CN1219793C - Method for renaturation of protein - Google Patents
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- CN1219793C CN1219793C CN 02156513 CN02156513A CN1219793C CN 1219793 C CN1219793 C CN 1219793C CN 02156513 CN02156513 CN 02156513 CN 02156513 A CN02156513 A CN 02156513A CN 1219793 C CN1219793 C CN 1219793C
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Abstract
The present invention relates to a method for renaturing protein by using a composite system of penetration matters and hydrophobic chromatography. The method comprises the following basic processes: denatured protein is absorbed to a hydrophobic chromatography column in a gradient mode by using salt with high concentration and a denaturing agent; then, the denatured protein is eluted by penetration matters with a certain concentration and is gradually renatured in the process of elution; protein with natural activity is formed after correct folding. The method has the advantages of simple and convenient operation and convenient large-scale application, has the capability for obtaining active protein with high concentration and keeps very high protein recovery rate and active recovery rate.
Description
Technical field
The present invention relates to a kind of method of recombinant protein matter, relate in particular to a kind of novel method that adopts the combined system of permeating matter and hydrophobic chromatography to make the natural protein renaturation of inclusion body protein matter or sex change.
Background technology
Current biotechnology has obtained development at full speed, and one of them key areas is exactly the protein with gene recombination technology mass production nature and trace thereof.Prokaryotic organism such as intestinal bacteria are owing to its fast growth, and nutritional requirement is low, normally the first-selected host bacterium of gene recombination technology.Yet, usually form the inclusion body of insoluble and non-activity with the exogenous protein of escherichia coli high-level expression, need people earlier with denaturing agent with solubilization of inclusion bodies, and then remove denaturing agent, make it recover its natural radioactivity, this operating process is called as the Protein Folding renaturation.There is a contradiction that is difficult to be in harmonious proportion in protein in folding renaturation, that be exactly Protein Folding and gathering be a competition process.When the combination of hydrophobic grouping occurs in the protein molecule, be folding, occur in and assemble exactly when intermolecular.The latter is an irreversible process, often forms insoluble precipitation.Protein Folding is monomolecular first order reaction, and gathering is bimolecular or multimolecular reaction, is generally secondary and even more high-grade reaction, therefore along with the raising accumulative tendency of protein concn will be greater than folding tendency.Engineered industrialization at first require can be under high density renaturation, protein concentration is crossed and lowly can be increased the loaded down with trivial details of aftertreatment undoubtedly, raises the cost; Secondly, guarantee high activity yield, promptly the product sex change and the natural structure of renaturation are identical.Once more, need high protein yield, reduce the aggregate and precipitate in the protein folding procedure as far as possible.
The classical way of protein renaturation generally adopts dilution method and dialysis method, the former directly adds renaturation buffer with denatured protein, often forms a large amount of precipitations under the high protein concentration, thereby requires very low protein concentration concentration during renaturation, and latter's complex operation is unfavorable for amplifying.The ultrafiltration process renaturation can be replaced denaturing agent with renaturation buffer continuously, denaturing agent concentration is slowly reduced, and protein concentration can obviously not reduce, and is convenient to automated operation, but shearing force may cause the sex change and reduce activity yield again of the good protein of renaturation.Other improved classical refolding method also has pulse renaturation, dropping renaturation and film pipe stream to add renaturation, and they all can not fundamentally solve the problem of aggregate and precipitate.
Document (M.Matsubrara .Nohara, E.Kurimoto, Y.Kuroda, T.Sakai, Chem.Pharm.Bull.41 (1993) 1207; S.Tandon, P.Horowitz, J Biol.Chem.261 (1986) 15615; J.L Cleland., S.E.Builder.J.R.Swartz, M.Winkler, J.Y.Chang, Bio/technol.10 (1992) 1013; Sharma, Ajit, Karuppiah, U.S.Patent. (1995) 5728804) in solution, add the solubility promoter of lower concentration during the report dilution refolding, as 2～3M urea, 1～2M Guanidinium hydrochloride, infiltration matter (comprising polyvalent alcohol, amino acid etc.), tensio-active agent (CATB, phosphatide etc.), can improve active the recovery, be only applicable to a few protein, and the protein concentration of renaturation is still lower but the result shows these methods.Someone is used for proteinic external folding renaturation from the folding in vivo principle of peptide chain with molecular chaperones and folding enzymes, obtained good effect, but this method has increased production cost undoubtedly, therefore is unfavorable for extensive use.
