CN1064968C - Process of extracting and purifying recombined protein - Google Patents
Process of extracting and purifying recombined protein Download PDFInfo
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- CN1064968C CN1064968C CN961067780A CN96106778A CN1064968C CN 1064968 C CN1064968 C CN 1064968C CN 961067780 A CN961067780 A CN 961067780A CN 96106778 A CN96106778 A CN 96106778A CN 1064968 C CN1064968 C CN 1064968C
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Abstract
The present invention belongs to the technical field of biologic medical preparation, which is used for extracting and purifying recombinant proteins from inclusion bodies, wherein the recombinant proteins form inclusion body shape and are expressed by genetic engineering bacteria (escherichia coli). The technology is characterized in that two systems of denaturation dissolving agents are used, and a step of denaturation dissolution and purification and a step that renaturation is carried out firstly and purification is carried out secondly are operated for two times. Compared with the prior art, the technology of the present invention enables the dissolvability of the recombinant proteins to increase 500 to 600 times, enables the use quantities of the denaturation dissolving agents to reduce about 100 times, enables the work efficiency to relevantly improve hundred times, enables the purity of the recombinant proteins to improve about 30% and enables the total yields of products to increase more than 24%; therefore, the technology as a new extracted and purified technology has the advantages of simple manufacturing process, obviously improved purity and yield of the products, obviously reduced cost and quite high economic effect.
Description
The invention belongs to field of biological pharmacy.
The expressed recombinant protein many (all) of many genetic engineering bacteriums (intestinal bacteria) exists with the inclusion body form, as: interleukin-2, the Filgrastim, interleukin-22-grain giant cells colony stimulating factor fusion protein ... Deng, therefore the technology of extracting purification of recombinant proteins from inclusion body attracts great attention, because the good and bad quality and the efficient that directly influences product of purifying process, thereby directly influence economic benefit.
In extracting and purifying recombined protein technology, must with protein denaturant (solvating agent) the recombinant protein sex change in the inclusion body be dissolved earlier, so that purifying is removed impurity, this is one of gordian technique.Under suitable condition, carry out renaturation then, to recapture natural radioactivity.Therefore recombinant protein is two of its gordian technique by sex change to renaturation.Above-mentioned two gordian techniquies mainly influence the purity and the efficient of product.
There is following problem in prior art to extracting purifying protein at present.
1. recombinant protein solubleness in Guanidinium hydrochloride or the single denaturing agent of urea is limited, and when reducing denaturing agent＜4mol/L, recombinant protein can not dissociate and be separated out with foreign protein, nucleic acid, has a strong impact on product recovery rate.
2. in order to improve the purity of recombinant protein, take the processing sequence of renaturation behind the first purifying, yet because the concentration height of denaturing agent, viscosity is big, consumption is also big, thereby operation easier is very big, the cost height.
3. because recombinant protein is long-time and the effect of high density denaturing agent, cause denaturing agent and product thereof the covalent modification to recombinant protein, cause the recombinant protein renaturation yield to reduce, product yield decreases.
In view of there are the problems referred to above in prior art.Consequently: the complex process operation easier is big, and the purity of product and yield are all low, and then causes the cost height, is unfavorable for suitability for industrialized production.
Purpose of the present invention seeks exactly that a technology is simple, and the purity of product and yield are all high, and the low extraction and purification process of cost.
Content of the present invention: attached process flow diagram
All recombinant proteins by the genetic engineering bacterium escherichia coli expression are to exist with the inclusion body form, and extract purification of recombinant proteins from inclusion body.All can produce by extraction and purification process flow process of the present invention.Extracting purifying procedure is with the engineering bacteria fragmentation, the inclusion body washing, and recombinant protein sex change purifying, recombinant protein secondary sex change purifying, the recombinant protein renaturation, ion exchange chromatography, sieve chromatography promptly gets recombinant protein, and its concrete technology is as follows:
One, engineering bacteria fragmentation
1. get engineering bacteria, by the wet bacterium of every gram add 5-10 times of damping fluid (1) (20mmol/LTris-HCl (Tutofusin tris-hydrochloric acid), pH8.0), the washing bacterium, in 4 ℃ centrifugal, 7000 (rpm) * 10min (minute), abandon supernatant liquor.
