CN1438243A - Method for preparing fiberbound protein - Google Patents
Method for preparing fiberbound protein Download PDFInfo
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- CN1438243A CN1438243A CN 03115784 CN03115784A CN1438243A CN 1438243 A CN1438243 A CN 1438243A CN 03115784 CN03115784 CN 03115784 CN 03115784 A CN03115784 A CN 03115784A CN 1438243 A CN1438243 A CN 1438243A
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Abstract
The invention refers to a method of making purified fibre combined protein, adopting alcohol deposition, PEG deposition and negative-ion layer separation to make thep rotein, at the same time making its preparation and original preparation of fibre protein, and improving use degree of cold deposition and the purity and stability of the final product.
Description
Technical field
The invention belongs to biological chemistry, hematology, biological products are learned the field, are specifically related to a kind of method for preparing Fiberonectin.
Background technology
Fiberonectin (being called for short Fn) is a high molecular glycoprotein.The Fiberonectin molecule is made up of two similar polypeptide chains, and a plurality of functional domains are arranged in its molecule, each structural domain respectively with collagen, scleroproein, heparin, DNA, and even the combination of cell generation specificity, these are specific in conjunction with character, have the cellular form of influence with Fn, keep the histoorgan globality, and adhere to, move propagation, differentiation, it is closely related function such as to engulf.Fiberonectin can be treated some ophthalmic diseases, and promotes wound healing, treatment burn and wound etc.
The purification of Fiberonectin starts from the seventies.Mosesson MW in 1970 etc. are raw material with the cryoprecipitate, with phosphoric acid buffer dissolving, add glycine and ethanol sedimentation respectively after, dissolving again, by the DEAE-cellulose column, the phosphoric acid buffer wash-out obtains Fiberonectin.The Fiberonectin of gained contains Fibrinogen, produces white flocks, makes the less stable of goods, and yield is lower, and purity is relatively poor.Axen in 1971 with the affinity column of the antibody of anti-Fiberonectin directly from fresh plasma adsorbing fiber conjugated protein, obtain with hydrochloric acid-glycine wash-out.This method at first needs the single-minded antibody of Fiberonectin, and its cost is very big, is not suitable for scale operation.Ammonium sulfate precipitation blood plasma such as Allen in 1973 carries out heparin-sepharose affinity chromatography after the isolating throw out dissolving, obtains Fiberonectin with the sodium chloride solution wash-out, and the Fiberonectin purity that this method obtains is relatively poor, and stability is not high.Eva Engvall in 1977 etc. are according to Fiberonectin and gelatin bonded characteristic, developed gelatin-protein-bonded simpler method of sepharose 4B affinity chromatography separated fiber, its utilize in the Fiberonectin molecule can with gelatin bonded functional area, gelatin is coupled on the Sepharose 4B with covalent linkage, blood plasma or cryoprecipitate lysate are directly crossed post, after carrying out affinity chromatography, again with initial damping fluid washing gelatin post, at last with urea with the Fiberonectin wash-out.But there is following defective:
Owing to contain volume Fibrinogen (Fg), and Fg is extremely unsettled albumen in the urea elutriant, in the process of purifying, the influence of temperature or other factors can constantly impel Fg to become scleroproein and separates out, and makes sample white flocks occur, affinity column is stopped up, influence purge process.Secondly, because of Fg and Fn mutually combine, so, when Fg becomes scleroproein and separates out, can make the form of Fn with co-precipitation, form the flocks of Fn and Fg white, thereby the rate of recovery of Fn is reduced.The 3rd, gelatin-Sephorose4B affinity column is not single-minded to the absorption of Fiberonectin, to Fg, or the like adsorption is also arranged, simultaneously because the interaction of Fg and Fn makes the purity of end article not high.Can not remove Fg fully in the end article, in containing the Fn goods of Fg,, make goods solution precipitation occur, can have a strong impact on quality of item because of Fg becomes scleroproein.The 4th, urea is influential to the biological activity of goods, and the urea of high density can influence some biological activity of Fn.