In recent years, the method for solid phase renaturation and chromatography renaturation is suggested and comes into one's own gradually.Because it has following superiority: (1) reduces the mutual accumulative chance of protein, improves protein renaturation concentration; (2) be convenient to remove denaturing agent; (3) break renaturation reaction of degeneration balance, make the renaturation reaction continue to carry out; (4) renaturation and purifying are carried out synchronously; (5) be convenient to automatization, large-scale production.
At present, the method of adopted solid phase and chromatography renaturation mainly contains: (1) chemical bonding fixing protein renaturation, finger with protein with being covalently bound on the chromatography substrate, make its renaturation with the renaturation buffer flushing then, this method is only applicable to experimental study, because bonded protein can't discharge under the active prerequisite of assurance; (2) affinity chromatography renaturation, comprise immobilized molecules companion, immobilized liposome body, sulfydryl aglucon covalent chromatography, substrate aglucon affinity chromatography, metal chelate chromatography etc., they or can induce effect in conjunction with unsettled folding intermediate or to proteinic renaturation, can quicken the formation in active centre, thereby improve activity yield; (3) gel-filtration renaturation can be easily separately make protein renaturation with protein and denaturing agent, and good its kinetics radius of protein of renaturation is often less than denatured protein, can enter more in the porous, thereby impel the renaturation reaction to be tending towards complete; (4) ion exchange chromatography renaturation utilizes proteinic electric charge that it is adsorbed, and makes its renaturation by the denaturing agent concentration that changes eluent, and its advantage also is to prevent proteinic gathering effectively.Wherein gel-filtration method is used at most, but it has the shortcoming similar to sepn process, and that is exactly that upward the sample volume can not be too big, thereby treatment capacity is less, is not suitable for scale operation equally.Ion exchange method be because the principle of electric charge absorption can adsorb relatively large protein, but along with the reduction of denaturing agent concentration in the chromatography column, the denatured protein of contiguous absorption can be assembled equally, and causes the decline of activity recovery.And other method is also very immature, only a few eggs white matter has been carried out exploratory study under lower concentration.
Hydrophobic chromatography is a kind of technology commonly used in the protein separation field, as document (S.Hjertdn (1978) .J.Chromatogr., 159,85) described, hydrophobic chromatography is that the isolating mixing protein of desire is under high salt concn combined with hydrophobic ligand, reduce the concentration of salt then gradually, mixing protein can according to the difference of its surface hydrophobicity by weak to by force successively wash-out carry out isolating a kind of chromatography method.Hydrophobic chromatography is owing to adopt the water salt system, avoided with an organic solvent that (reversed phase chromatography is owing to adopt organic solvent as eluent, often cause proteinic sex change), make more to help keeping activity of proteins, become and ion exchange chromatography strong favourable protein separation means of complementary mutually.The aglucon of hydrophobic chromatography generally includes ether base (Ether), butyl (Butyl), octyl group (Octyl), phenyl (Phenyl), sec.-propyl (Isopropyl) etc., the polarity of aglucon and in conjunction with density the hydrophobic intensity of hydrophobic chromatography is had material impact on matrix.
Summary of the invention
The objective of the invention is to overcome the shortcoming of prior art; Improve activity of proteins again in order to reach when improving the protein yield; And denatured protein can be in renaturation, the sedimentary purpose of difficult generation under the high density; Thereby provide a kind of simple to operate, method of combined system recombinant protein matter of utilizing hydrophobic chromatography and infiltration matter.
