CN104725464A - Protein product reuse method - Google Patents
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Abstract
The invention relates to a protein product reuse method. The method includes the steps of: conducting PEG (polyethylene glycol) modification on protein, when the activity decreases and the product is out of the period of validity, subjecting the product to thorough denaturation treatment, and performing refolding. The method provided by the invention has the advantages of enhancing the times of reutilization and reducing the cost. Also, in the refolding step, the concentration and the success rate of renatured protein can be effectively improved. The method has the advantages of simple process, no special additive requirement, and low cost, can be used for reuse of protein products in daily chemical, industry and other aspects so as to deal with harsh working conditions and higher cost reduction demand.
Description
Technical field
The invention belongs to biological technical field, be specifically related to the method for a kind of proteinaceous product recycling, comprise and protein PEG is modified, treat active reduction, after product exceedes validity period, it is thoroughly done denaturing treatment, and the step of refolding.Its advantage is, can improve the recycling number of times of proteinaceous product, reduce costs.And, in refolding steps, effectively can improve concentration and the success ratio of recombinant protein.
Technical background
Compared with chemical products, the problem that proteinaceous product all faces structural instability, reduces fast along with time lengthening is active, thus cause proteinaceous product validity period short, on the high side.
This unstable have from physical factor, have from chemical factor.Modal unstable is exactly under certain conditions, and the secondary of protein, three grades and quaternary structure may change, and this can cause protein aggregation.Protein aggregation can cause active loss or reduction, reduces solvability.Under many circumstances, the gathering of protein is the intermolecular cross-linking of the protein chain due to partially denaturing.There are some researches show that polymerization may betide protein specific transitions state conformation, instead of nonspecific combination.Accumulation process is divided into three steps substantially: cause, and transmits and stops.Protein aggregation thermodynamically can reduce the detrimental action between the hydrophobic residue of solvent and protein exposure.Hydrophobic interaction pair and protein folding and to assemble be all main motivating force.Folding and the gathering of protein all represents the one balance of the hydrophobic residue being exposed to protein surface and burying active site of protein.This balance very meticulous, the amino acid changed in protein may change its Assembling Behavior completely.
The gathering of protein can have a lot of physical factor inductions, such as temperature, ionic strength, eddy current, surface or interfacial adsorption etc.These factors can increase the hydrophobic surface of albumen, can cause the gathering of protein.Such as, recombinant human keratin cell growth factor (rhKGF) is the slow sex change of meeting in temperature-rise period, can cause protein aggregation and then sedimentation.Concentration is that the pig recombinant human growth hormone of 0.5mg/ml can precipitate 63 DEG C time.Vigorous stirring people recombinant growth factors solution (0.5mg/ml, pH7.4) has the protein aggregation of 67% to be soluble albumen after one minute.
Chemical degradation and modification can expose the hydrophobic surface of protein, also can cause the gathering of protein.Protein particulate directly forms the polymer of chemical bonding, as Regular Insulin; Also assemble after can there is chemical transformation, such as people's relaxin is assembled after His and Met is oxidized.Physics and chemistry polymerization also may occur simultaneously.Such as can there is the bonding of disulfide linkage and assemble in cryodesiccated beta galactose enzyme in storage, simultaneously because the interaction of non-chemically bonding also can form the aggregate of solubility.
The gathering of protein can by single molecule (single molecule or intramolecular process), also can by multiple molecule.Single molecular process comprises the dislocation of β-elimination and interchain disulfide bond, the interleukin-1 receptor antagonist in the such as aqueous solution.Polymolecular accumulation process comprises the sulfydryl disulfide exchange of bovine serum albumin, the disulfide exchange of the sulfydryl catalysis of Regular Insulin.The example forming covalency polymerization also has ribonuclease A, and the example of non-covalent polymerization has Toxoid,tetanus.If along with the increase of protein concentration, the ratio shared by aggregate is in increase, then this process may relate to polymolecular participation.
