CN100387614C - Method of inclusion body protein renaturation and purification at the same time - Google Patents
Method of inclusion body protein renaturation and purification at the same time Download PDFInfo
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Abstract
The present invention relates to a method for the protein renaturation of inclusion bodies and simultaneously purifying the inclusion bodies, more specifically to gradient elution of the concentration of a denaturing agent or gradient elution of pH value carried out in ion exchange chromatography columns by dissolved inclusion body protein or the gradient elution of the concentration of the denaturing agent and the gradient elution of the pH value simultaneously carried out, which comprises the specific steps: 1. an acidic / alkaline moving phase I or the inclusion body protein dissolved by other solutions enters the balanced ion exchange chromatography columns of the moving phase I, and the inclusion body protein can be reversibly absorbed on a chromatography media; 2. then, the gradient of an alkaline / acidic moving phase II replaces the gradient of the moving phase I to enable the length of the gradient to be from 1 to 5 column volume; the concentration of the denaturing agent in the moving phase II is lower than that of the denaturing agent in the moving phase I, and protein of the moving phase II can be recovered in a high activity mode under high concentration; simultaneously, the process has the purification function to impure inclusion body protein.
Description
Invention field
The invention belongs to biological technical field, the method for particularly a kind of inclusion body protein renaturation and while purifying.
Background technology
Development of biology makes the scale operation target protein become possibility.Some content rarenesses, the albumen that be difficult for to extract can efficiently express in host cell by transgenic technology, and the host cell of frequent use is intestinal bacteria.The escherichia coli expression target protein has fast, a large amount of, advantages of being cheap.But the great expression of foreign protein in intestinal bacteria often forms proteic aggregate, i.e. inclusion body.Inclusion body is the aggregate of random stretching, extension peptide chain, does not have biological activity, need dissolve renaturation then to inclusion body protein.
Proteinic annealing issues is difficult point and the focus in the biotechnology, not only relates to the great problem of molecular biology, cytobiology etc., and relates to the production and the cost of present gene engineering product, especially to the scale operation pharmaceutical protein.The shortcoming that general refolding method exists is: mainly be the low renaturation yield under the lower concentration, make whole technological process need a large amount of damping fluids, the container of large volume, a large amount of operating times, the efficient that these greatly reduce production has increased production cost.The formation of aggregate is the major cause of low activity yield.When albumen folded renaturation under high density, the peptide chain of stretching, extension was because exposing of hydrophobic grouping is more easy because the disulfide linkage of hydrophobic interaction or intermolecular mispairing forms aggregate.Because the formation of aggregate relates to two protein polypeptide chains at least, so be secondary or more high-grade reaction, proteic concentration is high more, and the formation of aggregate is just fast more, many more, and this has restricted the mass production of inclusion body protein greatly.
At present both at home and abroad in the industrial production the renaturation means of frequent use dilution refolding method, dialysis renaturation method etc. are arranged.The dilution method recombinant protein is that inclusion body protein is directly diluted with renaturation buffer, and reaches the purpose of its renaturation, exist the renaturation time long, the damping fluid consumption is big, container volume is big, is not suitable for scale operation; And the dialysis method recombinant protein is that inclusion body protein is placed in the dialysis tubing, renaturation buffer with continuous replacing progressively dialyses out with the denaturant in the bag, and reach its renaturation purpose, shortcoming is: cause the absorption between albumen and the dialysis membrane easily, and, operation steps and operating time have been increased owing to need often to change renaturation buffer; These two kinds of methods all exist the damping fluid volume big, make troubles not only for follow-up purifying process, have increased the usage quantity of damping fluid, have improved production cost, and will increase the treatment capacity of equipment, are unsuitable for industrial production;
Chromatography method is the separation means of frequent use in the biotechnology.Chromatography media and chromatography environment are relatively gentleer to albumen, and the proteic concentration of processing can be very high, simple to operate, and the time that complete operation needs is short, so also be used to carry out protein renaturation recently.Utilize gel permeation chromatography to carry out the renaturation means that renaturation is a newly-developed, albumen has been sloughed denaturing agent in by the gel permeation chromatography medium, and folding renaturation takes place albumen; But the moment of denaturing agent removes in this process, also causes the formation of aggregate easily, and insoluble aggregate can cause the obstruction of pillar; Gel permeation chromatography itself has limited the industrial application of this method equally to the requirement of applied sample amount restriction.
