CN1958600A - Method for enriching and purifying saccharide binding protein - Google Patents

Method for enriching and purifying saccharide binding protein Download PDF

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Publication number
CN1958600A
CN1958600A CN 200510096271 CN200510096271A CN1958600A CN 1958600 A CN1958600 A CN 1958600A CN 200510096271 CN200510096271 CN 200510096271 CN 200510096271 A CN200510096271 A CN 200510096271A CN 1958600 A CN1958600 A CN 1958600A
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binding protein
carbohydrate
micro
coupling agent
sugar
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CN1958600B (en
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李铮
陈超
杨刚龙
崔亚丽
惠文利
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Shaanxi Baimei Gene Co., Ltd.
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XIDA BEIMEI GENE CO Ltd SHANXI
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Abstract

This invention discloses a method for enriching and purifying glycoprotein. The coupling of sugar with magnetic micro- and nano-particles comprises: activating the coupling agent and coupling sugar onto amino-derived magnetic micro- and nano-particles or ethylene oxide derived magnetic micro- and nano-particles. The separation, enriching and purification of glycoprotein comprises: adding pretreated glycoprotein into sugar-coupled magnetic micro- and nano-particles for connection, washing and eluting to obtain enriched and purified glycoprotein. This invention solves the problems of complex process, inconvenient usage and low recovery rate faced by background technology. The method has such advantages as simple process, high speed, stable glycoprotein bioactivity, and high efficiency in separating, enriching and purifying glycoprotein.

Description

The method of enrichment and purifying saccharide binding protein
Technical field
The present invention relates to a kind of hemiacetal ROH by the reducing sugar reduction end, with the form covalent coupling of glycosidic link to hydroxyl (OH) on the micro-nano magnetic particle of derivatize, utilize thisly to be fixed on sugar on the micro-nano magnetic particle specifically in conjunction with carbohydrate-binding protein, to carbohydrate-binding protein separate, the method for enrichment and purifying.
Background technology
Carbohydrate-binding protein is a proteinoid, the non-covalent combination that can shift with carbohydrate, and its structure contains the carbohydrate differential threshold, and does not contain glycosyl.Carbohydrate-binding protein has important biological function, comprises intercellular communication and identification, the interaction of cell and pathogenic micro-organism, and the transportation of intracellular protein, interaction between protein, albumen combines or the like with the specificity of acceptor.Along with the researchdevelopment of genomics and proteomics, people recognize the importance of carbohydrate-binding protein gradually.But the research for carbohydrate-binding protein relatively lags behind at present.Major cause be lack high efficiencyly can separate carbohydrate-binding protein, the technology and the means of enrichment and purifying.
At present, carbohydrate-binding protein separation and concentration technology, the method for useful affinity column separation and concentration carbohydrate-binding protein, but affinity column is lower to the rate of recovery of carbohydrate-binding protein, and the method complexity, use inconvenience.
Summary of the invention
The object of the present invention is to provide a kind of method based on micro-nano magnetic technique separation, enrichment and purifying saccharide binding protein, it has solved method complexity in the background technology, uses inconvenience, the technical problem that the rate of recovery is lower.