2. add 5 milligrams of N,O-Diacetylmuramidases by the wet bacterium of every gram, (50mmol/L Tris-HCl, 1mmol/L EDTA (disodium ethylene diamine tetraacetate) pH8.0), stirred 1.5-2.5 hour in 4 ℃, spent the night-20 ℃ of placements to add the damping fluid (2) that 5-10 doubly measures simultaneously.
3. after the bacterium that will spend the night is thawed, use ultrasonication, 30 seconds * 10 times, each 1-2min at interval, till weakening, bubble to liquid, microscopy bacteria breaking rate is more than 98%.
Two, inclusion body washing
1. get ultrasonication bacterium liquid, centrifugal 4000rpm * 5min4 ℃, stay precipitation.
2. throw out (inclusion body) is carried out abundant agitator treating respectively successively with following damping fluid (3) (4) (5) (6) (7) (8),, wash 1-2 time, stay throw out (inclusion body) standby in 4 ℃ of centrifugal 12000rpm * 20min.Xi Di purpose is to remove and abandons the impurity such as plasmalogen, lipopolysaccharides, nucleic acid and foreign protein that are blended in inclusion body respectively.
Each damping fluid composition is as follows:
Damping fluid (3): 20mmol/L Tris-HCl, 2mmol/L EDTA, pH8.0
Damping fluid (4): 20mmol/L Tris-HCl, 10mmol/L EDTA, 2mmol/L 2-ME
(3-mercaptoethanol), 0.5% triton x-100, pH8.0
Damping fluid (5): identical with damping fluid (3)
Damping fluid (6): 2mol/L urea, 20mmol/L Tris-HCl, 2mmol/L EDTA, pH8.0
Damping fluid (7): 70% Virahol, 20mmol/L Tris-HCl, pH8.0
Damping fluid (8): 20mmol/L Tris-HCl, pH8.0
Three, recombinant protein sex change purifying
1. the inclusion body after will washing adds Guanidinium hydrochloride damping fluid (9) (the 6-8mol/L Guanidinium hydrochloride that 5-10 doubly measures by weight in wet base, 10mmol/L DTT (dithiothreitol (DTT)), 1mmol/L EDTA, 20mmol/L Tris-HCl, pH7.0-8.5), fully stir 25-35 minute after, recombinant protein sex change dissolving this moment, in 4 ℃ of centrifugal 12000rpm * 30min, get supernatant liquor and get recombinant protein solution.
Get guanidine hydrochloride dissolution liquid, add damping fluid (10) (10mmol/L DTT, 1mmol/L ED-TA, 20mmol/L Tris-HCl pH8.0) is diluted to 4-5mol/L with the Guanidinium hydrochloride buffer concentration, stirs, in 4 ℃ of centrifugal 12000rpm * 20min, abandon throw out.Supernatant liquor continues 10. concentration of guanidine hydrochloride to be diluted to 2-3mol/L with damping fluid again and stirs, in 4 ℃ of centrifugal 12000rpm * 20min, stay throw out (this throw out is a recombinant protein), then with throw out damping fluid (11) (20mmol/L Tris-HCl, 10mmol/L DTT, 1mmol/L EDTA, pH8.0) flushing, centrifugal 10000rpm * 10min washes and stays throw out 1-2 time.