To sum up, proteinaceous product itself can be produced physics different from storing link, the impact of chemical factor loses activity gradually.
For above problem; polyoxyethylene glycol (PEG) modification technique arises at the historic moment, by by polymer PEG chain, with protein bound; thus the impact of shielding and weakened part physics and chemistry effect, thus play the effect of protected protein matter, prolongation keeping life.PEG modification technique is full-fledged, and in biological medicine have trial and have launch, the PEG modified interferon of such as Schering Plough company more.But for aspects such as daily use chemicals and industry, because the working condition of harshness and the cost of Geng Gao reduce demand, need to explore the method for extending the expiration date or reusing further.
The present invention is then the angle from PEG modified protein and protein refolding processes, develops a kind of new method, comprises and being modified by protein PEG, treats active reduction, after product exceedes validity period, it is thoroughly done denaturing treatment, and the step of refolding.Its advantage is, can improve the recycling number of times of proteinaceous product, reduce costs.And, in refolding steps, effectively can improve concentration and the success ratio of recombinant protein.Concrete foundation is as follows:
Polyoxyethylene glycol (polyethylene glycol, PEG) is by the oxyethylene group (-CH much repeated
2-CH
2-O-) structural unit composition linear or branched neutralised inert high polymer, its CAS registration number is 25322-68-3, and chemical structure is as follows.
HO-(CH
2-CH
2-O)
n-1-CH
2-CH
2-OH
PEG has two activity hydroxies, for avoiding, in the process of protein modification, crosslinking reaction occurs, the polymkeric substance that people often select one end to be closed by methyl and mono methoxy polyethylene glycol (monomethoxyl polyethylene glycol, mPEG) carry out chemically modified to protein.But the end of mPEG is hydroxyl, and reactive behavior is more weak, can not be modified under mild conditions on the amino-acid residue of protein.So first derivatization treatment should be carried out to the terminal hydroxyl of mPEG, by hydroxy activated electroactive higher functional group of getting married, make it to react with the amino of protein or polypeptide, sulfydryl or other nucleophilic group under mild conditions, and do not damage its biologic activity.
When PEG modified protein experience sex change-refolding/renaturation process, PEG chain can effectively promote refolding efficiency, and suppress the rendezvous problem that this process is common, this is because (1) PEG hydration can occur in aqueous, each ethyl oxide unit (-CH2-CH2-O-) can tightly in conjunction with 2-3 water molecules.This hydration of PEG also can raise along with the increase of its molecular weight, when the continuation of molecular weight raises, can autofolding be there is in the solution in PEG chain, form the folding segment of more analogous protein or polypeptide secondary structure, these PEG chain segment by interact also can loosely in conjunction with water molecules free in bulk solution.Thus, make PEG modified protein in renaturation process, fast development becomes " independent individual "; (2) this hydration of PEG also shows larger spatial stability effect in the solution.This stereoeffect is in particular in elastic contribution aspect.When the protein surface that another macromolecular substance is modified close to PEG, because the motion of PEG chain segment is fettered, the loss of conformational entropy is caused to produce elastic repulsion force thus pushed open by macromole.The macromole that these power make other molecules in solution be difficult to modify with PEG fully collides and contacts, thus effectively suppresses the formation of aggregate in renaturation process.(3) the segment kindliness of PEG molecule is another important physical basis of its good nature.The fragment kindliness that PEG is good makes it constantly change conformation to adapt to the surface topology structure of protein, can promote the correct folding of target protein.
Although use, the mode of molecule mate to promote protein renaturation more report, first this proteinoid of molecular chaperones is found in 1989 by Horwich, Main Function is that slave part folds or incorrect folding protein, accelerates correct folding carrying out or provides the microenvironment needed for folding generation.But, because (1) molecular chaperones can only use in renaturation process, and limited to the facilitation effect of renaturation, cost is high; (2) in the normal life cycle of proteinaceous product; lack provide protection; after there is enzymolysis and chemical degradation, renaturation process cannot activity recovery, recycling, by contrast; the proteinaceous product of PEG covalent modification effectively can reduce the degraded to protein chain in life cycle; and denaturation renaturation process, do not need additive, renaturation concentration is high, yield significantly improves; so PEG modification being coupled with renaturation will be a kind of new way that proteinaceous product reuses.