Technical solution of the present invention is:
The method of a kind of enrichment and purifying saccharide binding protein, its special character is: the performing step of this method comprises
1) sugar and micro-nano magnetic particle coupling
(1) activation of coupling agent:
1. get coupling agent 4-hydroxy phenyl valeric acid, N-hydroxy-succinamide and dicyclohexylcarbodiimide by the ratio of 1: 5: 5 amount of substance, be dissolved in the organic solvent respectively;
2. earlier 4-hydroxy phenyl valeric acid is mixed with N-hydroxy-succinamide; Adding dicyclohexylcarbodiimide again mixes; Molten with organic solvent to making three's concentration be respectively 20mM, 100mM and 100mM;
3. in 35～40 ℃ Celsius, incubation 8～16 hours;
Described coupling agent is the coupling agent that contains a carboxyl and a hydroxyl;
(2) pre-treatment of the micro-nano magnetic particle of amino group derivatize: the micro-nano magnetic particle of getting the amino group derivatize adds the supernatant of activatory 4-hydroxy phenyl valeric acid solution in centrifuge tube, the ratio of material is 1: 10; In 35～40 ℃ Celsius, incubation 4～6 hours;
(3) clean: magnetic resolution, abandon supernatant; Clean 3 times with ethanol, use buffer solution for cleaning again 3 times;
(4) coupling: add the sugar soln of 2ml with the 100mM of coupling buffer preparation, in 35～40 ℃ Celsius, incubation 8～16 hours, sugar are connected on the micro-nano magnetic particle of amino group derivatize, must be connected with the micro-nano magnetic particle of sugar;
(5) clean: magnetic resolution, abandon supernatant, with buffer solution for cleaning 3 times;
(6) preserve: add and preserve damping fluid, in 2～6 ℃ of preservations;
2) to carbohydrate-binding protein separate, enrichment and purifying
(1) pre-treatment of carbohydrate-binding protein: the carbohydrate-binding protein of water preparation 10mg/ml is diluted to 0.5mg/ml with binding buffer liquid;
(2) association reaction: get be connected with sugar micro-nano magnetic particle in No. 1 centrifuge tube, add the carbohydrate-binding protein of 0.5mg/ml, in 35～40 ℃ of reactions Celsius 1～3 hour, magnetic resolution, abandon supernatant liquor;
(3) clean: in No. 1 centrifuge tube, add cleaning buffer solution 1 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing once;
(4) clean: in No. 1 centrifuge tube, add cleaning buffer solution 2 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing secondary again;
(5) wash-out: in No. 1 centrifuge tube, add cleaning buffer solution 3, cleaned 20～30 minutes, magnetic resolution, get supernatant liquor in No. 2 centrifuge tubes; Repeated washing is once got supernatant liquor and is incorporated in No. 2 centrifuge tubes again, obtains the carbohydrate-binding protein of enriching and purifying;
(6) preserve: in No. 1 centrifuge tube, add the preservation damping fluid, in 2～6 ℃ of preservations.
Above-mentioned organic solvent can adopt ethyl acetate or dimethyl formamide; Described coupling agent can adopt 4-hydroxy phenyl valeric acid or 16-hydroxyl ten hexacarboxylic acids.
Above-mentioned damping fluid and coupling buffer can adopt sodium acetate, yellow soda ash, phosphoric acid salt or Tris-HCl damping fluid etc.;
Described binding buffer liquid can adopt 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH7.0 damping fluid etc.;
Described cleaning buffer solution 1,2 all can adopt 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH6.5 damping fluid etc.;
Described cleaning buffer solution 3 can adopt 1mM CaCl 2, 1mM MnCl 2, 1mM KCl, 100mM Alpha-Methyl seminose, 10mM Tris-HCl pH7.0 damping fluid etc.;
Described preservation damping fluid is 1mM CaCl 2, 1mM MnCl 2, 0.02%NaN 3, 10mM Tris-HCl pH6.0 damping fluid etc.
In the priming reaction of above-mentioned coupling agent, be advisable with inflated with nitrogen protection gas during incubation.
In the priming reaction of above-mentioned coupling agent, heated culture temperature is advisable with 37 ℃ Celsius, and the incubation time was advisable with 12 hours; In the pre-treatment of the micro-nano magnetic particle of described amino group derivatize, heated culture temperature is advisable with 37 ℃ Celsius, and the incubation time was advisable with 5 hours; In the described association reaction, heated culture temperature is advisable with 37 ℃ Celsius, and the incubation time was advisable with 12 hours; The temperature of reaction that is connected with the micro-nano magnetic particle adding carbohydrate-binding protein of sugar in the described association reaction is advisable with 37 ℃, and the reaction times was advisable with 2 hours; Described storage temperature is advisable with 4 ℃ Celsius.