Four, recombinant protein secondary sex change purifying
Get throw out urea buffer solution (12) (the 6-8mol/L urea of purifying for the first time, 1mmol/L EDTA, 100mmol/2-ME, 20mmol/L NaAc (sodium-acetate), pH3.5-7.0) sex change dissolving, in 4 ℃ of centrifugal 12000rpm * 20min, the centrifugal insolubles of abandoning, staying supernatant liquor to adjust protein concentration is that the 8-10mg/ gram is standby.On this processing sequence, the difference of the present invention and prior art matter, be recombinant protein has been taked two kinds of sex change dissolution system, replace sex change dissolving purification process, can improve the solubleness (500-600 doubly) of recombinant protein significantly, and make full use of its solubleness and increase this characteristic greatly, painstakingly reduce the concentration (0.1mol/L) of (dilution) denaturing agent, its purpose is to make recombinant protein and foreign protein, nucleic acid to dissociate voluntarily, to reach purifying, improves yield.And existing technology is taked the processing of single denaturing agent, the concentration of denaturing agent can only be dropped to＞4mol/L, this concentration can not make recombinant protein and foreign protein, nucleic acid etc. dissociate voluntarily effectively, thereby recombinant protein is separated out with impurity, discard, so yield significantly reduces.
Five, recombinant protein renaturation
It is 8-10mg/ml solution that urea liquid is adjusted protein concentration, slowly add renaturation buffer (13) (1mmol/L EDTA, the 1mmol/L reduced glutathione, 0.1mmol/L oxidized form Triptide, the 20mmol/L sodium-acetate, pH4.0), promptly in 80-100 minute with the concentration of 6-8mol/L urea buffer solution progressively (gradient) be diluted to 0.1mol/L, spend the night in 4 ℃, use damping fluid (14) (10mmol/L sodium-acetate then, pH4.0) dialysis, or, get recombinant protein through ultrafiltration balance removal renaturation buffer.
The great technical characterictic of the present invention on this process procedure, when being to adopt renaturation buffer gradient dilution denaturing agent, progressively reduce the method for renaturation, its result:
1. simplified whole follow-up purifying process.
2. greatly reduce the consumption of denaturing agent, thereby reduced the trouble of under the denaturing agent condition, carrying out chromatographic separation.
3. recombinant protein and denaturing agent are very short action time in entire operation, thereby can avoid denaturing agent and product thereof hungry to the covalent modification of recombinant protein, and the recombinant protein renaturation yield can reach more than 95%.
Six, ion exchange chromatography (removing the nucleic acid pyrogen)
1. with CM Sepharose FastFlow (a kind of cation-exchange chromatography filler) glass chromatography column of packing into, with the 0.5mol/L sodium hydroxide wash-out of 2 times of column volumes once, wash chromatography column with apirogen water, to remove sodium hydroxide, pH7.0 to effluent liquid ends, then with the 20mmol/L sodium-acetate-acetic acid of 2-3 times of column volume, pH4.0 damping fluid balance chromatography column.
2. the recombinant protein of getting renaturation add damping fluid (15) (20mmol/L sodium-acetate-acetic acid, pH4.0), then with chromatography column on the flow velocity of 50-70 milliliter per hour.Use damping fluid (15) wash-out of 2 times of column volumes again, flow velocity is 25-30ml/ hour, uses sodium-chlor damping fluid (16) (0.3mol/L sodium-chlor, 20mmol/L sodium-acetate-acetic acid at last, pH4.0) wash-out stops to collect when the ultraviolet absorption value of ultraviolet monitoring instrument is zero.
Seven, sieve chromatography (removing isomer, pyrogen)
1. with gel exclusion chromatography filler (Sephacryl S-200) glaze chromatography column, wash the former processing of reducing phlegm and internal heat with 0.5mmol/L oxychlorination sodium.Use damping fluid (17) (0.004% tween-80,10mmol/L sodium-acetate-acetic acid, pH4.0) the balance chromatography column of 2 times of column volumes then.
2. the recombinant protein that the ion exchange chromatography of learning from else's experience is handled adds damping fluid (17), and by sample and wash-out on 10% column volume, flow velocity is 50-60ml/ hour, collects protein peak, stops to collect when the uv-absorbing of ultraviolet monitoring instrument is zero, can obtain recombinant protein.