Method of the present invention is to utilize the combined system recombinant protein matter of hydrophobic chromatography and infiltration matter, comprises the steps to realize;
(1) column equilibration: with the conventional hydrophobic chromatoghaphy medium of moving phase I balance; Moving phase I is that salt concn is the gradient buffering liquid of the damping fluid of 0～4M, the damping fluid of pH value 2～7, the damping fluid that contains reductive agent, denaturing agent～salt, damping fluid and the renaturation buffer that salt concentration is 0～4M and denaturing agent;
(2) absorption of denatured protein: with the 4～10M urea that contains 0～200mM reductive agent or 2～7M guanidine hydrochloride denaturation damping fluid dissolving inclusion body or natural protein, be adsorbed onto on the conventional hydrophobic chromatography post of moving phase I equilibrated, make the hydrophobic position of denatured protein combine and not combine, thereby avoid intermolecular gathering with other proteinic hydrophobic position with hydrophobic grouping on the chromatography media;
(3) the wash-out renaturation of denatured protein: after the natural protein of inclusion body protein matter lysate or sex change is adsorbed by the hydrophobic medium of above-mentioned selection in the step (2), with 0～5 column volume of moving phase I flushing, the elutriant with moving phase II and the infiltration matter that contains 1～70% (W/V or V/V) washes chromatography column then; Or the protein wash-out adopts the infiltration matter～denaturing agent of 0～10 column volume of elder generation's operation or the gradient of organic solvent (comprising urea, Guanidinium hydrochloride, ethylene glycol, methyl alcohol, ethanol etc.), the wetting ability of environment in the drainage column is from top to bottom strengthened (as shown in Figure 1) successively, change the denaturing agent～infiltration matter gradient of gradient direction again from 0～10 column volume, always with containing the moving phase II wash-out that permeates matter, make the protein desorb and form natural structure gradually then; Elution flow rate is 0.1～1CV/min (CV is a column volume); Wherein moving phase II can be the renaturation buffer (as phosphoric acid salt, Tris-hydrochloride etc.) of pH value between 7～9.
Preferred salt in the described method steps (1) is inorganic salt such as ammonium sulfate, sodium-chlor etc.; Denaturing agent is preferably 4～10M urea or 2～7M Guanidinium hydrochloride; Described reductive agent is the compound that contains free sulfhydryl groups, can be mercaptoethanol, dithiothreitol (DTT) or two sulphur erythrose alcohol etc.; Preferred hydrophobic medium is butyl-agarose medium (Butyl Sepharose FF), phenyl sepharose medium (Phenyl Sepharose FF), octyl sepharose medium (Octyl Sepharose FF) or phenyl ether organic polymer medium (Poros PE) etc.
Infiltration matter in the described method steps (3) is the water-soluble cpds that a class can be repelled the protein hydrophobic grouping, can be polyhydroxy-alcohol (as glycerine or N.F,USP MANNITOL), carbohydrate (as sucrose or seminose), high molecular weight water soluble polymer or amino acid such as polyoxyethylene glycol; When infiltration matter was solid, it accounted for 1～70% (W/V) at the elutriant of moving phase II and the infiltration matter that contains, and when infiltration matter was liquid state, it accounted for 1～70% (V/V) at the elutriant of moving phase II and the infiltration matter that contains.
The present invention also further comprises the regeneration of hydrophobic chromatography post, with the chromatography column among high density denaturing agent (comprising the organic solvent of 4～8M urea, 2～7M Guanidinium hydrochloride or 0～100% (V/V) etc.) the flushing above-mentioned steps B, the a small amount of absorption of wash-out is the protein in jail extremely, to recover its adsorption site.
Hydrophobic chromatography can be used for recombinant protein matter, is because outside the hydrophobic grouping of the natural protein of sex change all is exposed to.Have artificial molecule companion's characteristic when hydrophobic chromatography recombinant protein matter, it not only can adsorb the natural protein of sex change but also can adsorb folding intermediate.A defective when being used for protein renaturation, hydrophobic chromatography is arranged, that is exactly the variation of the hydrophobic aglucon existence around protein can causing system's free energy, because the protein interior hydrophobic grouping interosculate and the adsorption of protein and hydrophobic medium is an individual competition process, therefore it may destroy the protein folding thermodynamic(al)equilibrium, it is folding to lead to errors, and its degree is aggravated with the hydrophobic intensity of hydrophobic aglucon.Solving this contradiction has two kinds of methods, and a kind of is the weak hydrophobicity ligand medium of synthesizing new; Another is to adopt conventional hydrophobic medium, and manages to change the buffering system of protein folding renaturation, improves the extent of hydration of natural protein in renaturation process of sex change, makes its final activated protein that forms the tool natural structure.Adopt first method can improve use cost undoubtedly, and, be unfavorable for the mass-producing renaturation because the loading capacity of weak hydrophobic medium is lower.The key of second method is to seek good hydrated agent.Some proteinic stablizers have good aquation effect, and they mostly are infiltration matter.Proteinic stability with become, closely related between the renaturation, the forfeiture of general stability be since in bury exposing of hydrophobic grouping, cause aggregate and precipitate, this natural protein and folding intermediate thereof to sex change is similar.The principle of infiltration matter stabilizing protein is that hydrophobic grouping is had repellency, therefore impels water molecules to enter around the hydrophobic grouping, just aquation.Therefore if will permeate matter when being used for hydrophobic chromatography recombinant protein matter, may have the aquation effect equally, thus the activity recovery of raising renaturation.
