CN1083071A - A kind of method that from the solution that contains inactive protein, reclaims the biologically active form recombinant protein - Google Patents
A kind of method that from the solution that contains inactive protein, reclaims the biologically active form recombinant protein Download PDFInfo
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- CN1083071A CN1083071A CN93108052A CN93108052A CN1083071A CN 1083071 A CN1083071 A CN 1083071A CN 93108052 A CN93108052 A CN 93108052A CN 93108052 A CN93108052 A CN 93108052A CN 1083071 A CN1083071 A CN 1083071A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
- C07K1/1133—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure by redox-reactions involving cystein/cystin side chains
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormones [GH] (Somatotropin)
Abstract
The present invention relates to a kind of method of from the solution that contains inactive protein, producing natural bioactive form recombinant protein, inactive protein is with insoluble inclusion body or oxidation, existing of false folding with the useless albumen form of accumulative, this method comprises with first ealkaline buffer dilution inactive protein solution, add SDS and L-halfcystine to dissolve simultaneously and to reduce this albumen, it is about 70%-90% that relative second ealkaline buffer is filtered the density loss that makes SDS and L-halfcystine thoroughly, oxidation sex change and reductive albumen to form the disulfide linkage consistent with naturally occurring biological activity protein.
Description
The present invention generally speaking relates to a kind of method natural, the biologically active form recombinant protein that reclaims from the solution that contains inactive protein.
The known method for preparing recombinant protein in this area; Using recombinant technology can insert the allogeneic dna sequence DNA fragment of certain specific protein of coding in the host microorganism.Cultivating microorganism transformed under the condition of inducible protein expression, can produce as Regular Insulin, tethelin, interleukin, Interferon, rabbit, somatomedin etc. heterologous protein.
Regrettably, usually there is not biological activity, because these albumen are not folded into correct tertiary structure when transcribing in microorganism by the heterologous protein that transforms microorganisms.These heterologous proteins tend to form aggregate, and these aggregates can be identified as " inclusion body " (being also referred to as " refractile body " and/or " protein granules " sometimes) in cell.Connect the formation of disulfide linkage between the covalent molecule of several protein moleculars, produced insoluble complex body, this may also be the reason that these inclusion bodys form.Inclusion body generally contains the host microorganism albumen of most of heterologous protein and small portion pollution.
Now set up several methods, from microorganism, extracting inclusion body, and will wherein contained heterologous protein have changed into and have the albumen with natural bioactive consistent with natural parent protein or non-recombinant protein.These methods relate generally to: the disruption of microorganisms cell; From cell debris, isolate inclusion body; In the presence of denaturing agent and/or reductive agent, make inclusion body protein sex change and/or reduction, albumen is launched and himself and insoluble separated from contaminants; Remove the reductive agent in the solution or reduce its concentration; Oxidation has been reduced and the protein solution of sex change in the presence of denaturing agent, removes denaturing agent, thereby makes heterologous protein be folded into three grades of conformations again; From the contaminating protein that still exists in solution, isolate this heterologous protein.
Many recombinant proteins recovery and the purification scheme of following above-mentioned general step all are known in the art.
People's such as Yokoo United States Patent (USP) 4,985,544 discloses a kind of method that contains cysteine protein that activates natural form from inclusion body again.This method comprises: has again at existing denaturing agent to make protein dissolution in the presence of the reductive agent, removes reductive agent, and oxidation protein, the subsequent removal denaturing agent makes albumen be folded into its native conformation again.Disclosed possible denaturing agent is SDS, urea, Guanidinium hydrochloride, bronsted lowry acids and bases bronsted lowry, and the reductive agent that is proposed comprises unit price mercaptan, for example beta-mercaptoethanol, halfcystine, gsh and dithiothreitol (DTT) (DTT).In listed reductive agent, preferred especially DTT.Oxidation step can carry out atmospheric oxidation by inflation to be finished.
In people's such as Yokoo embodiment, illustrated and selected for use urea and DTT respectively as denaturing agent and reductive agent.The shortcoming of people's such as Yokoo method is that this method need carry out removing fully selectively this thorny step of reductive agent, the preferred gel filtration method of this step before oxidation step in the presence of denaturing agent.