The present invention has the following advantages compared with the prior art and sees the following form
The present invention compared with the prior art
Denaturing agent consumption denaturing agent recombinant protein action time purity OD280/260 finished product
(ml) (hour) 100 times of 80 above 30 0.5 2*OD280/260 value of (%) (absorbancy) * (%) prior art 1,500 85 60 1.0 1 the present invention 150 2＞90 1.5 3 differences are big more, product purity is high more, and it is few more to contain impurity such as nucleic acid.
As can be seen from the above table
1. the present invention using few 100 times of sex change dosage, and the working efficiency of removing denaturing agent will improve 100 times than prior art, and its cost significantly reduces.
2. the present invention lacks more than 80 hours than prior art denaturing agent and recombinant protein action time, causes the recombinant protein covalent modification to significantly reduce, thereby total yield of products improves.
3. the present invention improves 30% than prior art to recombinant protein on purity.
4. the present invention and prior art are respectively 34%, 10% in the recombinant protein total recovery, if purity is counted, then are respectively 30.6% and 6%, and with regard to product yield, the present invention of its economic benefit increases by 25% than prior art, or reduces cost more than 25%.
The extraction and purification process of embodiment one, recombinant methionyl human G-CSF
1. bacterial cell disruption
(1) get engineering bacteria 100 gram and add 1000ml 20mmol/L Tris-HCl (Tutofusin tris-hydrochloric acid), pH8.0, the washing bacterium, in 4 ℃ of centrifugal 7000rpm * 10min (minute), abandon supernatant.
(2) wet bacterium adds N,O-Diacetylmuramidase 500mg, 50mmol/L Tris-HCl, 1mmol/L EDTA (disodium ethylene diamine tetraacetate), pH8.0 damping fluid 1000ml, in 4 ℃ stir 2h (hour), spend the night-20 ℃ of placements.
(3) above-mentioned bacterium is thawed back is ultrasonic, 30sec (second) * 10, and each interval 1min is till weakening, bubble to liquid.Microscopy bacteria breaking rate＞98%.
2. the washing of inclusion body
(1) gets the centrifugal 4000rpm * 5min of bacterial cell disruption liquid, 4 ℃, stay precipitation.
(2) use 20mmol/L Tris-HCl, 2mmol/L EDTA, the washing of pH8.0 liquid.
(3) use 20mmol/L Tris-HCl, 10mmol/L EDTA, 2mmol/L2-ME (2 mercapto ethanol), 0.5%Tritonx-100 (triton x-100, a kind of washing agent), pH8.0 liquid is washed 1 time, degrease matter.
(4) use 20mmol/L Tris-HCl, 2mmol/L EDTA, pH8.0 liquid wash 2 times, to remove Tritonx-100.
(5) use 2mol/L urea, 20mmol/L Tris-HCl, 2mmol/L EDTA, pH8.0 liquid wash 1 time, remove foreign protein.
(6) 70% Virahols, 20mmol/L Tris-HCl, pH8.0 are washed one time, remove lipopolysaccharides.
(7) use 20mmol/L Tris-HCl, pH8.0 washes 2 times and removes Virahol.
Above step behind 4 ℃ of centrifugal 12000rpm * 20min, is abandoned supernatant with damping fluid 500ml.
3. recombinant protein sex change purifying
(1) inclusion body is dissolved in denaturing agent: get the inclusion body throw out, add the 7mol/L Guanidinium hydrochloride, and 10mmol/L DTT (dithiothreitol (DTT)), 1mmol/L EDTA, 20mmol/L Tris-HCl, pH8.0, concentration is 20%.Fully behind the stirring and dissolving 30min,, get supernatant, obtain the recombinant protein primary extract, and it is diluted to 50 μ g/ml in 4 ℃ of centrifugal 12000rpm * 30min.
(2) adding contains 10mmol/L DTT, 1mmol/L EDTA, 20mmol/L Tris-HCl in the inclusion body of 7mol/L guanidine hydrochloride dissolution, the pH8.0 damping fluid, concentration of guanidine hydrochloride is reduced at twice, all separate out 4 ℃ of centrifugal 12000rpm * 20min to recombinant protein, abandon supernatant, precipitation is used 20mmol/LTris-HCl, 10mmol/L DTT, 1mmol/L EDTA, the damping fluid of pH8.0 is washed 2 times, and 10000rpm * 10min is centrifugal must to be precipitated.