Summary of the invention
The invention provides a kind of protein recycling method, key step is:
Non-specific amido modified: target protein to be added damping fluid I and dissolve, regulate pH to 7-9, add polyethyleneglycol modified dose, albumen and polyethyleneglycol modified dose of mol ratio are 1: 5-1: 50.Described damping fluid I is 0.02-0.2mol/L Tris-Cl or phosphate buffered saline buffer (PB), and pH7-9, under temperature constant state, modifies for 4 DEG C-37 DEG C;
N Amino End Group is modified: target protein is added damping fluid II and dissolve, and regulate pH to 4-6, add polyethyleneglycol modified dose, albumen and modifier mol ratio are 1: 1-1: 10; Described damping fluid II is 0.02-0.2mol/L NaAc-HAc or phosphate buffered saline buffer, pH4-6, under temperature constant state, modifies for 4 DEG C-37 DEG C;
Or, sulfydryl modification: target protein is added damping fluid III and dissolve, regulate pH to 7-9, add polyethyleneglycol modified dose, albumen and modifier mol ratio are 1: 1-1: 10; Described damping fluid III is 0.02-0.2mol/L Tris-Cl or boric acid-borate buffer solution, pH7-9, under temperature constant state, modifies for 4 DEG C-37 DEG C;
Described PEG modifies in the method for target protein, and non-specific amido modified polyethyleneglycol modified dose used is mPEG-SC5000, mPEG-SC10000, mPEG-SC20000; It is mPEG-ALD5000, mPEG-ALD10000, mPEG-ALD20000 that N Amino End Group modifies polyethyleneglycol modified dose used; Or used polyethyleneglycol modified dose of sulfydryl modification is mPEG-MAL5000, mPEG-MAL10000, mPEG-MAL20000.
Described PEG modifies the method for target protein, and wherein modifying the time is 3-24 hour.
Described PEG modifies the method for target protein, albumen after wherein modifying adopts ion exchange chromatography DEAE Sepharose6FF or Q Sepharose6FF purifying to modify component, moving phase is 0.05-0.2mol/L Tris-HCl or phosphate buffered saline buffer pH6-9.5, adds 0-1.5M sodium-chlor.
Target protein Activity determination, active in after the company standard lowest limit, carry out denaturation renaturation operation.Denaturation method, uses 6M Guanidinium hydrochloride or 8M urea and excessive reductive agent as mercaptoethanol, makes protein molecule become unordered loose stretch-like structure from originally orderly curling imporosity.Refolding method, mixed with dilution refolding damping fluid by metaprotein, placement is spent the night, and by reducing the content of denaturing agent and reductive agent, promotes that target protein folds gradually.Whole renaturation process, as do not added any molecular chaperones renaturation being had to enhancement without illustrating.Only when contrasting the renaturation facilitation effect of PEGylation in covalent attachment PEG and classical molecular chaperones-solution, add in renaturation buffer with covalent attachment PEG equivalent and the PEGylation of same molecular amount.The activity change of target protein after monitoring renaturation, activity marks factory higher than enterprise and requires to be then qualified product.
The invention has the advantages that, the recycling number of times of proteinaceous product can be improved, reduce costs.And, in refolding steps, effectively can improve concentration and the success ratio of recombinant protein.This recycling operation is simple, and without special additive requirement, cost is low.Be proposed to be used in the recycling of the aspect proteinaceous product such as daily use chemicals and industry, reduce demand with the cost of tackling harsh working condition and Geng Gao.