The chromatography recombinant protein also comprises hydrophobic chromatography and ion chromatography, all is with the directly absorption on chromatography column of albumen inclusion body.The step of hydrophobic chromatography is: earlier with chromatography column high-salt buffer balance, (2) albumen advances post, adsorbs (1); (3) reduce salt concn, the albumen wash-out is come out; This method can be removed demodifier fully, and makes albumen folding renaturation on chromatography media, but this method only is suitable for the very strong albumen of surface hydrophobicity, otherwise the salt ion of the high density that hydrophobic chromatography uses causes proteic salt precipitation easily.And the activity of some albumen under the situation of high salt is not high, and the step of ion chromatography is: (1) earlier with chromatography column with less salt or there is not the salt buffer balance, (2) albumen advances post, adsorbs, (3) improve salt concn, and the albumen wash-out is come out; But this method generally all is the proteic purge process (US 4705848 for CN 1064968C, US4705845, EP 0505846A1, WO 00/03011) that is used for after the renaturation, not direct use ion exchange chromatography process renaturation.The patent (RU 2143492) of the recombinant protein of Russia's invention has been used ion exchange chromatography renaturation fusion rotein, but does not have the setting of two gradients of elution process, occurs the pairing of the disulfide linkage of aggregate and mistake unavoidably, has reduced the yield of renaturation.And when joining the metaprotein under the denaturing agent condition that is in high density in the pillar that does not have denaturing agent, because the instantaneous reduction of denaturing agent concentration has increased the possibility that aggregate forms greatly.Use the albumen after the damping fluid flushing renaturation do not contain denaturing agent, not only reduced the yield of renaturation, and some target proteins are adsorbed on and do not have wash-out to go out post on the chromatography media, make proteic mass recovery very low, and polluted chromatography column.Someone uses the natural antalzyme protein of this kind process renaturation sex change, and proteic mass yield is only about 10%.
Summary of the invention
The object of the present invention is to provide under a kind of high density the method for renaturation inclusion body protein matter efficiently, and purification of target albumen simultaneously.
Technical scheme of the present invention is as follows:
The method of a kind of inclusion body protein renaturation provided by the invention and while purifying, it is characterized in that this method is for carrying out the dissolved inclusion body protein gradient elution of the gradient elution of denaturing agent concentration or pH value or carrying out the concentration gradient wash-out of denaturing agent simultaneously and the gradient elution of pH value in ion exchange column; Its concrete steps are:
1) acidity/alkaline moving phase I or other solution dissolved inclusion body protein advance the ion exchange column after moving phase I balance, and inclusion body protein reversibly is attracted on the chromatography media;
2) replace moving phase I with alkalescence/acid moving phase II gradient then inclusion body protein solution is carried out gradient elution, gradient length is 1-5 column volume, and the concentration of the denaturing agent among the moving phase II is lower than the concentration of the denaturing agent among the moving phase I;
Described denaturing agent is urea, Triton or Tween; Concentration in moving phase I is 6～10mol/L; Concentration in moving phase II is 0.5～3mol/L; Described moving phase I is an acidic buffer, and pH is 3～7; Moving phase II is an ealkaline buffer, and pH is 8～11; Moving phase I is an ealkaline buffer, and pH is 8～11; Moving phase II is an acidic buffer, and pH is 3～7; Described acidic buffer is to contain the acidic buffer of single borate, acetate or carbonate or mix acidic buffer for it; Described ion-exchange chromatography media can be DEAE Sepharose Fast Flow, SP Sepharose Fast Flow, Q Sepharose Fast Flow or CM Sepharose Fast Flow.
The present invention provides the technology that can make quick, the easy renaturation of metaprotein or inclusion body protein on the basis of ion exchange chromatography renaturation, in protein renaturation, also can reach the purpose of carrying out separation and purification with other foreign proteins; Compare with other refolding method, operation steps has been simplified in the setting of two kinds of moving phases greatly, has improved the efficient of the renaturation of albumen under high density.