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing Fiberonectin.The present invention can simplify former purifying technique, shortens purification process, improves goods purity, is applicable to scale preparation.
The present invention is undertaken by following step,
1, with Tris-HCl damping fluid dissolving cryoprecipitate,
Its PH of described damping fluid is 7.0-8.5, contains trisodium citrate 0.5-2.0%, sodium-chlor 0.50-2.0%, and the dissolving ratio is 1: 3-1: 20, solvent temperature is 5-40 ℃, dissolution time is 1-3 hour.
2, alcohol precipitation
The cryoprecipitate lysate is centrifugal, and rotating speed is 1000-4000rpm, obtains supernatant, removes precipitation.Adding low temperature spirituous solution (containing trisodium citrate 0.5-2.0%, sodium-chlor 0.5-2.0%, 0～-30 ℃) to concentration in the supernatant is 3-30%, stirs 30-60min in the ice bath, leaves standstill 2-5h then, low-temperature centrifugation, rotating speed 3000-7000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation contains trisodium citrate 0.5-2.0% with Tris-HCl damping fluid (PH7.0-8.5), and sodium-chlor 0.50-2.0% is dissolved to original volume, and lysate can be controlled in 0-30 ℃.
3, the first step PEG precipitation
Add concentration in the alcohol precipitation lysate and be 40% PEG solution, to final concentration be 4-15%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation between 10-25 ℃, the PH of Controlling System between 7.0-8.5, stirring at room 30 minutes, leave standstill after 30-60 minute centrifugal, rotating speed 3000-5000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation (this resolution of precipitate is fibrinogen preparation).
4, the second step PEG precipitation
Add 40%PEG solution in the supernatant liquor that the first step PEG precipitation process obtains, add 5-25mlPEG solution in every 100ml supernatant liquor, the PH of Controlling System is between 7.0-8.5, temperature is between 10-25 ℃, stirred 30 minutes under the room temperature, left standstill 30-60 minute, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000-5000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is dissolved to original volume with Tris-HCl damping fluid (PH7.0-8.5).
5, ion exchange chromatography
Get QAE gel dress post, with Tris-HCl damping fluid (PH7.0-8.5) balance QAE gel chromatography system.Need three column volumes of Tris-HCl damping fluid (PH7.0-8.5) altogether.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, last sample flow velocity is 10-40ml/h, carries out the QAE anion chromatography; And then, wash QAE anion layer analysis system, with three column volumes with Tris-HCl damping fluid (PH7.0-8.5); Washing is for the second time washed with the Tris-HCl damping fluid (PH7.0-8.5) of sodium chloride-containing, and wherein sodium chloride concentration is (0.05-0.14M), washes foreign protein off; Use the Tris-HCl damping fluid (PH7.0-8.5) of sodium chloride-containing to carry out wash-out again, wherein sodium chloride concentration is (0.16-1.0M), and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Experiment material of the present invention and reagent: cryoprecipitate provides for Shanghai Vaccine and Serum Institute,
Ethanol, polyoxyethylene glycol (MW4000), QAE glue, the anti-people's Fiberonectin of rabbit serum, glycine (Gly), the Fiberonectin standard substance are commercial.
The result confirms that use method of purification provided by the invention, the purity of the Fiberonectin that obtains can reach about 90% (ultraviolet thin layer chromatography scannings), and its yield can reach more than 30.00%.Table 1 is the Fiberonectin purification result.