Step (3) principle is to change denaturing agent concentration and salt concn earlier, to weaken the intensity of protein and hydrophobic chromatoghaphy medium absorption, make proteinic hydrophobic position break away from the hydrophobic grouping of medium gradually, combination takes place in the hydrophobic position of same intramolecular difference, thereby starts the folding of protein molecule.In the folding initial stage, the hydrophobic grouping that protein exposes is more, easily assemble, but because the hydrophobic environment that hydrophobic aglucon causes has suppressed this tendency, thereby improved protein recovery.Contain the moving phase II conduct of permeating matter, make protein in post when mobile the wetting ability of environment strengthen gradually, the influence of hydrophobic aglucon weakens gradually, the protein in guiding renaturation later stage to correct folding pathway, thereby improve activity recovery.
The invention has the advantages that the natural protein absorption carrier of the conventional hydrophobic medium of the high carrying capacity of (1) employing, improved the renaturation treatment capacity of medium, reduced use cost as sex change; (2) natural protein of sex change can be in renaturation under the high density, and is difficult for producing aggregate and precipitate, has significantly reduced renaturation sample difficulty of post-processing, has improved protein yield simultaneously again; (3) employing contains the wash-out moving phase of permeating matter, has obtained the high reactivity yield in the high protein yield.(4) easy and simple to handle, be convenient to large-scale application.
Description of drawings
Fig. 1 is for after the sex change N,O-Diacetylmuramidase is adsorbed onto on the Poros PE 50 hydrophobic chromatography posts, with containing moving phase II and the denaturing agent gradient elution renaturation synoptic diagram that permeates matter.
Fig. 2 causes the active difference that reclaims for different glycerol concentration among the moving phase II among the inventive method embodiment 1.
Fig. 3 is the comparison of adopting renaturation N,O-Diacetylmuramidase of the present invention and dilution method renaturation among the inventive method embodiment 1.
Fig. 4 is for adopting the chromatography accompanying drawing of renaturation N,O-Diacetylmuramidase among the inventive method embodiment 1.
Fig. 5 is for adopting the chromatography accompanying drawing of renaturation human lysozyme inclusion body among the inventive method embodiment 2.
Embodiment 1:
The renaturation of sex change N,O-Diacetylmuramidase on the Poros of glycerine wash-out PE 50 hydrophobic chromatography posts
(128mm * 10mm I.D is 10mL) with moving phase I (3.6M (NH with Poros PE 50 perfusion hydrophobic chromatography posts
4)
2SO
4, 50mMTris-HCl, 1mM EDTA, 3mM GSH, 0.3mMGSSG, pH8.5) balance, operation moving phase I～denaturing agent A (8M urea, 50mMTris-HCl, 1mMEDTA, 3mM GSH, 0.3mM GSSG, pH8.5) gradient 2ml, last with denaturing agent B (6M Guanidinium hydrochloride, 50mM Tris-HCl, 1mMEDTA, the 1%2-mercaptoethanol, pH8.5) the N,O-Diacetylmuramidase 10mg of dissolving 50mg/ml concentration with moving phase I flushing, makes denatured protein absorption, change elutriant (50% glycerine, the 0.4M (NH of moving phase II and infiltration matter then
4)
2SO
4100mMTris-HCL, 1mMEDTA, 0.1%2-mercaptoethanol, pH8.5) column volume of flushing, successively move moving phase II～denaturing agent C (8M urea, 50mMTris-HCl, 1mMEDTA again, 0.1%2-mercaptoethanol, pH8.5) the gradient 4ml of gradient 4ml and denaturing agent C～moving phase II continues the elutriant wash-out with moving phase II and infiltration matter, and elution flow rate is 1ml/min (as shown in Figure 1).