In the protein renaturation process, removing of denaturing agent is the committed step that influences the protein renaturation yield.The present invention adopts the high density denaturing agent can make fine and close solubilization of inclusion bodies, and at 6～10mol/L, when the concentration of Guanidinium hydrochloride was 4～8mol/L, proteic polypeptide chain existed with the form that stretches as the concentration of urea; Under the denaturing agent condition of high density, can not form aggregate between the protein polypeptide chain because of hydrophobic interaction; In theory, remove denaturing agent and other the albumen that makes is separated folding factor, polypeptide chain can progressively fold renaturation.But, general traditional refolding method is to make the instantaneous reduction of denaturing agent concentration, even drops to zero point, like this, albumen can produce some folding intermediates when folding, these intermediates have stronger hydrophobic surface, because this moment, denaturing agent concentration was very low, no longer have the effect of dissolving aggregate, aggregate will produce in a large number, sometimes even observe in the renaturation solution precipitation of white.And gradient elution that the denaturing agent concentration that the present invention adopts reduces gradually or pH value reduce gradually or the gradient elution that raises or carry out denaturing agent simultaneously and reduce the gradient elution of concentration and the gradient elution that the pH value gradually reduces or raises gradually; Can in this process, progressively fold renaturation, satisfy the progressively needs of embedding of each hydrophobic position of protein polypeptide chain; Both can improve the protein renaturation yield, reduce the formation of aggregate; Simultaneously, (urea is 2～3mol/L, and Guanidinium hydrochloride is 1～2mol/L), can increase the snappiness of the protein peptide chain after the renaturation, obviously improves proteic renaturation yield to contain the denaturing agent of lower concentration in the final proteic damping fluid.
General albumen, the pH value in damping fluid is far away more from proteic iso-electric point, and forming aggregate or albumen between the albumen, to form sedimentary possibility just more little; Simultaneously, different albumen, the stability when difference folds degree has different requirements to pH, and the pH value of proteic optimal activity also has difference; Like this, select different pH values for use, under a pH value, make the protein stabilized of sex change, and help other operation, and the progressively variation of the pH value during protein folding, the folding conformation of stabilize proteins greatly, reduce the possibility of false folding, and the albumen after folding is under the suitableeest pH value.Special in the albumen that contains disulfide linkage, under the acidic conditions, can avoid the formation of disulfide linkage this moment, and the requirement difference of the pH value of the disulfide linkage of albumen different sites when forming, the graded of the pH value from acidity to alkalescence, can avoid the possibility of disulfide linkage mispairing, satisfy the requirement that the different positions disulfide linkage forms.If for proteic disulfide linkage is formed, albumen is in the high alkalinity environment, the disulfide linkage of some mispairing can not exchange rearrangement again, also can reduce the yield of renaturation.
With respect to the renaturation process of albumen in solution of sex change, the protein peptide chain of sex change is adsorbed on folding renaturation on the chromatography media, can avoid between the protein polypeptide chain because the hydrophobic interaction of folding intermediate forms aggregate.Because renaturation in solution, protein peptide chain are active, be easy to be in contact with one another the formation aggregate between the polypeptide chain.Be adsorbed on the chromatography media autonomous folding of each polypeptide chain, folding between rare and form aggregate.So the adsorption chromatography renaturation can reduce the formation of aggregate greatly.And in ion exchange chromatography, the protein polypeptide chain site of absorption is hydrophilic site, and hydrophobic site is in folding renaturation in the solution, can not disturb proteic folding.
The present invention (moving phase I) under the denaturing agent condition of high density makes the protein adsorption of sex change, has reduced the possibility that produces aggregate in the adsorption process owing to the unexpected reduction of denaturing agent concentration; The gradient of denaturing agent concentration or pH value or the gradient of denaturing agent concentration and pH value are set simultaneously to be changed simultaneously, make protein peptide chain folding renaturation in a stepping environment of stretching, extension, mild condition, reduced the possibility of false folding, disulfide linkage mispairing and formation aggregate, purpose is exactly to make target protein obtain high activity under high density to reclaim.The existence of (moving phase II) partially denaturing agent has not only improved active recovery in the final elutriant, and the albumen that helps to adsorb all washed, and has overcome that albumen in the patent in early stage reclaims and has actively reclaimed less shortcoming.Two gradient settings have simultaneously reduced operation steps, save the operating time, are suitable for scale operation.And, in renaturation, can reach separating of target protein and foreign protein.
