process for preparing uropoly acid-peptide composition |
| CN103740687B (en) * | 2013-11-30 | 2016-05-11 | 青岛康原药业有限公司 | A kind of method of preparing urokinase crude product |
| CN104531648A (en) * | 2014-12-23 | 2015-04-22 | 青岛康原药业有限公司 | Method for preparing crude product of urokinase |
-
1992
- 1992-05-26 KR KR1019920008884A patent/KR960009051B1/en not_active IP Right Cessation
- 1992-08-28 CN CN92108938A patent/CN1037103C/en not_active Expired - Fee Related
-
1996
- 1996-03-25 KR KR96008146A patent/KR960009052B1/en not_active IP Right Cessation
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064049C (en) * | 1995-10-05 | 2001-04-04 | 南京大学 | Thrombopoietin and its producing process |
| CN1113896C (en) * | 1998-01-04 | 2003-07-09 | 刘荣秀 | Preparation of epidermal growth factor with marine products and relevant discard |
| CN103509104A (en) * | 2013-08-23 | 2014-01-15 | 扬州艾迪生物科技有限公司 | LLarge scale human urine protein collection method |
| CN103694334A (en) * | 2013-12-23 | 2014-04-02 | 扬州艾迪生物科技有限公司 | Method for preparing hEGF (human Epidermal Growth Factor) crude product |
| WO2018133778A1 (en) * | 2017-01-20 | 2018-07-26 | 北京蛋白质组研究中心 | Preparation method for urine protein and detection method for urine proteome |
| CN112250753A (en) * | 2020-10-28 | 2021-01-22 | 宁波博睿瀚达生物科技有限公司 | Method for dissociative adsorption concentration of recombinant epidermal growth factor |
Also Published As
| Publication number | Publication date |
|---|---|
| KR960009052B1 (en) | 1996-07-10 |
| KR960009051B1 (en) | 1996-07-10 |
| CN1037103C (en) | 1998-01-21 |
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