In the pre-treatment of above-mentioned carbohydrate-binding protein, association reaction, twice cleaning and the elution of reactive, the detection step of joint efficiency, the rate of recovery comprises
1) surveys the OD value
(1) survey carbohydrate-binding protein pre-treatment in, carbohydrate-binding protein is OD with the OD value that binding buffer liquid is diluted to 0.5mg/ml 1
(1) survey in the association reaction, after the micro-nano magnetic particle that is connected with sugar added the carbohydrate-binding protein reaction, the OD value of supernatant liquor that magnetic resolution is abandoned was OD 2
(1) in twice cleaning reaction of survey, the OD value of supernatant liquor that magnetic resolution is abandoned is respectively OD 3, OD 4
(1) survey in the elution of reactive, the OD value of the carbohydrate-binding protein of gained enriching and purifying is OD 5
2) calculate
Joint efficiency %=(OD 1-OD 2-OD 3-OD 4)/OD 1
Rate of recovery %=OD 5/ OD 1
The method of a kind of enrichment and purifying saccharide binding protein, its special character is: the performing step of this method comprises
1) activation of sugar and micro-nano magnetic particle coupling (1) coupling agent:
1. coupling agent is dissolved in the organic solvent, is mixed with the coupling agent solution of 20～50mol/L;
2. add in the coupling agent solution by the ratio of 1: 10 amount of substance micro-nano magnetic particle the ethylene oxide group derivatize;
3. hatched under the room temperature 2～4 hours;
Described coupling agent is the coupling agent that contains an amino and a hydroxyl;
(2) clean: magnetic resolution, abandon supernatant; Clean 3 times with ethanol, use the buffer solution for cleaning 3 times of pH5.6 again;
(3) coupling: add the sugar soln of 2ml with the 100mM of pH5.6 coupling buffer preparation, in 35～40 ℃ Celsius, incubation 8～16 hours, then sugar is connected on the micro-nano magnetic particle of ethylene oxide group derivatize, must be connected with the micro-nano magnetic particle of sugar;
(4) clean: magnetic resolution, abandon supernatant; With the buffer solution for cleaning of pH5.6 3 times;
(5) preserve: add and preserve damping fluid, in 2～6 ℃ of preservations;
2) to carbohydrate-binding protein separate, enrichment and purifying
(1) pre-treatment of carbohydrate-binding protein: the carbohydrate-binding protein of water preparation 10mg/ml is diluted to 0.5mg/ml with binding buffer liquid;
(2) association reaction: get be connected with sugar micro-nano magnetic particle in No. 1 centrifuge tube, add the carbohydrate-binding protein of 0.5mg/ml, in 35～40 ℃ of reactions Celsius 1～3 hour, magnetic resolution, abandon supernatant liquor;
(3) clean: in No. 1 centrifuge tube, add cleaning buffer solution 1 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing once;
(4) clean: in No. 1 centrifuge tube, add cleaning buffer solution 2 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing secondary again;
(5) wash-out: in No. 1 centrifuge tube, add cleaning buffer solution 3, cleaned 20～30 minutes, magnetic resolution, get supernatant liquor in No. 2 centrifuge tubes; Repeated washing is once got supernatant liquor and is incorporated in No. 2 centrifuge tubes again, obtains the carbohydrate-binding protein of enriching and purifying;
(6) preserve: in No. 1 centrifuge tube, add the preservation damping fluid, in 2～6 ℃ of preservations.
Above-mentioned organic solvent can adopt dimethyl formamide or ethyl acetate etc.; Described coupling agent can adopt 4-hydroxybenzamide or 4 hydroxybutyric acid hydrazine etc.
Above-mentioned damping fluid and coupling buffer can adopt sodium acetate, yellow soda ash, phosphoric acid salt or Tris-HCl etc.;
Described binding buffer liquid is 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH7.0 damping fluid etc.;
Described cleaning buffer solution 1,2 is 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH6.5 damping fluid etc.;
Described cleaning buffer solution 3 is 1mM CaCl 2, 1mM MnCl 2, 1mM KCl, 100mM Alpha-Methyl seminose, 10mM Tris-HCl pH7.0 damping fluid etc.;
Described preservation damping fluid is 1mM CaCl 2, 1mM MnCl 2, 0.02%NaN 3, 10mM Tris-HCl pH6.0 damping fluid etc.