Table 1
Stoste alcohol precipitation PEG precipitation PEG precipitation QAE chromatography
1 2Fn concentration, 1.25 0.93 0.60 0.49 2.02 mg/ml volume (ml) 50 50 57 50 12 yield (%), 100.0 74.4 54.72 39.2 38.78 purity (%) 11 19 50 71 90
The operational path that the inventive method is set up can obtain Fiberonectin from cryoprecipitate.The outward appearance of its freeze-dried products of Fiberonectin preparation of preparation is a white, solubleness height, transparent clarification.Present method has been established the technology of the preparation Fiberonectin that can be used for large-scale production.Present method has following advantage: 1) the present invention improves the purity and the stability of end article, the viral inactivation treatment step can be incorporated into wherein.2) present method is applicable to large-scale production, and intermediate steps is few, and is simple to operation.3) preparation of the purification Fiberonectin that provides of the inventive method, the cycle is short, helps aseptic control in the production process.4) present method has been introduced the anion chromatography route, compares with existing affinity chromatography technology of preparing, and the present invention utilizes reinforcing yin essence ion column QAE, and the protein-bonded ability of its adsorbing fiber is apparently higher than the gelatin affinity column; The Fiberonectin purity height of gained of the present invention; Active high; The present invention activates Sepharose 4B because of not adopting activators such as cyanogen bromide, so environmentally safe.5) present method can make Fiberonectin preparation and fibrinogen preparation simultaneously, has improved the availability of cryoprecipitate, saves cost.Present method is used the former and Fiberonectin of PEG separating fibrin after adopting alcohol precipitation Fibrinogen and Fiberonectin again, can make Fiberonectin preparation and fibrinogen preparation simultaneously.
Embodiment
Embodiment 1
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 100g was dissolved in the Tris-HCl damping fluid with 1: 3, and PH7.0 contains trisodium citrate 0.60%, sodium-chlor 0.50%, and solvent temperature is 15 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (0 ℃) to concentration in the supernatant is 4%, stirs 40min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 5000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.0 contains trisodium citrate 0.60%, and sodium-chlor 0.50% is dissolved to original volume, and the lysate temperature is 0 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 5%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 12 ℃, and the PH of system is controlled at 7.0, and stirring at room 30 minutes leaves standstill 30 minutes, and is centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 7mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.0, temperature is controlled at 12 ℃, stirred 30 minutes under the room temperature then, left standstill 30 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.0 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.0, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 10ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.0, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.0 of sodium chloride-containing, and wherein sodium chloride concentration is 0.05M, washes foreign protein off; Tris-HCl buffer solution ph 7.0 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.16M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 2
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 100g was dissolved in the Tris-HCl damping fluid with 1: 5, and PH7.10 contains trisodium citrate 0.70%, sodium-chlor 0.90%, and solvent temperature is 8 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 2000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (3 ℃) to concentration in the supernatant is 6%, stirs 40min in the ice bath, leaves standstill 2h then, low-temperature centrifugation, rotating speed 6000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.10 contains trisodium citrate 0.70%, and sodium-chlor 0.90% is dissolved to original volume, and the lysate temperature is 3 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 7%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 10 ℃, and the PH of system is controlled at 7.10, and stirring at room is 30 minutes then, leaves standstill 40 minutes, and is centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 9mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.10, temperature is controlled at 10 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.10 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.10, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 12ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.10, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.10 of sodium chloride-containing, and wherein sodium chloride concentration is 0.06M, washes foreign protein off; Tris-HCl buffer solution ph 7.10 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.18M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 3
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 100g was dissolved in the Tris-HCl damping fluid with 1: 7, and PH7.20 contains trisodium citrate 1.00%, sodium-chlor 0.90%, and solvent temperature is 37 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (5 ℃) to concentration in the supernatant is 8%, stirs 40min in the ice bath, leaves standstill 4h then, low-temperature centrifugation, rotating speed 6000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.20 contains trisodium citrate 1.00%, and sodium-chlor 0.90% is dissolved to original volume, and the lysate temperature is 5 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 8%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 14 ℃, and the PH of system is controlled at 7.2, and stirring at room is 30 minutes then, leaves standstill 60 minutes, and is centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 10mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.2, temperature is controlled at 14 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.20 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.20, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 15ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.20, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.20 of sodium chloride-containing, and wherein sodium chloride concentration is 0.07M, washes foreign protein off; Tris-HCl buffer solution ph 7.20 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.20M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 4
