CN100348302C - Method for preparing water-soluble affinity ultrafiltration carrier - Google Patents
Method for preparing water-soluble affinity ultrafiltration carrier Download PDFInfo
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- CN100348302C CN100348302C CNB2004100271917A CN200410027191A CN100348302C CN 100348302 C CN100348302 C CN 100348302C CN B2004100271917 A CNB2004100271917 A CN B2004100271917A CN 200410027191 A CN200410027191 A CN 200410027191A CN 100348302 C CN100348302 C CN 100348302C
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- ovomucin
- glucan
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- trapped fluid
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Abstract
The present invention relates to a method for preparing a water-soluble affinity ultrafiltration carrier. A glucan-ovomucin water-soluble macromolecular carrier is prepared by an epoxy chloropropane activating method to be used for an ultrafiltration process. Because the molecular weight of trypsin approximates to that of chymotrypsin, the trypsin is specifically adsorbed through ovomucin, the chymotrypsin is filtered, the trypsin and the chymotrypsin are effectively separated, and then the adsorbed trypsin is purified through elution; the purification multiple of the obtained trypsin is 93 times, and the recovery rate is more than 80%.
Description
Technical field
The present invention relates to the organic chemical industry field, specifically is a kind of preparation method of water-soluble affinity ultrafiltration carrier.
Background technology
Hyperfiltration technique is mainly used in the separation and purification of large biological molecule at present, has the material of maintenance activity, easily the advantage of industry amplification.But the isolated ultrafiltration isolation technics is difficult to separately the close composition of molecular weight.Adopt affine-hyperfiltration technique at this problem, promptly synthetic a kind of water-soluble macromolecule amount affiliation carrier, special absorption target molecule makes target molecule by membrane retention, filters impurity molecule, and the ideal molecule obtains purifying by wash-out.The present invention adopts the glucan of solubility 2,000,000 molecular weight to activate by epoxychloropropane, and covalently bound ovomucin forms soluble large molecule amount affiliation carrier, is applied to prepare high-purity trypsase.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of water-soluble affinity ultrafiltration carrier, prepare the big molecule affiliation carrier of a kind of water dissolvable, be applied to affine-ultra-filtration process, the close composition of molecular weight is separated, make target molecule obtain purifying.For example because the molecular weight of trypsase and chymotrypsin is close, by the specific absorption trypsase of ovomucin, filter chymotrypsin, trypsase and chymotrypsin are effectively separated, the trypsase of absorption is able to purifying by wash-out, obtain the tryptic purification multiple and reach 93 times, reclaim more than 80%.
Method of the present invention is to adopt the method for epoxychloropropane activation, prepares the water-soluble affinity ultrafiltration macromolecular carrier of a kind of glucan-ovomucin.
The preparation method of water-soluble affinity ultrafiltration carrier of the present invention may further comprise the steps particularly:
The first step, add glucan and NaOH in batching cup, make glucan concentration reach 3.5～4.5% weight ratios, naoh concentration reaches 0.4～0.5mol/L, is stirred to dissolving fully, handles 30～40 minutes; Described dextran molecule amount 150-300 ten thousand, the best is 2,000,000;
Second step, adding 4～5% volume epoxychloropropane;
The 3rd step, second feed liquid that obtain of step is put into 55～60 ℃ of constant temperature shaking tables, shaking speed 100～110r/min, lucifuge reaction 1.8～2 hours;
The 4th step, the feed liquid that the 3rd step was obtained move in the milipore filter separator, and retaining molecular weight is 100,000, and remaining epoxychloropropane in the solution is removed in ultrafiltration, the little molecule of NaOH; Earlier be washed till pH7.8～8.2 with distilled water, use 0.1M again, the pH9.5 sodium carbonate buffer is washed, and is long-pending 1.2 ± 0.1 times of primary liquid in the batching cup to the trapped fluid volume;
The 5th step, ovomucin is dissolved in the 4th trapped fluid that obtain of step, making ovomucin concentration is 6 ± 0.1g/L; Move to 35～40 ℃ of constant temperature shaking tables, shaking speed 100～110r/min, lucifuge was handled 23～24 hours;
The 6th step, solution after the processing moves in the milipore filter separator, retaining molecular weight is 140,000, remove not by the ovomucin of coupling, earlier with the distillation washing, use 0.1M formic acid-0.5M potassium chloride then, pH2.5 formic acid is washed, be washed till pH6.2～6.5 with distilled water again, use 0.5M potassium chloride-0.05M calcium chloride-0.1M at last, the pH7.8Tris-HCl buffer solution is washed, be dissolved in the Tris-HCl buffer solution to glucan, to the trapped fluid volume for long-pending 1.2 ± 0.1 times of primary liquid in the batching cup, stirred the water-soluble affinity ultrafiltration carrier of acquisition glucan-ovomucin 1～1.5 hour.