As shown in Figure 2, the present invention adopts infiltration matter to improve hydrophobic chromatography renaturation activity of lysozyme significantly as eluent and reclaims, and active the recovery reaches more than 85% when 50% glycerol concentration, and active recovery of hydrophobic chromatography has only 50.9% when not having glycerine.
As shown in Figure 3, adopt renaturation N,O-Diacetylmuramidase of the present invention to compare with dilution method, the present invention is not subjected to the influence of initial protein concentration substantially, so the present invention can adopt concentration up to the direct upper prop renaturation of the denatured protein of 50mg/ml.
Fig. 4 is for adopting the chromatography accompanying drawing of renaturation N,O-Diacetylmuramidase among the inventive method embodiment 1.
Table 1 is the comparison of the gel-filtration renaturation N,O-Diacetylmuramidase of the present invention and bibliographical information.Adopt renaturation N,O-Diacetylmuramidase of the present invention, initiation protein concentration, recombinant protein final concentration and annealing efficiency (amount of unit volume chromatography media recombinant protein matter) show that apparently higher than the latter the present invention has great using value.
Table 1
| Chromatography column volume (ml) | Initial protein concentration (mg/ml) | Applied sample amount (mg) | Recombinant protein final concentration (mg/ml) | Annealing efficiency (mg albumen/ml medium) | |
| Refolding method gel-filtration refolding method of the | 10 467.21 | 50 9.6 | 10 14.5 | 1.2 0.18 | 1 0.032 |
Embodiment 2:
The renaturation of human lysozyme solubilization of inclusion bodies liquid on the Poros of glycerine wash-out PE 50 hydrophobic chromatography posts
(128mm * 10mm I.D is 10mL) with moving phase I (3.6M (NH with Poros PE 50 chromatography columns
4)
2SO
4, 50mMTris-HCl, 1mM EDTA, 0.1% 2 mercapto ethanol, pH8.5) balance, operation moving phase I～denaturing agent A (8M urea, 50mMTris-HCl, 1mMEDTA, 3mMGSH, 0.3mM GSSG, pH8.5) gradient 2ml, on contain denaturing agent B (6M Guanidinium hydrochloride, 50mMTris-HCl, 1mMEDTA, 1%2-mercaptoethanol, pH8.5) human lysozyme lysate 2mg is with moving phase I flushing, make denatured protein absorption, change moving phase II (50% glycerine, 0.4M (NH then
4)
2SO
4100mM Tris-HCL, 1mMEDTA, 0.1%2-mercaptoethanol, pH8.5) column volume of flushing, successively move moving phase II～denaturing agent C (8M urea, 50mMTris-HCl, 1mMEDTA again, 0.1%2-mercaptoethanol, pH8.5) the gradient 5ml of gradient 5ml and denaturing agent C～moving phase II continues the wash-out with moving phase II, and flow velocity is 1ml/min.
Its elution chromatography spectrogram as shown in Figure 5, wherein N,O-Diacetylmuramidase elution peak protein recovery reaches 85%, than the rate of recovery 90% alive.
Illustrate that the present invention can be used for reorganization inclusion body protein matter is carried out renaturation.
Embodiment 3:
The renaturation of recombinant human alpha-2b Interferon, rabbit on the Phenyl of N.F,USP MANNITOL wash-out Sepharose FF hydrophobic chromatography post
With Phenyl Sepharose FF chromatography column (25mm * 16mm I.D, 5mL) with moving phase I (0.1N acetic acid) balance, on use the 6M Guanidinium hydrochloride, 50mM Tris-HCl, 1mMEDTA, 1%2-mercaptoethanol, pH8.5 sex change reductive recombinant alpha-2b Interferon, rabbit inclusion body is with moving phase I flushing, make denatured protein absorption, change moving phase II (20% N.F,USP MANNITOL, 0.2MNaCl, 20mMNa then
2HPO
4, 0.01%2-mercaptoethanol) and column volume of flushing, successively move moving phase II～denaturing agent C (8M urea, 10mM Na again
2HPO
4, 0.01%2-mercaptoethanol) gradient 5ml and the gradient 5ml of denaturing agent C～moving phase II, continue wash-out with moving phase II, flow velocity is 2ml/min, the α of wash-out-2b Interferon, rabbit is 1.5 * 10 than work
8IU/mg, the gross activity yield reaches 80%.
Illustrate that the present invention can use on various hydrophobic chromatography posts.