People's such as the United States Patent (USP) 4,518,526 of K.Olson, Builder United States Patent (USP) 4,511,502 and 4,620,948 and people's such as Wetzel United States Patent (USP) 4,599,197, all relate to the dissolving and the purifying of refrangibility heterologous protein, wherein, albumen is handled with denaturing soln, and this solution can comprise the SDS as the possibility denaturing agent.Then, choose wantonly in the presence of reductive agent, make albumen folding again by the dilution denaturing soln.The material of included conduct possibility reductive agent is a beta-mercaptoethanol.
The United States Patent (USP) 4,766,205 of P.Ghosh-Dastidar relates to the method that a kind of protein that contains a plurality of disulfide linkage that makes natural and recombinant sources produces the native conformation of biologically active.After institute's protein of interest matter being handled with denaturing agent and reductive agent, reduce the concentration of reductive agent, introduce simultaneously and contain compound disulfide linkage, that can form adducts.Contain the compound and the reaction of reductive cysteine residues of disulfide linkage, when removing reductive agent, formed stable middle adduct.This patent disclosure to contain the single, double or multi-functional sulfhydryl reagent that contains be appropriate reductant, as beta-mercaptoethanol or dithiothreitol (DTT).Most preferred reductive agent is a beta-mercaptoethanol.The suitable compound that can form the disulfide linkage adducts comprises cystamine, Sleep-promoting factor B, Gelucystine, S-WAT etc.
After stable adducts forms, can form natural Gelucystine disulfide linkage again, in the oxidation/reducing environment of gentleness, protein can be folded into its native conformation again, and the oxidation/reducing environment of this gentleness comprises weak reductant and the weak oxidant under the appropriate pH.The weak reductant that is applicable to this processing comprises halfcystine, and is applicable to that the weak oxidant of this processing comprises atmosphericoxygen.
Need to improve productive rate in this area by containing the biological activity protein that reclaims in solution non-activity, incorrect folded protein.
The invention describes the novel method of the biological activity protein productive rate that a kind of raising obtains from the solution that contains inactive protein.Inactive protein can be to exist with insoluble inclusion body form, also can existing with the useless proteic form of accumulative with oxidation, false folding.
The inventive method is to begin with first ealkaline buffer dilution this step of inactive protein.The inactive protein that has diluted is used as the SDS of denaturing agent and handles, to dissolve simultaneously and to go back crude protein as the L-halfcystine of reductive agent.To contain to have dissolved with part diafiltration (partial diafiltration) and filter thoroughly, thereby reduce the concentration of SDS and L-halfcystine in the solution with respect to second ealkaline buffer that does not contain denaturing agent and reductive agent with the proteic solution of reductive.Sex change and the albumen that has reduced are oxidized then, have formed the disulfide linkage consistent with naturally occurring biological activity protein.
In a preferred embodiment, solution dilution to the protein concentration that will contain inactive protein with first ealkaline buffer is about 0.1-1.0mg/ml.The inactive protein that has diluted to the L-halfcystine processing of about 25mM with about 0.05% to about 1.0% SDS and about 5mM then.To contain to have dissolved and filter thoroughly, make the SDS in the solution and the density loss about 70% to about 90% of L-halfcystine with respect to second ealkaline buffer that does not contain denaturing agent and reductive agent of about 0.5 to 3 times of volume with the proteic solution of reductive.Sex change and the albumen oxidation by air then of having reduced form the disulfide linkage consistent with naturally occurring biological activity protein.
Method of the present invention can be used for effectively and economically improving the rate of recovery of the biological activity protein that produces with the active inclusion body form of insoluble lifeless matter in transforming microorganism, this conversion microorganism is meant the recombinant DNA carrier microorganism transformed that those have been expressed with the guidance of heterologous protein encoding gene.Method of the present invention is particularly useful for that lifeless matter is active, oxidation, false folding change into useful products with the useless albumen of accumulative, the desirable proteins that exists with its natural bioactive form especially.The present invention further can be used for directly successfully reclaiming the monomer of oxidation from inclusion body.
" useless albumen " be meant resemble recovery technology defined known in the art after the dissolving and reductive solution of oxidation inclusion body protein, non-activity, oxidation, the false folding that together forms with correct folding biological activity protein with accumulative albumen.Specifically, useless albumen is to produce owing to the non-covalent gathering of the inclusion body protein that is reclaimed and/or intermolecular or intramolecularly covalency poly.The present invention can be used for the useless albumen of recirculation, to prepare correct folding monomeric products.