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 200g was dissolved in the Tris-HCl damping fluid with 1: 9, and PH7.30 contains trisodium citrate 1.00%, sodium-chlor 1.00%, and solvent temperature is 35 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (8 ℃) to concentration in the supernatant is 10%, stirs 50min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 6000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.30 contains trisodium citrate 1.00%, and sodium-chlor 1.00% is dissolved to original volume, and the lysate temperature is 6 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 9%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 15 ℃, and the PH of system is controlled at 7.3, and stirring at room is 30 minutes then, leaves standstill 40 minutes, and is centrifugal, rotating speed 4000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 11mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.3, temperature is controlled at 15 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.30 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.30, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 20ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.30, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.30 of sodium chloride-containing, and wherein sodium chloride concentration is 0.08M, washes foreign protein off; Tris-HCl buffer solution ph 7.30 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.25M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 5
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 200g was dissolved in the Tris-HCl damping fluid with 1: 11, and PH7.40 contains trisodium citrate 1.30%, sodium-chlor 1.20%, and solvent temperature is 20 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (9 ℃) to concentration in the supernatant is 12%, stirs 40min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 5000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.40 contains trisodium citrate 1.30%, and sodium-chlor 1.20% is dissolved to original volume, and the lysate temperature is 7 ℃.
3. add 40PEG% solution in the alcohol precipitation lysate, to final concentration be 10%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 16 ℃, and the PH of system is controlled at 7.40, and stirring at room is 30 minutes then, leaves standstill 50 minutes, and is centrifugal, rotating speed 4000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 12mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.40, temperature is controlled at 16 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.40 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.40, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 30ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.40, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.40 of sodium chloride-containing, and wherein sodium chloride concentration is 0.09M, washes foreign protein off; Tris-HCl buffer solution ph 7.40 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.28M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 6
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 200g was dissolved in the Tris-HCl damping fluid with 1: 13, and PH7.50 contains trisodium citrate 1.50%, sodium-chlor 1.0%, and solvent temperature is 20 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (10 ℃) to concentration in the supernatant is 14%, stirs 50min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 5000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.50 contains trisodium citrate 1.50%, and sodium-chlor 1.00% is dissolved to original volume, and the lysate temperature is 10 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 11%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 18 ℃, and the PH of system is controlled at 7.50, and stirring at room is 50 minutes then, leaves standstill 50 minutes, and is centrifugal, rotating speed 4000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 14mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.50, temperature is controlled at 20 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.50 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.50, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 40ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.50, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.50 of sodium chloride-containing, and wherein sodium chloride concentration is 0.10M, washes foreign protein off; Tris-HCl buffer solution ph 7.50 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.33M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 7
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 200g was dissolved in the Tris-HCl damping fluid with 1: 15, and PH7.60 contains trisodium citrate 1.30%, sodium-chlor 1.50%, and solvent temperature is 9 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (12 ℃) to concentration in the supernatant is 16%, stirs 30min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 4000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.60 contains trisodium citrate 1.30%, and sodium-chlor 1.50% is dissolved to original volume, and the lysate temperature is 15 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 12%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 20 ℃, and the PH of system is controlled at 7.60, and stirring at room is 30 minutes then, leaves standstill 40 minutes, and is centrifugal, rotating speed 4000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 16mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.60, temperature is controlled at 25 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.60 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.60, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 35ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.60, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.60 of sodium chloride-containing, and wherein sodium chloride concentration is 0.11M, washes foreign protein off; Tris-HCl buffer solution ph 7.60 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.35M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 8