Accompanying drawing explanation
Fig. 1 is that Interferon alfacon-1 preparation process detects picture group, and A. tunning detects, and (1-2 is respectively the forward and backward full bacterium of induction; 3-4 is respectively lysis supernatant, precipitation), the ionic energy transfer of B. renaturation product, C.SDS-PAGE detects the disulfide formation situation of product;
Fig. 2 is the picture group of Interferon alfacon-1 PEG covalent modification, the separating spectrum of D.PEG pointed decoration product, and (P1, P2 distinguish chromatographic separation peak in corresponding diagram D in the SDS-PAGE detection of E. sepn process; R is modification reaction mixture), the mass spectroscopy different modifying degree of F.PEG modified outcome;
Fig. 3 denaturation renaturation process monitoring figure, A. Interferon alfacon-1, B. Interferon alfacon-1+PEGylation (molecular chaperones), C.PEG modification Interferon alfacon-1 three test group are contrasted, detect after traditional dilution refolding process, protein recovery and activity recovery;
Fig. 4 recombinant protein concentration affects surveillance map, A. Interferon alfacon-1 to renaturation effect, and C.PEG modifies Interferon alfacon-1;
Fig. 5 covalent attachment PEG promotes folding principle analysis chart, and A. tradition dilution refolding adds molecular chaperones, B. covalent attachment PEG
Embodiment
The present invention relates to the method for a kind of proteinaceous product recycling, comprise and protein PEG is modified, treat active reduction, after product exceedes validity period, it is thoroughly done denaturing treatment, and the step of refolding.For the recycling of the aspect proteinaceous products such as daily use chemicals and industry, reduce demand with the cost of tackling harsh working condition and Geng Gao.
Specifically, method of the present invention is as follows:
(1) intestinal bacteria of fermentation restructuring target protein, after bacterial cell disruption, collect supernatant liquor direct purification, or collect inclusion body by purifying acquisition target protein after renaturation.
(2) PEG modification is carried out to target protein.Wherein, non-specific amido modified: target protein to be added damping fluid I and dissolve, regulate pH to 7-9, add polyethyleneglycol modified dose, albumen and polyethyleneglycol modified dose of mol ratio are 1: 5-1: 50.Described damping fluid I is 0.02-0.2mol/L Tris-Cl or phosphate buffered saline buffer (PB), and pH7-9, under temperature constant state, modifies for 4 DEG C-37 DEG C; N Amino End Group is modified: target protein is added damping fluid II and dissolve, and regulate pH to 4-6, add polyethyleneglycol modified dose, albumen and modifier mol ratio are 1: 1-1: 10; Described damping fluid II is 0.02-0.2mol/L NaAc-HAc or phosphate buffered saline buffer, pH4-6, under temperature constant state, modifies for 4 DEG C-37 DEG C; Or, sulfydryl modification: target protein is added damping fluid III and dissolve, regulate pH to 7-9, add polyethyleneglycol modified dose, albumen and modifier mol ratio are 1: 1-1: 10; Described damping fluid III is 0.02-0.2mol/L Tris-Cl or boric acid-borate buffer solution, pH7-9, under temperature constant state, modifies for 4 DEG C-37 DEG C; Described PEG modifies in the method for target protein, and non-specific amido modified polyethyleneglycol modified dose used is mPEG-SC5000, mPEG-SC10000, mPEG-SC20000; It is mPEG-ALD5000, mPEG-ALD10000, mPEG-ALD20000 that N Amino End Group modifies polyethyleneglycol modified dose used; Or used polyethyleneglycol modified dose of sulfydryl modification is mPEG-MAL5000, mPEG-MAL10000, mPEG-MAL20000.The modification reaction time is 3-24 hour.
(3) albumen after modifying adopts ion exchange chromatography DEAE Sepharose6FF or Q Sepharose6FF purifying to modify component, and moving phase is 0.05-0.2mol/L Tris-HCl or phosphate buffered saline buffer pH6-9.5, adds 0-1.5M sodium-chlor.
(4) SDS-PAGE detection is carried out to the component that purifying obtains, determine that the PEG of target protein modifies situation, the activity of the protein modified front and back of testing goal.