The selected ion-exchange chromatography media of the present invention can be DEAE Sepharose Fast Flow, SPSepharose Fast Flow, Q Sepharose Fast Flow or CM Sepharose Fast Flow etc.; For the albumen of macromolecule, need the particle diameter of medium and aperture all bigger, with the protein polypeptide chain folding that provides enough spaces to make stretching, extension; For micromolecular albumen, excessive particle diameter causes the waste of volume easily; The dissolved inclusion body protein generally contains negative charge under the alkaline condition, can select the anion-exchange chromatography medium for use, as the media such as SP Sepharose Fast Flow of Pharmacia company; The dissolved inclusion body protein generally contains positive charge under the acidic conditions, can select the cation-exchange chromatography medium for use, as the DEAESepharose Fast Flow of Pharmacia company, and media such as Q Sepharose Fast Flow.
Select suitable gradient length equally very important.The gradient of a column volume is best selection to folding easily albumen, and protein requirement that be difficult to fold or polymeric increases the length of gradient, can to several column volumes from two column volumes.
Select suitable flow velocity, should save time, make target protein fully folding again.Be difficult to the albumen that folds, then folding rate is slow more; Perhaps multimeric protein needs the process of polymer assembling, and then flow velocity is low more good more.
The denaturing agent such as the urea of non-ionic type are selected in the setting of denaturing agent gradient, and protein adsorption can be avoided the formation of protein aggregation body at this moment to medium in the environment (moving phase I) of the denaturing agent (as the 6mol/L urea) of high density.Elution buffer contains the denaturing agent (as the 1mol/L urea) of lower concentration, the selection of concentration washes out opportunity according to recombinant protein, as N,O-Diacetylmuramidase being adopted the 1mol/L urea, this moment the renaturation that wash-out goes out protein solution in urea concentration about 2mol/L, reclaim also the highest for this protein-active this moment.
The setting of pH value gradient (is example with natural N,O-Diacetylmuramidase), the pH acidity (3～6) during absorption is better, can increase proteic adsorptive capacity on the one hand, can avoid between protein molecular this moment again and the formation of intramolecular disulfide bond.During wash-out, the pH value is that alkalescence (8.7～10) is better, and be beneficial to the formation of disulfide linkage this moment, can not increase the probability of the mispairing of disulfide linkage again.High pH value helps the wash-out of this kind recombinant protein equally.
Select suitable temperature range, for general inclusion body protein, suitable temperature range is spent to 25 degree 4.When temperature is higher, can promote the reaction of protein folding, but also improve the speed of side reaction in the protein folding process simultaneously.Various speed of response reduce under the low temperature, can influence the speed of protein renaturation.Generally speaking, for the albumen that is easy to fast fold, under 25 degree, can obtain higher yield.And slow for protein folding, perhaps need the albumen of polymeric assembling process, better at low temperatures.
Embodiment
Further describe the present invention below in conjunction with drawings and Examples:
Accompanying drawing 1 is the wash-out collection of illustrative plates of the white N,O-Diacetylmuramidase of natural ovum gallinaceum of renaturation sex change;
Accompanying drawing 2 is the wash-out collection of illustrative plates of the inclusion body protein Fe-SOD of renaturation and while purifying;
Accompanying drawing 4 is the wash-out collection of illustrative plates of the inclusion body protein human lysozyme of renaturation and while purifying;
Accompanying drawing 6 is the comparison collection of illustrative plates of embodiment 6 different renaturation modes.