The biological activity protein that available the inventive method obtains comprises animal growth hormone, for example ox, pig, bird, sheep or people's tethelin.
Should be appreciated that, this paper loosely mentions protein or mentions concrete protein such as ox and Porcine somatotropin is not the molecule that will be confined to contain the complete amino-acid sequence of native protein, but also comprise the protein fragments that has removed different piece in the order, and different displacements and modification (being analogue) in its natural order, have been carried out but not saboteur's bioactive protein or its fragment.
The first step of this method comprises with first ealkaline buffer dilution inactive protein, to reduce protein concn and to adjust the pH value of protein soln.The preferred damping fluid yellow soda ash (Na of weight such as serve as reasons
2CO
3) and sodium bicarbonate (NaHCO
3) carbonate buffer solution formed, its concentration generally is preferably about 35mM to about 60mM, and more preferably about 40mM is about 50mM extremely, most preferably is about 46mM.The pH of first damping fluid is preferably about 9 to about 11, more preferably about 9.6 to about 10, most preferably is about 9.8.The proteinic concentration in damping fluid dilution back is preferably about 0.1 to about 1.0mg/ml, and more preferably about 0.3 to about 0.6mg/ml.This may require extent of dilution is about 1: 1.1 to about 1: 40, and when inactive protein existed with oxidation, false folding and the useless albumen form of accumulative, extent of dilution was generally about 1: 5; When inactive protein existed with insoluble inclusion body form, extent of dilution was generally about 1: 40.
Initial dilution step is that present method has been created advantageous environment.Proteinic concentration as if very doughtily influence subsequently dissolving/reduction and the efficient of re-oxidation step.Dilution reduces protein concn, with and increased distance between protein molecular.Specifically, the protein concn reduction makes covalency and non-covalent molecular interaction all be reduced to minimum level.Thereby the solution after the dilution has promoted correct intramolecular interaction, causes forming correct disulfide linkage.
Next step handles the inactive protein of dilution with denaturing agent SDS and reductive agent L-halfcystine, to dissolve simultaneously and to reduce desirable proteins.Adding SDS is in order to destroy noncovalent interaction, is to become sulfydryl for the intermolecular and intramolecular disulfide bond that makes the covalency mispairing dissociates and add the L-halfcystine.The concentration of SDS is preferably about 0.05% to about 1.0%, most preferably is about 0.2%(weight/volume), and the concentration of L-halfcystine is preferably about 5mM to about 25mM, most preferably is about 15mM.
Other reductive agent, especially dithiothreitol (DTT) (DTT), beta-mercaptoethanol (β ME) and reduced form and Sleep-promoting factor B also are widely used for control and have reduced proteinic reoxidizing.Yet DTT is costliness (563.50 dollars of per 100 grams of Sigma company product) very, and stores difficulty owing to it has water absorbability to cause, and makes this reagent be not suitable for being used for scale operation.On the other hand, though β ME cheap (32.15 dollars every liter of Sigma company products) because undesirable characteristic odor is arranged, also is out of favour.The more important thing is, in the method with β ME(20mM) when using as reductive agent, it usually has disadvantageous effect owing to form unusual stable folding again intermediate product to folding process again.This folding again intermediate product reduces monomer (RM) with a large amount of remnants, even in oxidation also usually existence still after 48 hours.
As reductive agent, avoided above-mentioned all problems with halfcystine (L-halfcystine).Its reasonable price (77.00 dollars every kilogram of aginomoto company products).In addition, halfcystine is a natural amino acid, and is prepared into dried crystals solution soluble in water, stable under alkaline pH.And, can obtain a large amount of food chemistry pharmacopeia level halfcystines.Halfcystine also is easy to be oxidized to generally acknowledged oxygenant Gelucystine, under the alkaline pH condition that especially occurs in claimed method (pKa of sulfydryl is 8.3).Because any residue problems can be reduced to minimum level.In addition, the existence of Gelucystine has improved protein soln oxidation subsequently.
Carry out the required time of above-mentioned reaction, depend on that inactive protein is to exist with insoluble inclusion body form, still with oxidation, the existing of false folding with the useless albumen form of accumulative.For inclusion body, the reaction times generally needs 2-3 hour, and for useless albumen, 45-60 minute generally just enough.