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 200g was dissolved in the Tris-HCl damping fluid with 1: 17, and PH7.70 contains trisodium citrate 1.10%, sodium-chlor 1.00%, and solvent temperature is 40 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (16 ℃) to concentration in the supernatant is 18%, stirs 40min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 3000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.70 contains trisodium citrate 1.10%, and sodium-chlor 1.00% is dissolved to original volume, and the lysate temperature is 20 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 13%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 20 ℃, and the PH of system is controlled at 7.70, and stirring at room is 30 minutes then, leaves standstill 50 minutes, and is centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 18mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.70, temperature is controlled at 10 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 4000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.7 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.70, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 25ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.70, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.70 of sodium chloride-containing, and wherein sodium chloride concentration is 0.12M, washes foreign protein off; Tris-HCl buffer solution ph 7.70 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.40M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 9
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 200g was dissolved in the Tris-HCl damping fluid with 1: 19, and PH7.50 contains trisodium citrate 1.70%, sodium-chlor 0.80%, and solvent temperature is 14 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (20 ℃) to concentration in the supernatant is 20%, stirs 40min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 4000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.50 contains trisodium citrate 1.70%, and sodium-chlor 0.80% is dissolved to original volume, and the lysate temperature is 18 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 15%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 22 ℃, and the PH of system is controlled at 7.50, and stirring at room is 30 minutes then, leaves standstill 50 minutes, and is centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 20mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 7.50, temperature is controlled at 22 ℃, stirred 30 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH7.50 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH7.50, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 30ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH7.50, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 7.50 of sodium chloride-containing, and wherein sodium chloride concentration is 0.13M, washes foreign protein off; Tris-HCl buffer solution ph 7.50 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.35M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Embodiment 10
Purifying Fiberonectin from cryoprecipitate
1. cryoprecipitate 200g was dissolved in the Tris-HCl damping fluid with 1: 20, and PH8.00 contains trisodium citrate 1.60%, sodium-chlor 1.60%, and solvent temperature is 17 ℃, dissolution time is about 1 hour.
2. the cryoprecipitate lysate is centrifugal, and rotating speed is 3000rpm, obtains supernatant, removes precipitation.Adding low temperature alcohol (22 ℃) to concentration in the supernatant is 21%, stirs 40min in the ice bath, leaves standstill 3h then, low-temperature centrifugation, rotating speed 4000rpm, centrifugal 30min.Supernatant discarded obtains precipitation.Precipitation is used the Tris-HCl damping fluid, and PH8.00 contains trisodium citrate 1.60%, and sodium-chlor 1.60% is dissolved to original volume, and the lysate temperature is 17 ℃.
3. add 40%PEG solution in the alcohol precipitation lysate, to final concentration be 14%.The temperature of adjusting alcohol precipitation lysate and PEG during precipitation is 25 ℃, and the PH of system is controlled at 8.0, and stirring at room is 40 minutes then, leaves standstill 50 minutes, and is centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Take out supernatant, discard precipitation.
4. add 40%PEG solution in the supernatant liquor that the PEG precipitation process obtains for the first time, add 17mlPEG solution in every 100ml supernatant liquor, the PH of system is controlled at 8.0, temperature is controlled at 25 ℃, stirred 40 minutes under the room temperature then, left standstill 60 minutes, carry out the PEG precipitation process second time.Centrifugal, rotating speed 3000rpm, centrifugation time 30 minutes.Supernatant discarded is got precipitation.Precipitation is used the Tris-HCl damping fluid, and PH8.00 is dissolved to original volume.
5. get QAE gel dress post, use the Tris-HCl damping fluid, PH8.00, balance QAE gel chromatography system.With the resolution of precipitate liquid that the second time, the PEG precipitation obtained, last sample, flow velocity are 40ml/h, carry out the QAE anion chromatography; And then use the Tris-HCl damping fluid, and PH8.00, washing QAE anion layer analysis system is with three column volumes; Washing is for the second time washed with the Tris-HCl buffer solution ph 8.0 of sodium chloride-containing, and wherein sodium chloride concentration is 0.14M, washes foreign protein off; Tris-HCl buffer solution ph 8.0 with sodium chloride-containing carries out wash-out again, and wherein sodium chloride concentration is 0.45M, and the albumen that obtains is Fiberonectin, and sodium-chlor is removed in dialysis.