4. recombinant protein secondary sex change purifying
8.0mol/L urea, 1mmol/L EDTA, 100mmol/L2-ME, 20mmol/LNaAc, pH4.0 dissolve above-mentioned precipitation, and it is centrifugal that (12000rpm * 20min) remove insolubles, adjusting protein concentration is the 8-10 mg/ml.
5. recombinant protein renaturation
In urea buffer solution, slowly add renaturation solution (1mmol/L EDTA, 1mmol/L reduced glutathione (GSH), 0.1mmol/L oxidized form Triptide (CSSG), 20mmol/L NaAc, pH4.0), promptly in 80min, reduce urea concentration to 1.0mol/L, be diluted to 0.1mol/L again, the renaturation thing is crossed liquid for 4 ℃, removes the renaturation agent with dialysis or ultrafiltration balance among the 10mmol/L NaAc pH4.0 then.
6. ion exchange chromatography (stoning acid, thermal source)
(1) dress CM Sepharose FF (a kind of cation-exchange chromatography filler) chromatography column, with 2 times of column volume 0.5mol/L NaOH (sodium hydroxide) wash-out once (pyrogen-free processing), be washed till the pH7.0 of effluent liquid with apirogen water, use 20mmol/L NaAc-HAC (sodium-acetate-acetic acid) pH4.0 balance then.
(2) 60ml/h with on the sample in chromatography column, use the 20mmol/L NaAc-HAC pH4.0 wash-out of 2 times of column volumes again, flow velocity 30ml/h is again with the 20mmol/L NaAc-HAC that contains 1.0mol/L NaCl (sodium-chlor), the pH4.0 gradient elution is collected main protein peak.When being zero, the ultraviolet absorption value of ultraviolet monitoring instrument stops to collect.
7. sieve chromatography (remove isomer, pyrogen, change system)
(1) Sephacryl S-200 (a kind of gel exclusion chromatography filler) the chromatography column former processing of reducing phlegm and internal heat.
2) with 2 times of column volume 0.004%Tween-80,10mmol/L NaAc-HAC, pH4.0 balance chromatography column.
(3) by sample and wash-out on 10% column volume is in above-mentioned buffer system, flow velocity 60ml/h.Collect protein peak, when the ultraviolet absorption value of ultraviolet monitoring instrument is zero, stop to collect.
Embodiment two, rhIL2-PE66 (gene recombination Ro 24-7472/000 II-Pseudomonas aeruginosa extracellular toxin 66, molecular weight 80KD) extraction and purification process
1. bacterial cell disruption
(1) get engineering bacteria 100 gram and add 1000 milliliters of 20mmol/L Tris-HCl (tris-HCI buffer), pH8.0, the washing bacterium, in 4 ℃ of centrifugal 7000rpm * 10min (minute), abandon supernatant.
(2) add N,O-Diacetylmuramidase 500mg, 50mmol/L Tris-HCl, 1mmol/L EDTA (disodium ethylene diamine tetraacetate), pH8.0 damping fluid 1000ml in 4 ℃ of stirring 2h, spends the night-20 ℃ of placements.
(3) above-mentioned bacterium is thawed back is ultrasonic, 30sec (second) * 10, and each interval 1min is till weakening, bubble to liquid.The microscopy bacterium is cracked rate＞98%.
2. the washing of inclusion body
(1) the centrifugal 4000rpm * 5min of supernatant, stays precipitation by 4 ℃.
(2) use 20mmol/L Tris-HCl, 2mmol/L EDTA, the washing of pH8.0 liquid.
(3) use 20mmol/L Tris-HCl, 10mmol/L EDTA, 2mmol/L2-ME (2 mercapto ethanol), 0.5%Tritonx-100 (triton x-100, a kind of washing agent), pH8.0 liquid is washed 1 time, degrease matter.