Embodiment 4:
The renaturation of recombinant methionyl human G-CSF (rhG-CSF) on the Poros ET hydrophobic chromatography post of polyoxyethylene glycol (PEG) wash-out
With Poros ET chromatography column (50mm * 16mm I.D, 10mL) with moving phase I (0.1N acetic acid) balance, on use the 6M Guanidinium hydrochloride, 50mM Tris-HCl, 1mMEDTA, 1%2-mercaptoethanol, pH8.5 sex change reductive reorganization rhG-CSF inclusion body is with moving phase I flushing, make denatured protein absorption, change moving phase II (5% (W/V) PEG4000,0.2M NaCl, 20mMNa then
2HPO
4, 0.01%2-mercaptoethanol) and flushing, flow velocity is 5ml/min, the rhG-CSF of wash-out is 1.0 * 10 than work
8IU/mg, the gross activity yield reaches 90%.
Claims (4)
1. the method for a recombinant protein matter utilizes the combined system of hydrophobic chromatography and infiltration matter to carry out, and comprises the steps:
(1) column equilibration: with the conventional hydrophobic chromatoghaphy medium of moving phase I balance; Wherein said moving phase I is 3.6M (NH
4)
2SO
4, 50mM Tris-HCl, 1mM EDTA, 3mM GSH, 0.3mM GSSG, pH8.5, or 3.6M is (NH
4)
2SO
4, 50mM Tris-HCl, 1mMEDTA, 0.1%2-mercaptoethanol, pH8.5, or 0.1N acetic acid;
(2) absorption of denatured protein: sex change damping fluid dissolving inclusion body or natural protein with the 4～10M urea that contains 0～200mM reductive agent or 2～7M Guanidinium hydrochloride are adsorbed onto on the moving phase I equilibrated hydrophobic chromatography post;
(3) the wash-out renaturation of denatured protein: when the inclusion body protein matter of adsorbing in the elution step (2) or the natural protein of sex change, adopt the elutriant flushing chromatography column of moving phase II and the infiltration matter that contains 1～70% (V/V) earlier, move 0.4 column volume of gradient of moving phase II～denaturing agent C then, the wetting ability of environment in the drainage column is from top to bottom strengthened successively, change 0.4 column volume of gradient of gradient direction operation denaturing agent C～moving phase II again, continue at last to make the protein desorb and form natural structure gradually with moving phase II and the elutriant wash-out that contains the infiltration matter of 1～70% (V/V); Elution flow rate is 0.1 column volume/min;
Described denaturing agent is the 8M urea, 50mM Tris-HCl, 1mM EDTA, 3mM GSH, 0.3mM GSSG, pH8.5; Or the 6M Guanidinium hydrochloride, 50mM Tris-HCl, 1mM EDTA, 1%2-mercaptoethanol, pH8.5; The 8M urea, 50mMTris-HCl, 1mMEDTA, 0.1%2-mercaptoethanol, pH8.5; Or the 8M urea, 10mM Na
2HPO
4, the 0.01%2-mercaptoethanol;
Described moving phase II is 0.4M (NH
4)
2SO
4, 100mM Tris-HCL, 1mMEDTA, 0.1%2-mercaptoethanol, pH8.5; Or 0.2M is NaCl, 20mM Na
2HPO
4, the 0.01%2-mercaptoethanol;
Described infiltration matter is polyhydroxy-alcohol, polyoxyethylene glycol or amino acid.
2. the method for recombinant protein matter according to claim 1 is characterized in that reductive agent is the compound that contains free sulfhydryl groups.
3. the method for recombinant protein matter according to claim 2, the compound that it is characterized in that containing free sulfhydryl groups is mercaptoethanol or reduced glutathion.
4. the method for recombinant protein matter according to claim 1 is characterized in that polyhydroxy-alcohol is glycerine or N.F,USP MANNITOL.
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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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| CN1309826C (en) * | 2004-10-27 | 2007-04-11 | 北京北大维信生物科技有限公司 | Nattokinase purification process and microcapsule formulation process |
| 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 |
| DE102010026094B4 (en) * | 2010-07-05 | 2012-01-12 | Sigeng Han | A new method for characterizing and multidimensionally representing the folding process of proteins |
| 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 |
| CN116174044B (en) * | 2023-02-21 | 2024-07-02 | 集美大学 | New preparation method and application of artificial metalloenzyme with protein framework |
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