In the priming reaction of above-mentioned coupling agent, the micro-nano magnetic particle of ethylene oxide group derivatize adds the incubation time of coupling agent and was advisable with 3 hours; Heated culture temperature in the described association reaction is advisable with 37 ℃ Celsius, and the incubation time was advisable with 12 hours; The temperature of reaction that is connected with the micro-nano magnetic particle adding carbohydrate-binding protein of sugar in the described association reaction is advisable with 37 ℃, and the reaction times was advisable with 2 hours; Described storage temperature is advisable with 4 ℃ Celsius.
The present invention has the following advantages:
1. utilizing the reduction end of sugar is hemiacetal ROH, with the form covalent coupling of glycosidic link to hydroxyl (OH) on the micro-nano magnetic particle of derivatize, like this mode of connection of immobilization sugar simply, quick, and cost is lower.
2. the biological activity of carbohydrate-binding protein is stable, and the prolonged preservation biological activity is constant at ambient temperature.
To carbohydrate-binding protein separate, the efficient height of enrichment and purifying.
Description of drawings
Fig. 1,2 is respectively principle schematic of the present invention.
Embodiment
The present invention has adopted two kinds of methods that micro-nano magnetic particle is carried out hydroxyl (OH) derivatize, with reducing sugar is the hemiacetal ROH of monose, oligosaccharides or polysaccharide reduction end, with the form covalent coupling of glycosidic link to hydroxyl (OH) on the micro-nano magnetic particle of derivatize, utilize thisly to be fixed on sugar on the micro-nano magnetic particle specifically in conjunction with carbohydrate-binding protein, to carbohydrate-binding protein separate, enrichment and purifying.
Referring to Fig. 1.Embodiment 1 be the micro-nano magnetic particle that utilizes the amino group derivatize to carbohydrate-binding protein separate, enrichment and purifying.Because one deck amino group is contained on the surface of the micro-nano magnetic particle of amino group derivatize, can with coupling agent through N-hydroxy-succinamide (NHS) and dicyclohexylcarbodiimide (DCC) activatory acidic group covalent coupling, the hydroxyl of the coupling agent the other end (can be that reduction end generation aldol reaction forms glycosidic link with 1 aldehyde radical of sugar OH), sugar is connected on the micro-nano magnetic particle in the mode of covalency, with carbohydrate-binding protein is separated, enrichment and purifying.Coupling agent is the coupling agent that contains a carboxyl and a hydroxyl, as: 4-hydroxy phenyl valeric acid, 16-hydroxyl ten hexacarboxylic acids etc.
The performing step of embodiment 1 is as follows:
1) activation of sugar and micro-nano magnetic particle coupling (1) coupling agent:
1. get coupling agent 4-hydroxy phenyl valeric acid, N-hydroxy-succinamide and dicyclohexylcarbodiimide by the ratio of 1: 5: 5 amount of substance, be dissolved in respectively in the organic solvent, organic solvent can adopt ethyl acetate, dimethyl formamide (DMF) etc.
2. earlier 4-hydroxy phenyl valeric acid is mixed with N-hydroxy-succinamide, all dissolving; Adding dicyclohexylcarbodiimide again mixes; Fixed molten with ethyl acetate to 25ml, make three's concentration be respectively 20mM, 100mM and 100mM.
3. inflated with nitrogen protection, in 37 ℃ Celsius, incubation 12 hours, the adularescent precipitation generates.
(2) pre-treatment of the micro-nano magnetic particle of amino group derivatize: the micro-nano magnetic particle of amino group derivatize of getting 15.58mg/ml adds the supernatant of the ethyl acetate solution of activatory 4-hydroxy phenyl valeric acid, ratio 1: 10 in the 5ml centrifuge tube; In 37 ℃ Celsius, incubation reaction 5 hours.
(3) clean: magnetic resolution, abandon supernatant; Clean 3 times with ethanol, the sodium acetate buffer with pH5.6 cleans 3 times again.
(4) coupling: add the D-mannose solution of 2ml with the 100mM of the sodium acetate buffer preparation of pH5.6, in 37 ℃ Celsius, incubation 12 hours, then sugar is connected on the micro-nano magnetic particle of amino group derivatize, must be connected with the micro-nano magnetic particle of sugar.