After the enough time is carried out in above-mentioned reaction, with diafiltration, preferred part diafiltration, make contain dissolve with the proteic solution of reductive in the concentration of SDS and L-halfcystine reduce, this diafiltration generally is to carry out with respect to about 0.5 second ealkaline buffer that does not contain denaturing agent and reductive agent to about 3 times of volumes (preferred about 1 times of volume).The buffer salinity of second ealkaline buffer is preferably about 35mM to about 60mM, and more preferably from about 40mM is to about 50mM, most preferably from about 46mM; Its pH value preferred about 9 is to about 11, and more preferably from about 9.6 to about 10, and most preferably from about 9.8.The saturating filter operation of part makes the concentration of SDS and L-halfcystine all reduce about 70% to about 90%, preferred about 80% before oxidation step.Shown in embodiment 4, filter thoroughly with respect to the damping fluid that is higher than volume described in the claimed method and will cause required oxidation protein monomer productive rate significantly to reduce.
The advantage of diafiltration is to make the concentration of low molecular weight substance such as SDS and L-halfcystine to reduce, and high molecular weight material such as proteinic concentration are then unaffected.
The re-oxidation step of carrying out subsequently that is reduced to of SDS and L-semicystinol concentration has been created advantageous environment.In addition, be preferably about 9.8 alkaline pH value, can make the halfcystine of staying in the solution be converted into Gelucystine naturally by protein solution is adjusted to, thus improved subsequently oxidizing reaction and the ultimate yield of required oxidation protein monomer.
Next step is oxidized to dissolving of being produced and reductive protein monomer correct folding oxidation monomer, preferably about 24 hours of reaction times in the presence of air.This oxidation monomer contains the disulfide linkage consistent with naturally occurring biological activity protein.As mentioned above, because under as desired alkaline pH condition in the claimed method, the halfcystine spontaneous oxidation becomes known oxygenant Gelucystine, so do not need other oxidizer.After oxidizing reaction takes place by the enough time, can make gained solution by resin such as Amberlite are housed
The ion exchange column of IRA-400 is to remove all remaining SDS.Then, can carry out required subsequent processing steps to the effluent liquid that contains the aggregate derivative monomer.
The present invention has carried out general description, and the following examples have provided specific embodiments of the present invention, and its purpose is to illustrate enforcement of the present invention and advantage thereof.Be appreciated that embodiment provides in illustrational mode, will limit specification sheets and claims anything but.
46mM carbonate buffer solution with pH9.8, with the useless protein solution of 1: 5 dilution, described damping fluid contains 21mM yellow soda ash and 25mM sodium bicarbonate, and described useless protein solution then contains covalency and non-covalent aggregate and a spot of pST oxidation monomer of Porcine somatotropin (pST).Next step adds 0.2%(W/V) SDS and 15mM halfcystine (L-halfcystine), make useless protein dissolution and reduction.Use the SDS and the halfcystine of above-mentioned concentration, dissolving/reduction reaction was finished in about 1 hour basically.Next step is 10,000 dalton with polysulfone membrane PM10(molecular weight cut-off at room temperature), carry out the saturating filter of 1 times of volume with respect to another 46mM carbonate buffer solution, the concentration of SDS and halfcystine is reduced rapidly.After saturating filter, the environment of solution is at pH value, pST concentration and SDS and halfcystine/aspects such as Gelucystine concentration, for spontaneous folding again the best that is of reduction pST.
Then, make and contain the solution that dissolves with reductive pST and carried out the ambient air oxidation about 24 hours, then make solution pass through Amberlite
The IRA-400 post is to remove remaining SDS.Then, the effluent liquid that contains the aggregate derivative monomer is carried out follow-up treatment step, comprise ultrafiltration and chromatography, with the monomer pST of preparation purifying.
Figure 1A-1E has shown the color atlas of the high pressure liquid chromatography (HPLC) of each step of present method.Figure 1A representative is with the useless protein solution of dilution in 1: 5.As among Figure 1B color atlas drew, after 1 hour, the peak that was present in representative oxidation monomer (OM) among Figure 1A originally disappears, and the bigger peak of representative reduction monomer (RM) occurred in 0.2%SDS sex change and the reduction of 15mM halfcystine.This shows that the pST aggregate and the oxidation monomer that exist are all effectively reduced in useless protein solution.Shown in Fig. 1 C, after the saturating filter of 1 times of volume, the OM peak enlarges markedly, and the RM peak reduces.Reoxidizing 13(Fig. 1 D) and 24(Fig. 1 E) hour after, the RM peak disappears substantially, and the OM peak roughly increases to about 3 times of initial OM peak.This shows successfully to have finished and reoxidizes.As indicated in the data shown in Fig. 1 E, 1.55 grams per liter OM are derived from 0.53 grams per liter OM in the useless protein solution (shown in Figure 1A) that originally is present in dilution.This increase is converted into the pST monomer owing to the pST aggregate.