The present invention compared with prior art, have following advantage and beneficial effect: solved the low difficult problem of component purification efficiency close during isolated ultrafiltration separates to molecular weight, utilize ovomucin to tryptic special affine suction-operated, ovomucin-glucan affinity ultrafiltration the carrier that obtains carries out purifying to thick trypsase, obtains good purification effect.Synthetic water-soluble affiliation carrier has many advantages: affiliation carrier and target protein reaction speed in homogeneous system is fast, and it is short to reach absorption or wash-out balance time, and adsorption capacity is big.
The inventor is to the present invention---and the preparation of glucan-ovomucin affinity ultrafiltration carrier has many successful embodiment through long-term creative research and experiment, lifts several specific embodiments below.In implementation process, the retaining molecular weight of ultrafiltration apparatus is 100,000 and 140,000, and the ultrafilter specification is determined by process lot size.
The specific embodiment
Embodiment 1:
Glucan 1 gram of the molecular weight 2,000,000 of buying Sigma company is put in the beaker, added water 15mL, be uniformly dispersed, glucan is dissolved fully, add 0.4 gram NaOH then, add water 10mL, use magnetic stirrer 35 minutes with magnetic stirring apparatus; Add epoxychloropropane 1mL, magnetic agitation is uniformly dispersed rapidly; Above-mentioned stirring liquid is moved in 57 ± 2 ℃ of constant temperature shaking tables shaking speed 100～110r/min, lucifuge reaction 1.8 hours rapidly; Above-mentioned reactant liquor is moved in the ultrafiltration cup, and retaining molecular weight is 100,000, removes remaining NaOH, the little molecule of epoxychloropropane.Earlier be washed till pH8.0 ± 0.2 with distilled water, and to trapped fluid be 40 ± 5mL, and then use 0.1M, it is 30 ± 2.5mL that pH9.5 sodium carbonate buffer 100 ± 5mL is washed till trapped fluid, entire process process need lucifuge; Ovomucin 180 ± 2mg is dissolved in the above-mentioned trapped fluid, and magnetic agitation is even, move to 37 ± 2 ℃ the constant temperature shaking table (in 100～110r/min), lucifuge 23 hours; Solution ultra-filtration and separation after the processing, retaining molecular weight is 140,000, remove not by the ovomucin of coupling, earlier with the distillation washing of 140mL, to trapped fluid be 40 ± 5mL; Use 0.1M formic acid-0.5M potassium chloride then, pH2.5 formic acid 80mL washes, to trapped fluid be 35 ± 5mL, being washed till trapped fluid pH with distilled water again is 6.2～6.5, and the trapped fluid volume is 30 ± 5mL, wash with 0.5M potassium chloride-0.05M calcium chloride-0.1M, pH7.8Tris-HCl buffer solution 120mL at last, to the trapped fluid volume be 30 ± 2.5mL, stop ultrafiltration, pour out trapped fluid, magnetic agitation 1.5 hours obtains the water-soluble affinity ultrafiltration carrier of glucan-ovomucin, preserves to 3.5～4.5 ℃ of refrigerators.
By the specific absorption trypsase of the ovomucin of the water-soluble affinity ultrafiltration carrier of glucan-ovomucin, filter chymotrypsin, trypsase and chymotrypsin are effectively separated, the trypsase of absorption is able to purifying by wash-out, obtain the tryptic purification multiple and reach 91 times, reclaim 85%.