(5) clean: magnetic resolution, abandon supernatant.Sodium acetate cleaning buffer solution with pH5.6 cleans 3 times.
(6) preserve: add 1mM CaCl 2, 1mM MnCl 2, 0.02%NaN 3, 10mM Tris-HCl pH6.0 preserves damping fluid, in 4 ℃ of preservations.
2) to carbohydrate-binding protein separate, enrichment and purifying
(1) pre-treatment of carbohydrate-binding protein:, use 1mM CaCl with the carbohydrate-binding protein of ultrapure water preparation 10mg/ml 2, 1mM MnCl 2, 10mM Tris-HCl pH7.0 binding buffer liquid be diluted to 0.5mg/ml, survey its OD value for OD 1The water of preparation carbohydrate-binding protein also can use high purity water, ionized water, pure water etc.
(2) association reaction: get 0.5ml and be connected with sugared micro-nano magnetic particle in No. 1 centrifuge tube of 1.5ml, add the carbohydrate-binding protein of 300 μ l 0.5mg/ml, as ConA, in 37 ℃ of reactions Celsius 1～3 hour.Magnetic resolution, get supernatant in No. 2 centrifuge tubes, survey its OD value and be OD 2
(3) clean: the 1mM CaCl that in No. 1 centrifuge tube, adds 0.5ml 2, 1mM MnCl 2, 10mM Tris-HCl, pH6.5 cleaning buffer solution 1, on shaking table, shook 2 minutes, on magnetic separator, separated 3 minutes, get supernatant liquor and place centrifuge tube No. 3.Repeated washing is once surveyed its OD value and is OD 3
(4) clean: the 1mM CaCl that in No. 1 centrifuge tube, adds 500 μ l 2, 1mM MnCl 2, 1mM KCl, 10mMTris-HCl, pH6.5 cleaning buffer solution 2, cleaned 2 minutes, on magnetic separator, separated 3 minutes, get supernatant and place centrifuge tube No. 4.The repeated washing secondary is surveyed its OD value and is OD again 4Cleaning buffer solution 1,2 is used to remove the albumen of non-specific binding.
(5) wash-out: in No. 1 centrifuge tube, add 1mM CaCl 2, 1mM MnCl 2, 1mM KCl, 100mM Alpha-Methyl seminose (a-methyl mannose), 10mM Tris-HCl, pH7.0 cleaning buffer solution 3, cleaned 20～30 minutes.On magnetic separator, separated 3 minutes, get supernatant and place centrifuge tube No. 5.Again repeated washing once, supernatant is incorporated in No. 5 centrifuge tubes, is the carbohydrate-binding protein of enriching and purifying.Survey its OD value and be OD 5
(6) detection of efficient:
Joint efficiency %=(OD 1-OD 2-OD 3-OD 4)/OD 1
Rate of recovery %=OD 5/ OD 1
(7) preserve: in No. 1 centrifuge tube, add 1mM CaCl 2, 1mM MnCl 2, 0.02%NaN 3, 10mM Tris-HClpH 6.0 the preservation damping fluid, in 4 ℃ of preservations.
This micro-nano magnetic particle that is connected to sugar repeatedly is reusable, and the number of times of enriching and purifying glycoprotein is at least 10 times.
Carbohydrate-binding protein with SDS-PAGE electrophoresis detection enriching and purifying.
The Alpha-Methyl seminose is arranged in the washing lotion cleaning buffer solution 3, and its specific combination ability with ConA is stronger than D-seminose and ConA specific combination ability.At this competitive combination has taken place, and ConA has been washed.Specifically be the dissociating buffer 1 that in No. 5 centrifuge tubes, adds the 10mM Tris-HCl pH2.0 of 1ml, on shaking table, shook 30 minutes in 25 ℃ Celsius that all solution are packed in the dialysis tubing with 1L water behind 4 ℃ of 2h that dialysed Celsius, change water and repeat to dialyse three times.