Embodiment 2
Fig. 2 A has provided the color atlas that two width of cloth derive from gel permeation chromatography (GPC), and one of them is to finish before the aggregate process recycling of embodiment 1, and another width of cloth is then finished thereafter.Solid line is represented undiluted useless protein solution, and the dotted line representative derives from Amberlite
The product effluent liquid of IRA-400 post.The numeral that Fig. 2 B is reported has provided ppm number and pST polymkeric substance or the high molecular impurity and the monomeric ratio of pST of GPC monomer (GM).The ratio that this is called the P/M ratio, the purity that is used to measure sample.The P/M ratio is more little, and sample is pure more.
Fig. 2 A and 2B show, when containing 174ppm GM and a large amount of pST aggregates, P/M ratio is 42.4 useless protein solution, when handling according to the aggregate method for recycling of embodiment 1, GM increases by 10 times and reach 1695ppm, and the P/M ratio reduces to 4.1.This acute variation of GM and P/M is because the pST aggregate changes into the pST monomer.
Embodiment 3
To contain δ 7-pST(at the N-terminal of amino-acid sequence disappearance 7 amino acid whose pST) inclusion body from coli strain HB101, separate, this bacterial strain is at United States Patent (USP) 4, cultivate under 788, the 144 disclosed δ 7-pST preparation conditions.With cell centrifugal come out from fermented liquid, and resuspending is in the 0.1M sodium phosphate buffer that contains 20mM EDTA of pH7.8,8,000-10, under 000 pound/square inch the pressure, make twice in cell by the Manton-Gaulin homogenizer with lysing cell.Rough inclusion body recentrifuge is come out, and, carry out centrifugal after each washing with pH7.5, the 0.176M sodium phosphate buffer washed twice again that contains 10mM EDTA.The 46mM carbonate buffer solution that the inclusion body of process washing is used pH9.8 was with 1: 40 dilution, and this damping fluid contains 21mM yellow soda ash and 25mM sodium bicarbonate.Next step adds 0.2%(W/V) SDS and 15mM L-halfcystine, make solubilization of inclusion bodies and reduction.At room temperature stirred the mixture 2 hours 15 minutes.After dissolving and reduction end, HPLC analysis revealed RM is 16.8 grams per liters, and OM disappears.At room temperature mixture is filtered another 46mM carbonate buffer solution of 1 times of volume thoroughly with the PM10 film then.The saturating filtrate of HPLC analysis revealed is contained 14.4 grams per liter RM and 0.96 grams per liter OM.
Then, the solution through saturating filter was carried out the ambient air oxidation about 20 hours.After oxidizing reaction finished, OM roughly increased to 10.0 grams per liters, and RM reduces to 0.52 grams per liter.This shows that oxidizing reaction finishes in fact.
These results show that the present invention can be used for reclaiming the monomer of oxidation from inclusion body.
Embodiment 4
46mM carbonate buffer solution with pH9.8, with the useless protein solution of 1: 5 dilution, described damping fluid contains 21mM yellow soda ash and 25mM sodium bicarbonate, and described useless protein solution then contains the covalency of pST and the oxidation monomer of non-covalent aggregate and pST.The concentration of OM is determined as 0.26 grams per liter through HPLC in the useless protein solution of dilution.Next step adds 0.16%(W/V) SDS and 13mM L-halfcystine, make useless protein solution dissolving and reduction.At room temperature stirred the mixture about 3 hours, and at room temperature mixture was filtered another 46mM carbonate solution of 15 times of volumes thoroughly with the PM10 film then, to remove SDS and L-halfcystine fully.
After 2 times, 4 times and 15 times of volume buffer-exchanged, from sample, get aliquots containig.They represented respectively " part " (70-90%), " substantially " (90-99%) and " all " (100%) removed solvating agent (SDS) and reductive agent (L-halfcystine).At room temperature there are oxidation down 24 hours in all samples with air, analyze OM concentration with the HPLC method then.The HPLC OM of 2 times, 4 times and 15 times volume samples is respectively 0.38,0.29 and 0.075 grams per liter.All peaks all oxidations of disappearance confirmation with RM are all finished.