Embodiment 2:
Glucan weighing 3 grams of the molecular weight 2,000,000 of buying Pharmacia company are put in the beaker, added water 50mL, be uniformly dispersed with magnetic stirring apparatus, and glucan is dissolved fully, add NaOH 1.3 grams then, water 15mL uses magnetic stirrer 30 minutes; Add epoxychloropropane 3 ± 0.2mL then, magnetic agitation is uniformly dispersed rapidly; Above-mentioned stirring liquid is moved in the constant temperature shaking table 58 ± 2 ℃ of temperature, shaking speed 100～110r/min, lucifuge reaction 2 hours rapidly; Above-mentioned reactant liquor is moved in the ultrafiltration cup, retaining molecular weight is 100,000, remove remaining NaOH, the little molecule of epoxychloropropane, earlier be washed till pH8.0 ± 0.2 with distilled water, and to trapped fluid be 80 ± 5mL, and then use 0.1M, it is 80 ± 5mL that pH9.5 sodium carbonate buffer 300 ± 5mL is washed till the trapped fluid volume, and the entire process process needs lucifuge; Ovomucin 490 ± 2mg is dissolved in the above-mentioned trapped fluid, and magnetic agitation is even, move to 37 ± 2 ℃ the constant temperature shaking table (in 100～110r/min), lucifuge 24 hours; Solution ultra-filtration and separation after the processing, retaining molecular weight is 140,000, remove not by the ovomucin of coupling, earlier with the distillation washing of 400mL, to trapped fluid be 90 ± 5mL.Use 0.1M formic acid-0.5M potassium chloride then, pH2.5 formic acid 240mL washes, to trapped fluid be 90 ± 5mL, being washed till trapped fluid pH with distilled water again is 6.2～6.5, and the trapped fluid volume is 90 ± 5mL, wash with 0.5M potassium chloride-0.05M calcium chloride-0.1M, pH7.8Tris-HCl buffer solution 350mL at last, to the trapped fluid volume be 80 ± 5mL, stop ultrafiltration, pour out trapped fluid, magnetic agitation 1.5 hours obtains the water-soluble affinity ultrafiltration carrier of glucan-ovomucin, puts to 4 ℃ of refrigerators and preserves.
By the specific absorption trypsase of the ovomucin of the water-soluble affinity ultrafiltration carrier of glucan-ovomucin, filter chymotrypsin, trypsase and chymotrypsin are effectively separated, the trypsase of absorption is able to purifying by wash-out, obtain the tryptic purification multiple and reach 90 times, reclaim 90%.
Embodiment 3:
Glucan 1.5 grams of the molecular weight 2,000,000 of buying Pharmacia company are put in the beaker, added water 25mL, be uniformly dispersed with magnetic stirring apparatus, and glucan is dissolved fully, add NaOH 0.6 gram then, add entry 8mL again, use magnetic stirrer 40 minutes; Add epoxychloropropane 1.5mL then, magnetic agitation is uniformly dispersed rapidly; Above-mentioned stirring liquid is moved in the constant temperature shaking table 58 ± 2 ℃ of temperature, shaking speed 100～110r/min, lucifuge reaction 1.9 hours rapidly; Above-mentioned reactant liquor is moved in the ultrafiltration cup, retaining molecular weight is 100,000, remove remaining NaOH, the little molecule of epoxychloropropane, earlier be washed till pH7.8 ± 0.2 with distilled water, and to trapped fluid be 40 ± 5mL, and then use 0.1M, it is 40 ± 2mL that pH9.5 sodium carbonate buffer 150 ± 5mL is washed till the trapped fluid volume, and the entire process process needs lucifuge; Ovomucin 238 ± 2mg is dissolved in the above-mentioned trapped fluid, and magnetic agitation is even, move to 37 ± 2 ℃ the constant temperature shaking table (in 100～110r/min), lucifuge 23～24 hours; Solution ultra-filtration and separation after the processing, retaining molecular weight is 140,000, remove not by the ovomucin of coupling, earlier with the distillation washing of 180mL, to trapped fluid be 40 ± 5mL.Use 0.1M formic acid-0.5M potassium chloride then, pH2.5 formic acid 100mL washes, to trapped fluid be 45 ± 5mL, being washed till trapped fluid pH with distilled water again is 6.2～6.5, and the trapped fluid volume is 40 ± 5mL, wash with 0.5M potassium chloride-0.05M calcium chloride-0.1M, pH7.8Tris-HCl buffer solution 180mL at last, to the trapped fluid volume be 40 ± 2mL, stop ultrafiltration, pour out trapped fluid, magnetic agitation 1.5 hours gets the water-soluble affinity ultrafiltration carrier of glucan-ovomucin, puts to 3.5～4 ℃ of refrigerators and preserves.