Referring to Fig. 2.Embodiment 2 are micro-nano magnetic particles of utilizing the ethylene oxide group derivatize to carbohydrate-binding protein separate, enrichment and purifying.Because one deck ethylene oxide group is contained on the micro-nano magnetic particle surface of ethylene oxide group derivatize, can with coupling agent the amino covalence coupling; The hydroxyl of the coupling agent the other end (can be that reduction end generation aldol reaction forms glycosidic link with 1 aldehyde radical of sugar OH), sugar is connected on the micro-nano magnetic particle in the mode of covalency.With carbohydrate-binding protein is separated, enrichment and purifying.Coupling agent is the coupling agent that contains an amino and a hydroxyl, as 4-hydroxybenzamide, 4 hydroxybutyric acid hydrazine etc.
The performing step of embodiment 2 is as follows:
1) activation of coupling (1) coupling agent of sugar and micro-nano magnetic particle:
1. coupling agent 4-hydroxybenzamide or 4 hydroxybutyric acid hydrazine are dissolved in the organic solvent, are mixed with the coupling agent solution of 20～50mol/L.Organic solvent can adopt dimethyl formamide (DMF) or ethyl acetate etc.
2. add in the coupling agent solution by the ratio of 1: 10 amount of substance micro-nano magnetic particle the ethylene oxide group derivatize.
3. hatched under the room temperature 3 hours.
(2) clean: magnetic resolution, abandon supernatant.Clean 3 times with ethanol, the sodium acetate cleaning buffer solution with pH5.6 cleans 3 times again.
(3) coupling: add the D-mannose solution of 2ml with the 100mM of the sodium acetate buffer preparation of pH5.6, in 37 ℃ Celsius, incubation 12 hours, then sugar is connected on the micro-nano magnetic particle of ethylene oxide group derivatize, must be connected with the micro-nano magnetic particle of sugar.
(4) clean: magnetic resolution, abandon supernatant.Sodium acetate cleaning buffer solution with pH5.6 cleans 3 times.
(5) preserve: add 1mM CaCl 2, 1mM MnCl 2, 0.02%NaN 3, 10mM Tris-HCl pH6.0 the preservation damping fluid, in 4 ℃ of preservations.
2) to carbohydrate-binding protein separate, enrichment and purifying
With embodiment 1.

Claims (10)

1. the method for enrichment and purifying saccharide binding protein, it is characterized in that: the performing step of this method comprises
1) activation of sugar and micro-nano magnetic particle coupling (1) coupling agent:
1. get coupling agent 4-hydroxy phenyl valeric acid, N-hydroxy-succinamide and dicyclohexylcarbodiimide by the ratio of 1: 5: 5 amount of substance, be dissolved in the organic solvent respectively;
2. earlier 4-hydroxy phenyl valeric acid is mixed with N-hydroxy-succinamide; Adding dicyclohexylcarbodiimide again mixes; Molten with organic solvent to making three's concentration be respectively 20mM, 100mM and 100mM;
3. in 35～40 ℃ Celsius, incubation 8～16 hours;
Described coupling agent is the coupling agent that contains a carboxyl and a hydroxyl;
(2) pre-treatment of the micro-nano magnetic particle of amino group derivatize: the micro-nano magnetic particle of getting the amino group derivatize adds the supernatant of activatory 4-hydroxy phenyl valeric acid solution in centrifuge tube, the ratio of material is 1: 10; In 35～40 ℃ Celsius, incubation 4～6 hours;
(3) clean: magnetic resolution, abandon supernatant; Clean 3 times with ethanol, use buffer solution for cleaning again 3 times;
(4) coupling: add the sugar soln of 2ml with the 100mM of coupling buffer preparation, in 35～40 ℃ Celsius, incubation 8～16 hours, sugar are connected on the micro-nano magnetic particle of amino group derivatize, must be connected with the micro-nano magnetic particle of sugar;
(5) clean: magnetic resolution, abandon supernatant, with buffer solution for cleaning 3 times;
(6) preserve: add and preserve damping fluid, in 2～6 ℃ of preservations;
2) to carbohydrate-binding protein separate, enrichment and purifying
(1) pre-treatment of carbohydrate-binding protein: the carbohydrate-binding protein of water preparation 10mg/ml is diluted to 0.5mg/ml with binding buffer liquid;
(2) association reaction: get be connected with sugar micro-nano magnetic particle in No. 1 centrifuge tube, add the carbohydrate-binding protein of 0.5mg/ml, in 35～40 ℃ of reactions Celsius 1～3 hour, magnetic resolution, abandon supernatant liquor;
(3) clean: in No. 1 centrifuge tube, add cleaning buffer solution 1 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing once;
(4) clean: in No. 1 centrifuge tube, add cleaning buffer solution 2 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing secondary again;
(5) wash-out: in No. 1 centrifuge tube, add cleaning buffer solution 3, cleaned 20～30 minutes, magnetic resolution, get supernatant liquor in No. 2 centrifuge tubes; Repeated washing is once got supernatant liquor and is incorporated in No. 2 centrifuge tubes again, obtains the carbohydrate-binding protein of enriching and purifying;
(6) preserve: in No. 1 centrifuge tube, add the preservation damping fluid, in 2～6 ℃ of preservations.