This result shows that removing solvating agent and reductive agent fully can have a negative impact to the monomeric high yield recovery of desirable proteins.
Embodiment 5
46mM carbonate buffer solution with pH9.8, with the useless protein solution of 1: 5 dilution, described damping fluid contains 21mM yellow soda ash and 25mM sodium bicarbonate, and described useless protein solution then contains the covalency of pST and the oxidation monomer of non-covalent aggressiveness and pST.OM concentration is 0.15 grams per liter in the useless protein solution of HPLC analysis revealed dilution.Next step adds 0.5(W/V) SDS, 60 mg/litre EDTA and 40mM L-halfcystine, make useless protein solution dissolving and reduction.At room temperature stirred the mixture 1 hour.The HPLC analysis revealed, after dissolving and reduction end, RM is 0.68 grams per liter.
Under the condition that air existence and room temperature are arranged, oxidation mixture 24 hours is not removed solvating agent or reductive agent in this process.HPLC analysis revealed RM still has 0.42 grams per liter, and OM does not then measure.
This result shows the concentration that does not reduce solvating agent and reductive agent, and the just oxidation that can not hit pay dirk can not reach the monomeric high yield of desirable proteins and reclaim.
Claims (15)
1, a kind of method of producing natural bioactive form recombinant protein from the solution that contains the lifeless matter activated protein, this method comprises the following steps:
A) with first ealkaline buffer dilution inactive protein solution;
B) handle albumen after the dilution with the denaturing agent SDS of capacity and reductive agent L-halfcystine, to dissolve simultaneously and to reduce said albumen;
C), make the density loss about 70% to about 90% of SDS and L-halfcystine in the resulting solution by filtering thoroughly with respect to second ealkaline buffer;
The sex change and the reductive albumen of d) oxidation (c) step are to form the disulfide linkage consistent with naturally occurring biological activity protein.
2, according to the process of claim 1 wherein step (a) and (c) in the concentration of damping fluid be about 35mM about 60mM extremely, the pH value is about 9 to about 11.
3, according to the method for claim 2, wherein step (a) and (c) in the concentration of damping fluid be about 40mM about 50mM extremely, the pH value is about 9.6 to about 10.
4, according to the process of claim 1 wherein that after the damping fluid dilution of step (a), the concentration of inactive protein is that about 0.3mg/ml is to about 0.6mg/ml.
5, according to the process of claim 1 wherein that denaturing agent is about 0.05% to about 1.0% SDS.
6, according to the method for claim 5, wherein denaturing agent is about 0.2%SDS.
7, according to the process of claim 1 wherein that reductive agent is the L-halfcystine of about 5mM to about 25mM.
8, according to the method for claim 7, wherein reductive agent is the L-halfcystine of about 15mM.
9, according to the process of claim 1 wherein that the decline of said SDS and L-semicystinol concentration is to take place by filtering thoroughly to said second damping fluid of about 3 times of volumes with respect to about 0.5.
10, according to the process of claim 1 wherein that the oxidation of step (d) takes place in the presence of air.
11, according to the process of claim 1 wherein that protein is tethelin.
12, according to the method for claim 11, wherein tethelin is ox, pig, bird, sheep or human growth hormone.
13, according to the method for claim 12, wherein tethelin is Porcine somatotropin.
14, according to the process of claim 1 wherein that said inactive protein exists with insoluble inclusion body form.