Claims (12)
1. a method for preparing Fiberonectin is characterized in that being undertaken by following step,
1) with Tris-HCl damping fluid dissolving cryoprecipitate,
2) alcohol precipitation,
3) the first step PEG precipitation,
4) the second step PEG precipitation,
5) anion-exchange chromatography.
2. by the described method for preparing Fiberonectin of claim 1, but it is characterized in that described arbitrary step can be carried out separately or wherein 1,2,3,5 step arbitrary combination carry out.
3, by the described method for preparing Fiberonectin of claim 1, it is characterized in that described cryoprecipitate dissolving damping fluid contains trisodium citrate and sodium-chlor, its PH is 7.0-8.5, and cryoprecipitate dissolving ratio is 1: 3-1: 20, solvent temperature is 5-40 ℃, and dissolution time is 1-3 hour.
4, by the described method for preparing Fiberonectin of claim 3, it is characterized in that the trisodium citrate concentration of described cryoprecipitate dissolving damping fluid is 0.50-2.0%, sodium chloride concentration is 0.50-2.0%.
5, by the described method for preparing Fiberonectin of claim 1, it is characterized in that spirituous solution contains Trisodium Citrate and sodium-chlor in the described alcohol precipitation, its alcohol final concentration is 3-30%, spirituous solution temperature 0～-30 ℃, PH is 7.0-8.5.
6, by the described method for preparing Fiberonectin of claim 5, it is characterized in that sodium citrate concentration is 0.5-2.0% in the described spirituous solution, sodium chloride concentration is 0.5-2.0%.
7, by the described method for preparing Fiberonectin of claim 1, it is characterized in that described alcohol precipitation lysate contains Trisodium Citrate and sodium-chlor, the lysate temperature is 0-30 ℃, and PH is 7.0-8.5.
8, by the described method for preparing Fiberonectin of claim 7, it is characterized in that sodium citrate concentration is 0.5-2.0% in the described alcohol precipitation lysate, sodium chloride concentration is 0.5-2.0%.
9, by the described method for preparing Fiberonectin of claim 1, it is characterized in that the PEG concentration in the described the first step PEG precipitation is 4-15%, service temperature is 10-25 ℃, PH is 7.0-8.5.
10, by the described method for preparing Fiberonectin of claim 1, it is characterized in that in the second step PEG precipitation of described step 4, the PEG that adds concentration 40% at the sedimentary supernatant of the first step PEG, add-on is to add 5-25ml in every 100ml supernatant, service temperature is 10-25 ℃, PH7.0-8.5, resolution of precipitate damping fluid are Tris-HCl, PH7.0-8.5.
11, by the described method for preparing Fiberonectin of claim 1, it is characterized in that the system that described anion-exchange chromatography adopts is QAE, wherein level pad, washings and elutriant are Tris-HCl, PH7.0-8.5.
12, by the described method for preparing Fiberonectin of claim 11, it is characterized in that described second time of washings sodium chloride-containing, its concentration is 0.05-0.14M, elutriant sodium chloride-containing, its concentration are 0.16-1.0M.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102295696A (en) * | 2011-08-16 | 2011-12-28 | 山东泰邦生物制品有限公司 | Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation |
| CN101703763B (en) * | 2009-11-05 | 2012-07-11 | 绿十字(中国)生物制品有限公司 | Production method of human fibrinogen |
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2003
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101703763B (en) * | 2009-11-05 | 2012-07-11 | 绿十字(中国)生物制品有限公司 | Production method of human fibrinogen |
| CN102295696A (en) * | 2011-08-16 | 2011-12-28 | 山东泰邦生物制品有限公司 | Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation |
| CN102295696B (en) * | 2011-08-16 | 2013-06-26 | 山东泰邦生物制品有限公司 | Method for preparing coagulation factor VIII, fibrinogen and fibronectin from cryoprecitation |
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