(4) use 20mmol/L Tris-HCl, 2mmol/L EDTA, pH8.0 liquid wash 2 times, to remove Tritonx-100.
(5) use 2mol/L urea, 20mmol/L Tris-HCl, 2mmol/L EDTA, pH8.0 liquid wash 1 time, remove foreign protein.
(6) 70% Virahols, 20mmol/L Tris-HCl, pH8.0 are washed one time, remove lipopolysaccharides.
(7) use 20mmol/L Tris-HCl, pH8.0 washes 2 times and removes Virahol.
Above step all adds the washing of 500ml damping fluid, abandons supernatant behind 4 ℃ of centrifugal 12000rpm * 20min.
3. recombinant protein sex change purifying
With 8mol/L guanidine hydrochloride dissolution inclusion body, add then and contain 10mmol/L DTT, 1mmol/LEDTA, 20mmol/L Tris-HCl, the pH8.0 damping fluid, concentration of guanidine hydrochloride is reduced to 2mol/L at twice, centrifugal 12000rpm * 20min behind the stirring 10min, recombinant protein is all separated out, and 12000rpm * 20min abandons supernatant, precipitation (contains 10mmol/LDTT with 20mmol/L Tris-HCl, 1mmol/L EDTA), the damping fluid of pH8.0 is washed 2 times, and 10000rpm * 10min is centrifugal must to be precipitated.
4. recombinant protein secondary sex change purifying
Use 8.0mol/L urea, 1mmol/L EDTA, 100mmol/L2-ME, 20mmol/LTris-HCl, pH8.0 dissolving, it is centrifugal that (12000rpm * 20min) stays supernatant liquor, and to adjust protein concentration be that 8-10mg/ml is standby.
5. the renaturation of recombinant protein
2 sex change lysates of above-mentioned recombinant protein are slowly added renaturation solution (1mmol/LEDTA, 1mmol/L reduced glutathione (GSH), 0.1mmol/L oxidized form Triptide (GSSG), 20mmol/L Tris-HCl pH8.0), reduces urea concentration to 1.0mol/L in 90min, be diluted to 0.1mol/L again, the renaturation thing spends the night for 4 ℃, uses 20mmol/L Tris-HCl then, and pH8.0 dialysis or ultrafiltration balance are removed the renaturation agent.
6. ion exchange chromatography (stoning acid, thermal source)
(1) with CM Sepharose FastFlow (a kind of cation-exchange chromatography filler) glass chromatography column of packing into, the record admission space, with 0.5mol/L NaOH (sodium hydroxide) wash-out of 2 times of column volumes once (pyrogen in the removal system pollutes), with the pH7.0 of apirogen water washing chromatography column (removing NaOH), then with 20mmol/L NaAc-HAC (sodium-acetate-acetic acid) the pH4.0 damping fluid balance chromatography column of 2-3 times of column volume to effluent liquid.
(2) with the flow velocity of 60ml/h with sample on 20mmol/L NaAc-HAC pH4.0 damping fluid in chromatography column, use the 20mmol/L NaAc-HACpH4.0 wash-out of 2 times of column volumes again, flow velocity 30ml/h, again with the 20mmol/L NaAc-HAC that contains 0.3mol/L NaCl (sodium-chlor), pH4.0 wash-out, purpose are to replace the hG-CSF recombinant protein that is adsorbed on the positively charged ion chromatography post with the NaCl with suitable ionic strength.Under the ultraviolet monitoring condition, to collect protein peak and when 0.3mol/L NaCl wash-out, be a symmetric simple spike, ultraviolet absorption value stops wash-out when being zero.
7. sieve chromatography (remove isomer, pyrogen, change system)
(1),, and handles through the 0.5mol/L NaOH protoplasm that reduces phlegm and internal heat with the above-mentioned method glass chromatography column chromatography column of packing into gel exclusion chromatography filler (Sephacryl S-200).
(2) with 2 times of column volume 0.004%Tween-80,10mmol/L NaAc-HAC, pH4.0 balance chromatography column.