15, according to the process of claim 1 wherein said inactive protein be oxidation, false folding with the useless albumen of accumulative.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US90814692A | 1992-07-02 | 1992-07-02 | |
| US908,146 | 1992-07-02 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1083071A true CN1083071A (en) | 1994-03-02 |
| CN1039330C CN1039330C (en) | 1998-07-29 |
Family
ID=25425274
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN93108052A Expired - Fee Related CN1039330C (en) | 1992-07-02 | 1993-07-01 | A process for recovering a recombinant protein, in biologically active form, from a solution containing inactive protein |
Country Status (7)
| Country | Link |
|---|---|
| EP (1) | EP0650497A1 (en) |
| CN (1) | CN1039330C (en) |
| AU (1) | AU4665193A (en) |
| PL (1) | PL306858A1 (en) |
| TW (1) | TW235965B (en) |
| WO (1) | WO1994001453A1 (en) |
| ZA (1) | ZA934780B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318443C (en) * | 2000-05-16 | 2007-05-30 | 博尔德生物技术公司 | Methods for refolding proteins containing free cysteine residues |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080076706A1 (en) | 1997-07-14 | 2008-03-27 | Bolder Biotechnology, Inc. | Derivatives of Growth Hormone and Related Proteins, and Methods of Use Thereof |
| US6753165B1 (en) | 1999-01-14 | 2004-06-22 | Bolder Biotechnology, Inc. | Methods for making proteins containing free cysteine residues |
| US7153943B2 (en) | 1997-07-14 | 2006-12-26 | Bolder Biotechnology, Inc. | Derivatives of growth hormone and related proteins, and methods of use thereof |
| EP1881005B1 (en) | 1997-07-14 | 2013-04-03 | Bolder Biotechnology, Inc. | Derivatives of G-CSF and related proteins |
| US7270809B2 (en) | 1997-07-14 | 2007-09-18 | Bolder Biotechnology, Inc. | Cysteine variants of alpha interferon-2 |
| BRPI9913018B8 (en) * | 1998-08-27 | 2021-07-06 | Lg Chemical Ltd | process for the preparation of active somatotropin from inclusion bodies. |
| US8288126B2 (en) | 1999-01-14 | 2012-10-16 | Bolder Biotechnology, Inc. | Methods for making proteins containing free cysteine residues |
| WO2008076933A2 (en) | 2006-12-14 | 2008-06-26 | Bolder Biotechnology, Inc. | Long acting proteins and peptides and methods of making and using the same |
| WO2017019776A1 (en) * | 2015-07-27 | 2017-02-02 | Purdue Research Foundation | Tandem folding methods to improve protein folding yield |
| EP3344651B1 (en) | 2015-09-02 | 2022-03-02 | Merck Sharp & Dohme Corp. | A process for obtaining insulin with correctly formed disulfide bonds |
| CN111610324B (en) * | 2020-06-06 | 2022-10-14 | 北京均大检测科技有限公司 | RIPA lysate and preparation method thereof |
| CN114878728A (en) * | 2022-05-06 | 2022-08-09 | 浙江大学 | Novel antibody peptide pattern detection reduction method |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4931543A (en) * | 1987-05-11 | 1990-06-05 | Cetus Corporation | Process for recovering microbially produced interleukin-2 |
| AU2257488A (en) * | 1987-07-29 | 1989-03-01 | Schering Biotech Corporation | Purification of human interleukin-4 expressed in escherichia coli |
| JPH01132598A (en) * | 1987-10-14 | 1989-05-25 | Pitman Moore Inc | Method for promoting production of intramolecular disulfide bond in recominant protein contained in modifier solution |
| US5064943A (en) * | 1988-12-16 | 1991-11-12 | American Cyanamid Company | Method for solubilization and naturation of somatotropin |
| AU664021B2 (en) * | 1990-09-05 | 1995-11-02 | Natinco Nv | Solubilization of proteins in active forms |
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1993
- 1993-07-01 WO PCT/US1993/006351 patent/WO1994001453A1/en not_active Application Discontinuation
- 1993-07-01 AU AU46651/93A patent/AU4665193A/en not_active Abandoned
- 1993-07-01 PL PL93306858A patent/PL306858A1/en unknown
- 1993-07-01 CN CN93108052A patent/CN1039330C/en not_active Expired - Fee Related
- 1993-07-01 EP EP93916974A patent/EP0650497A1/en not_active Ceased
- 1993-07-02 TW TW082105281A patent/TW235965B/zh active
- 1993-07-02 ZA ZA934780A patent/ZA934780B/en unknown
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1318443C (en) * | 2000-05-16 | 2007-05-30 | 博尔德生物技术公司 | Methods for refolding proteins containing free cysteine residues |
Also Published As
| Publication number | Publication date |
|---|---|
| AU4665193A (en) | 1994-01-31 |
| EP0650497A1 (en) | 1995-05-03 |
| ZA934780B (en) | 1994-03-17 |
| TW235965B (en) | 1994-12-11 |
| PL306858A1 (en) | 1995-04-18 |
| CN1039330C (en) | 1998-07-29 |
| WO1994001453A1 (en) | 1994-01-20 |
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