The ion exchange column that present embodiment uses is SP Sepharose Fast Flow prepacked column, column volume Be 5mL;
The damping fluid that dissolves the white N,O-Diacetylmuramidase of natural ovum gallinaceum is the Tris-HCL of 0.05mol/L, and pH 6.0, and contains the plain and 0.1mol/L DTT of 8mol/L urea;
Balance, last sample and dcq buffer liquid (moving phase I) are 0.05mol/L Tris-HCL, and pH 6.0, and contain the plain and 3mmol/L GSD of 6mol/L urea, 0.3mmol/L GSSG;
Elution buffer (moving phase II) is 0.1mol/L Tris-HCl, pH9.5, and contain 1mol/L urea, 0.3mol/L NaCl, 3mmol/L GSH, 0.3mmol/L GSSG;
The white N,O-Diacetylmuramidase of the natural ovum gallinaceum of last sample 8mg sex change, the flow velocity that uses is 0.4mL/min, the gradient of a column volume (5mL); Urea concentration reduces gradient (being reduced to 1mol/L by 6mol/L) and pH value gradient (pH is elevated to 9.5 from 6) in sex change and the while purge process, and the activity that is obtained is recovered as 95%, and albumen is recovered as 98%.Accompanying drawing 1 is its wash-out collection of illustrative plates, and by collection of illustrative plates as can be known: the white N,O-Diacetylmuramidase of the natural ovum gallinaceum of sex change is through after the method renaturation of the present invention, and wash-out goes out two peaks, and peak 1 is the absorption peak that contains reductive agent DTT in the metaprotein; Peak 2 is white N,O-Diacetylmuramidase elution peaks of natural ovum gallinaceum after the sex change.
The ion exchange column that present embodiment uses is Q Sepharose Fast Flow prepacked column, volume 5mL;
The damping fluid of dissolving inclusion body protein Fe-SOD is a 0.05mol/L PBS damping fluid, and pH 8.5, and contain 10mol/L urea and 3%Triton X-100;
Balance and sample introduction damping fluid (moving phase I) are 0.05mol/L PBS, and pH 8.5, and contain the 6mol/L urea, 0.01mol/L FeCl
3With 3%Triton X-100;
Elution buffer (moving phase II) is 0.1mol/L PBS, and pH 6.5, and contains the 1mol/L urea, 0.01mol/L FeCl
3, 0.2%Triton X-100,0.2mol/L NaCl;
Last sample 4mg inclusion body protein Fe-SOD renaturation solution, the flow velocity of 0.3mL/min, the gradient of two column volumes.
This process is used the elution buffer (moving phase II) that contains combined degeneration agent (urea and TritonX-100), increased the solvability of inclusion body protein Fe-SOD, finally obtained 92.5% albumen recovery, activity is recovered as 48%, complete operation within 60 minutes.And dilution refolding needed more than 40 hours, and final concentration of protein is 40 μ g/mL, and activity is recovered as 12%;
The wash-out collection of illustrative plates of accompanying drawing 2 present embodiments, accompanying drawing 3 are electrophorograms of refolded protein, and it is that electrophoresis is pure that SDS-PAGE analyzes the albumen that shows after the renaturation.By accompanying drawing 2 and 3 as can be known: the activity after the Fe-SOD inclusion body process method renaturation of the present invention (ion exchange chromatography renaturation), more a lot of than the activity yield raising of dilution refolding, and have purification simultaneously, the sample of collection is pure product.
Embodiment 3: renaturation and while purifying inclusion body protein human lysozyme
The ion exchange column that present embodiment uses is SP Sepharose Fast Flow 7mL;
The damping fluid of dissolving inclusion body protein is 0.05mol/L Tris-HCl, and pH 5.5, and contain 8mol/L urea and 0.1mol/L dithiothreitol (DTT);
Sample-loading buffer (moving phase I) is 0.05mol/L Tris-HCl, and pH 5.5, and contains 6mol/L urea, 3mmol/L GSH and 0.3mmol/L GSSG;
Elution buffer (moving phase II) is 0.1mol/L Tris-HCl, and pH 10.0, and contains 1mol/L urea, 0.2mol/L (NH
4)
2SO
4, 3mmol/L GSH and 0.3mmol/L GSSG, flow velocity is 0.4mL/min, the gradient of a column volume, last sample albumen 8mg;
Refolded protein than average out to 42618U/mg alive, protein yield 98%, whole protein concentration 1-1.2mg/mL; The time of renaturation shortens to from 8.5 hours of dilution refolding in 2 hours of ion exchange chromatography; The wash-out collection of illustrative plates of accompanying drawing 4 present embodiments, the electrophoretogram of accompanying drawing 5 present embodiments, it is same that to reach electrophoresis pure; From accompanying drawing 4 and accompanying drawing 5 as can be seen, the renaturation yield of providing and the proteic dual function of purification of target are provided method of the present invention, accompanying drawing 6 present method are compared with traditional dilution refolding, simple ion-exchange techniques or single gradient ion exchange chromatography method, have bigger advantage.