(3) by sample and wash-out on 10% column volume is in above-mentioned buffer system, flow velocity 60ml/h.Collect protein peak, ultraviolet monitoring stops wash-out when absorbing zero.
Claims (8)
1. the purification process of the expressed recombinant protein of a genetic engineering bacterium is characterized in that with the engineering bacteria fragmentation inclusion body washing, recombinant protein sex change purifying, recombinant protein secondary sex change purifying, recombinant protein renaturation, ion exchange chromatography, sieve chromatography promptly get the recombinant protein of purifying.
2. method according to claim 1 is characterized in that engineering bacteria staying precipitation with damping fluid washing, centrifugal, add bacteriolyze enzyme buffer liquid in 4 ℃ of stirrings, place-20 ℃ and spend the night, thaw the back with ultrasonication to microscopy bacteria breaking rate greater than 98%, can get the inclusion body mixed solution.
3. method according to claim 1, it is characterized in that with the inclusion body mixture in 4 ℃ centrifugal, its throw out washs successively with multiple damping fluid, 4 ℃ centrifugal 1-2 time, the inclusion body throw out.
4. method according to claim 1, it is characterized in that inclusion body is dissolved in the 6-8mol/L Guanidinium hydrochloride damping fluid (9), damping fluid (9) is the 6-8mol/L Guanidinium hydrochloride, 10mmol/L DDT, 1mol/L EDTA, 20mmol/L Tris-HCt pH7.0-8.5, centrifugal then, to get supernatant liquor and concentration of guanidine hydrochloride is diluted to 4-5mol/L with damping fluid (10), damping fluid (10) is 10mmol/LDTT, 1mmol/L EDTA, 20mmol/L, Tris-HCt, pH8.0, centrifugal then, get supernatant liquor, with damping fluid (10) Guanidinium hydrochloride is diluted to 2-3mol/L again, centrifugal, throw out washes twice with damping fluid (11), stay throw out, damping fluid (11) is 20mmol/L Tris-HCt, 10mmol/LDTT, 1mmol/L EDTA, pH8.0.
5. method according to claim 1, it is characterized in that the throw out of recombinant protein is added 6-8mol/L urea buffer solution (12) sex change dissolving, the centrifugal supernatant liquor that stays, damping fluid (12) is a 6-8mol/L urea, 1mmol/L EDTA, 100mmol/L, 2-ME, the 20mmol/L sodium-acetate, pH3.5-7.0.
6. method according to claim 1, it is characterized in that the sex change recombinant protein is dissolved in urea buffer solution, levelling concentration is the 8-10 mg/ml, slowly add renaturation buffer then, promptly in 80-100 minute, urea concentration is diluted to 0.1mmol/L, removes renaturation buffer in 4 ℃ with damping fluid dialysis or ultrafiltration balance at last.
7. method according to claim 1 is characterized in that renaturation recombinant protein is added damping fluid, and last chromatography column is used buffer solution elution then, at last with buffer solution elution during to ultraviolet absorption value zero till.
8. method according to claim 1 is characterized in that upper prop and wash-out after adding damping fluid through the recombinant protein of ion exchange chromatography, stops during to ultraviolet absorption value zero.
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| CN100387614C (en) * | 2001-09-27 | 2008-05-14 | 中国科学院过程工程研究所 | Method of inclusion body protein renaturation and purification at the same time |
| CN101220081B (en) * | 2008-01-28 | 2011-01-05 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN104558100A (en) * | 2015-02-04 | 2015-04-29 | 吉林农业大学 | Inclusion body pretreatment method |
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| CN105968182A (en) * | 2016-06-03 | 2016-09-28 | 黄文林 | Production process of recombinant human cryptochrome protein I (hCRY1) and composition thereof |
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| CN100336912C (en) * | 2005-03-25 | 2007-09-12 | 深圳国家生化工程技术开发中心 | Method for preparing recombined human atrial natriuretic peptide rhANP by using ferment in high density |
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| CN1169998A (en) | 1998-01-14 |
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