By the specific absorption trypsase of the ovomucin of the water-soluble affinity ultrafiltration carrier of glucan-ovomucin, filter chymotrypsin, trypsase and chymotrypsin are effectively separated, the trypsase of absorption is able to purifying by wash-out, obtain the tryptic purification multiple and reach 93 times, reclaim 82%.
Claims (1)
1, a kind of preparation method of water-soluble affinity ultrafiltration carrier is characterized in that may further comprise the steps:
The first step, add glucan and NaOH in batching cup, make glucan concentration reach 3.5～4.5% weight, naoh concentration reaches 0.4～0.5mol/L, is stirred to dissolving fully, handles 30～40 minutes; Described dextran molecule amount 150-300 ten thousand; The epoxychloropropane that adds 4～5% volumes;
Second step, the feed liquid that the first step is obtained are put into 55～60 ℃ of constant temperature shaking tables, shaking speed 100～110r/min, lucifuge reaction 1.8～2 hours;
The 3rd step, the feed liquid that second step was obtained move in the milipore filter separator, and retaining molecular weight is 100,000, and remaining epoxychloropropane in the solution is removed in ultrafiltration, the little molecule of NaOH; Earlier be washed till pH7.8～8.2 with distilled water, use 0.1M again, the pH9.5 sodium carbonate buffer is washed, and is long-pending 1.2 ± 0.1 times of primary liquid in the batching cup to the trapped fluid volume;
The 4th step, ovomucin is dissolved in the 3rd trapped fluid that obtain of step, making ovomucin concentration is 6 ± 0.1g/L; Move to 35～40 ℃ of constant temperature shaking tables, shaking speed 100～110r/min, lucifuge was handled 23～24 hours;
The 5th step, solution after the processing moves in the milipore filter separator, retaining molecular weight is 140,000, remove not by the ovomucin of coupling, earlier with the distillation washing, use 0.1M formic acid-0.5M potassium chloride then, pH2.5 formic acid is washed, be washed till pH6.2～6.5 with distilled water again, use 0.5M potassium chloride-0.05M calcium chloride-0.1M at last, the pH7.8Tris-HCl buffer solution is washed, be dissolved in the Tris-HCl buffer solution to glucan, to the trapped fluid volume for long-pending 1.2 ± 0.1 times of primary liquid in the batching cup, stirred the water-soluble affinity ultrafiltration carrier of acquisition glucan-ovomucin 1～1.5 hour.
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| CNB2004100271917A CN100348302C (en) | 2004-05-14 | 2004-05-14 | Method for preparing water-soluble affinity ultrafiltration carrier |
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| CNB2004100271917A CN100348302C (en) | 2004-05-14 | 2004-05-14 | Method for preparing water-soluble affinity ultrafiltration carrier |
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| CN101633687B (en) * | 2009-08-28 | 2012-06-20 | 华南理工大学 | Preparation method and application of affinity-membrane filtration carrier for separating gene recombinant protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1199643A (en) * | 1998-06-08 | 1998-11-25 | 南开大学 | Globular cellulose DNA immunoadsorbent |
| CN1271621A (en) * | 1999-04-26 | 2000-11-01 | 中国科学院大连化学物理研究所 | Process for synthesizing protein immunoadsorbent medium for removing pathogenic antibody and its compounds from plasm |
| CN1413760A (en) * | 2002-09-17 | 2003-04-30 | 天津大学 | Preparation method of nylon-shell glycan compound film for affinity microfilter |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1199643A (en) * | 1998-06-08 | 1998-11-25 | 南开大学 | Globular cellulose DNA immunoadsorbent |
| CN1271621A (en) * | 1999-04-26 | 2000-11-01 | 中国科学院大连化学物理研究所 | Process for synthesizing protein immunoadsorbent medium for removing pathogenic antibody and its compounds from plasm |
| CN1413760A (en) * | 2002-09-17 | 2003-04-30 | 天津大学 | Preparation method of nylon-shell glycan compound film for affinity microfilter |
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