Claims (4)
1. plant the method for inclusion body protein renaturation and while purifying, it is characterized in that this method is for carrying out the dissolved inclusion body protein gradient elution of the gradient elution of denaturing agent concentration or pH value or carrying out the concentration gradient wash-out of denaturing agent simultaneously and the gradient elution of pH value in ion exchange column; Its concrete steps are:
3) acidity/alkaline moving phase I or other solution dissolved inclusion body protein advance the ion exchange column after moving phase I balance, and inclusion body protein reversibly is attracted on the chromatography media;
4) replace moving phase I with alkalescence/acid moving phase II gradient then inclusion body protein solution is carried out gradient elution, gradient length is 1-5 column volume, and the concentration of the denaturing agent among the moving phase II is lower than the concentration of the denaturing agent among the moving phase I;
Described denaturing agent is urea, Triton or Tween: its concentration in moving phase I is 6～10mol/L; Concentration in moving phase II is 0.5～3mol/L; Described moving phase I is an acidic buffer, and pH is 3～7; Moving phase II is an ealkaline buffer, and pH is 8～11.
2. by the inclusion body protein renaturation of claim 1 and the method for while purifying, it is characterized in that described moving phase I is an ealkaline buffer, pH is 8～11; Moving phase II is an acidic buffer, and pH is 3～7.
3. by the inclusion body protein renaturation of claim 1 and the method for while purifying, it is characterized in that described damping fluid is to contain the damping fluid of single borate, acetate, phosphoric acid salt or carbonate or be its cocktail buffer.
4. by the inclusion body protein renaturation of claim 1 and the method for while purifying, it is characterized in that described ion-exchange chromatography media can be DEAE Sepharose Fast Flow, SP Sepharose Fast Flow, Q Sepharose Fast Flow or CM Sepharose Fast Flow.
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| CN102807599A (en) * | 2011-06-02 | 2012-12-05 | 复旦大学 | Method for purifying and renaturing inclusion body protein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100336824C (en) * | 2005-12-19 | 2007-09-12 | 百奥泰生物科技(广州)有限公司 | Recombinant protein efficient renaturation method |
| CN102399291B (en) * | 2010-09-10 | 2014-11-05 | 中国科学院过程工程研究所 | Affinity chromatography renaturation method of anticoagulation thrombolysis bifunction fusion protein |
| CN102876645A (en) * | 2012-10-26 | 2013-01-16 | 西北大学 | Double-function chromatographic medium assisted lysozyme in-vitro renaturation method |
| CN107098959B (en) * | 2017-05-27 | 2019-10-18 | 中国农业科学院蜜蜂研究所 | The preparation method of bumblebee peptidoglycan recognition protein |
| CN111423486A (en) * | 2020-03-31 | 2020-07-17 | 艾柏森(江苏)生物科技有限公司 | Renaturation method of new type coronavirus recombinant protein inclusion body |
| CN111777659B (en) * | 2020-07-09 | 2023-11-24 | 向开军 | Universal electric field driven protein solid phase renaturation method |
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| CN1169998A (en) * | 1996-07-10 | 1998-01-14 | 广州南方生物技术有限公司 | Process of extracting and purifying recombined protein |
| RU2143492C1 (en) * | 1998-07-23 | 1999-12-27 | Навашин Петр Сергеевич | Recombinant plasmid encoding fused protein as precursor of human insulin (variants), strain of bacterium escherichia coli - producer of fused protein as precursor of human insulin (variants), method of human insulin preparing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1169998A (en) * | 1996-07-10 | 1998-01-14 | 广州南方生物技术有限公司 | Process of extracting and purifying recombined protein |
| RU2143492C1 (en) * | 1998-07-23 | 1999-12-27 | Навашин Петр Сергеевич | Recombinant plasmid encoding fused protein as precursor of human insulin (variants), strain of bacterium escherichia coli - producer of fused protein as precursor of human insulin (variants), method of human insulin preparing |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102807599A (en) * | 2011-06-02 | 2012-12-05 | 复旦大学 | Method for purifying and renaturing inclusion body protein |
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