2. the method for enrichment according to claim 1 and purifying saccharide binding protein is characterized in that: described organic solvent is ethyl acetate or dimethyl formamide; Described coupling agent is 4-hydroxy phenyl valeric acid or 16-hydroxyl ten hexacarboxylic acids.
3. the method for enrichment according to claim 1 and 2 and purifying saccharide binding protein is characterized in that:
Described damping fluid and coupling buffer are sodium acetate, yellow soda ash, phosphoric acid salt or Tris-HCl;
Described binding buffer liquid is 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH7.0 damping fluid;
Described cleaning buffer solution 1,2 is 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH6.5 damping fluid etc.;
Described cleaning buffer solution 3 is 1mM CaCl 2, 1mM MnCl 2, 1mM KCl, 100mM Alpha-Methyl seminose, 10mM Tris-HCl pH7.0 damping fluid etc.;
Described preservation damping fluid is 1mM CaCl 2, 1mM MnCl 2, 0.02%NaN 3, 10mM Tris-HCl pH6.0 damping fluid.
4. the method for enrichment according to claim 3 and purifying saccharide binding protein is characterized in that: in the priming reaction of described coupling agent, be filled with nitrogen protection gas during incubation.
5. the method for enrichment according to claim 4 and purifying saccharide binding protein is characterized in that: in the priming reaction of described coupling agent, heated culture temperature is 37 ℃ Celsius, and the incubation time is 12 hours; In the pre-treatment of the micro-nano magnetic particle of described amino group derivatize, heated culture temperature is 37 ℃ Celsius, and the incubation time is 5 hours; In the described association reaction, heated culture temperature is 37 ℃ Celsius, and the incubation time is 12 hours; The temperature of reaction that is connected with the micro-nano magnetic particle adding carbohydrate-binding protein of sugar in the described association reaction is 37 ℃, 2 hours reaction times; Described storage temperature is 4 ℃ Celsius.
6. the method for enrichment according to claim 5 and purifying saccharide binding protein is characterized in that: in the pre-treatment of described carbohydrate-binding protein, association reaction, twice cleaning and the elution of reactive, the detection step of joint efficiency, the rate of recovery comprises
1) surveys the OD value
(1) survey carbohydrate-binding protein pre-treatment in, carbohydrate-binding protein is OD with the OD value that binding buffer liquid is diluted to 0.5mg/ml 1
(1) survey in the association reaction, after the micro-nano magnetic particle that is connected with sugar added the carbohydrate-binding protein reaction, the OD value of supernatant liquor that magnetic resolution is abandoned was OD 2
(1) in twice cleaning reaction of survey, the OD value of supernatant liquor that magnetic resolution is abandoned is respectively OD 3, OD 4
(1) survey in the elution of reactive, the OD value of the carbohydrate-binding protein of gained enriching and purifying is OD 5
2) calculate
Joint efficiency %=(OD 1-OD 2-OD 3-OD 4)/OD 1
Rate of recovery %=OD 5/ OD 1
7. the method for enrichment and purifying saccharide binding protein, it is characterized in that: the performing step of this method comprises
1) activation of sugar and micro-nano magnetic particle coupling (1) coupling agent:
1. coupling agent is dissolved in the organic solvent, is mixed with the coupling agent solution of 20～50mol/L;
2. add in the coupling agent solution by the ratio of 1: 10 amount of substance micro-nano magnetic particle the ethylene oxide group derivatize;
3. hatched under the room temperature 2～4 hours;
Described coupling agent is the coupling agent that contains an amino and a hydroxyl;
(2) clean: magnetic resolution, abandon supernatant; Clean 3 times with ethanol, use the buffer solution for cleaning 3 times of pH5.6 again;
(3) coupling: add the sugar soln of 2ml with the 100mM of pH5.6 coupling buffer preparation, in 35～40 ℃ Celsius, incubation 8～16 hours, then sugar is connected on the micro-nano magnetic particle of ethylene oxide group derivatize, must be connected with the micro-nano magnetic particle of sugar;
(4) clean: magnetic resolution, abandon supernatant; With the buffer solution for cleaning of pH5.6 3 times;
(5) preserve: add and preserve damping fluid, in 2～6 ℃ of preservations;
2) to carbohydrate-binding protein separate, enrichment and purifying
(1) pre-treatment of carbohydrate-binding protein: the carbohydrate-binding protein of water preparation 10mg/ml is diluted to 0.5mg/ml with binding buffer liquid;
(2) association reaction: get be connected with sugar micro-nano magnetic particle in No. 1 centrifuge tube, add the carbohydrate-binding protein of 0.5mg/ml, in 35～40 ℃ of reactions Celsius 1～3 hour, magnetic resolution, abandon supernatant liquor;
(3) clean: in No. 1 centrifuge tube, add cleaning buffer solution 1 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing once;
(4) clean: in No. 1 centrifuge tube, add cleaning buffer solution 2 and clean, magnetic resolution, abandon supernatant liquor; Repeated washing secondary again;
(5) wash-out: in No. 1 centrifuge tube, add cleaning buffer solution 3, cleaned 20～30 minutes, magnetic resolution, get supernatant liquor in No. 2 centrifuge tubes; Repeated washing is once got supernatant liquor and is incorporated in No. 2 centrifuge tubes again, obtains the carbohydrate-binding protein of enriching and purifying;
(6) preserve: in No. 1 centrifuge tube, add the preservation damping fluid, in 2～6 ℃ of preservations.
8. the method for enrichment according to claim 7 and purifying saccharide binding protein is characterized in that: described organic solvent is dimethyl formamide or ethyl acetate; Described coupling agent is 4-hydroxybenzamide or 4 hydroxybutyric acid hydrazine.
9. according to the method for claim 7 or 8 described enrichments and purifying saccharide binding protein, it is characterized in that:
Described damping fluid and coupling buffer are sodium acetate, yellow soda ash, phosphoric acid salt or Tris-HCl;
Described binding buffer liquid is 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH7.0 damping fluid;
Described cleaning buffer solution 1,2 is 1mM CaCl 2, 1mM MnCl 2, 10mM Tris-HCl pH6.5 damping fluid etc.;
Described cleaning buffer solution 3 is 1mM CaCl 2, 1mM MnCl 2, 1mM KCl, 100mM Alpha-Methyl seminose, 10mM Tris-HCl pH7.0 damping fluid etc.;
Described preservation damping fluid is 1mM CaCl 2, 1mM MnCl 2, 0.02%NaN 3, 10mM Tris-HCl pH6.0 damping fluid.
10. the method for enrichment according to claim 9 and purifying saccharide binding protein is characterized in that: in the priming reaction of described coupling agent, the incubation time that the micro-nano magnetic particle of ethylene oxide group derivatize adds coupling agent is 3 hours; Heated culture temperature in the described association reaction is 37 ℃ Celsius, and the incubation time is 12 hours; The temperature of reaction that is connected with the micro-nano magnetic particle adding carbohydrate-binding protein of sugar in the described association reaction is 37 ℃, 2 hours reaction times; Described storage temperature is 4 ℃ Celsius.
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CN101481402B (en) * 2008-01-10 2011-12-28 陕西北美基因股份有限公司 Method for separating and purifying glycopeptide

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101481402B (en) * 2008-01-10 2011-12-28 陕西北美基因股份有限公司 Method for separating and purifying glycopeptide

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