(5) active in after the company standard lowest limit, carry out denaturation renaturation operation.Denaturation method, uses 6M Guanidinium hydrochloride or 8M urea and excessive reductive agent as mercaptoethanol, makes protein molecule become unordered loose stretch-like structure from originally orderly curling imporosity.Refolding method, metaprotein mixes with dilution refolding damping fluid, and placement is spent the night, and by reducing the content of denaturing agent and reductive agent, promotes that target protein folds gradually.Whole renaturation process, as do not added any molecular chaperones renaturation being had to enhancement without illustrating.Only when contrasting the renaturation facilitation effect of PEGylation in covalent attachment PEG and classical molecular chaperones-solution, add in renaturation buffer with covalent attachment PEG equivalent and the PEGylation of same molecular amount.The activity change of target protein after monitoring renaturation, activity marks factory higher than enterprise and requires to be then qualified product.
Being described further for embodiment below in conjunction with accompanying drawing, is modify Interferon alfacon-1 (bacterial classification is provided by Beijing Baichuanfeihong Biology Science Co., Ltd, patent No. ZL200810101309.4) for mPEG-MAL20000 in embodiment.
Embodiment one
The cultivation of recombinant bacterium, renaturation and purifying:
Bacterial strain is kept at-80 DEG C.Scrape the glycerine tube-surface freezed with the inoculating needle of sterilizing, then immediately the bacterium sticked on inoculating needle is drawn in the LB agar plate surface containing 100 μ g/ml Amp, in 37 DEG C of overnight incubation.Picking list bacterium colony, being inoculated in 3 is respectively equipped with in the 500ml shaking flask of 100mlLB nutrient solution, 37 DEG C, shaking table 240rpm cultivates, survey switching 3L LB liquid medium (adding 100 μ/ml Amp before switching) when OD600 reaches 1.0, cultivate under similarity condition, when OD600 reaches 1.0, proceed to 100L fermentor tank as secondary seed solution.37 DEG C of environment bottom fermentations, in fermenting process, adjusting rotary speed and air flow keep oxygen saturation at 20%-60%, induce with 1mM IPTG when OD600 is 1.0 to 2.0, and period keeps oxygen saturation at 20%-60%, pH at 7.0-7.4, (ammoniacal liquor and hydrochloric acid regulate).If there is a large amount of bubble in fermenting process, with the soybean oil 0.Sml-1.0ml froth breaking of defoamer or sterilizing.When simple dependence rotating speed and air flow can not regulate oxygen saturation at 20%-60%, then feed supplement.Induce lower tank after about 4-8 hour, OD600 is 6.0-20.0.5000rpm, 30min collected by centrifugation thalline, freezen protective or directly carry out following experiment.
Get a certain amount of thalline, fully suspend with the broken bacterium liquid of the ratio of 1: 6 (W: V), ultrasonication in ice bath, ultrasonic power 300W, ultrasonic 4 points (surpassing 5 seconds, 5 seconds, interval), stop 3 points, 5 times repeatedly.After broken bacterium, 12000rpm, 30min are centrifugal, collecting precipitation.Supernatant and precipitation all sample electrophoresis.Above-mentioned precipitation is fully suspended in the ratio inclusion body washings of 1: 5 (W/V) and forms even turbid solution, in the centrifugal 20min of 12000rpm, collecting precipitation.Same method composition washs 2 times, and the inclusion body finally washed is weighed.Each supernatant and precipitation all sample electrophoresis.After taking washing, inclusion body suspends in the ratio denaturing agent of 1: 5 (W/V) and stirs 3 hours.Then in the centrifugal 20min of 12000rpm, collect supernatant, supernatant and precipitation all sample electrophoresis.A certain amount of metaprotein, the ratio being 0.1mg/ml in final protein concentration slowly adds in corresponding renaturation buffer.All add rear room temperature and place a night.
With the buffer A balance chromatography column of 2 column volumes (CV); Whole chromatography process flow velocity is 10mL/min, 280nm monitoring.After recombinant protein dialysis desalting, adjust pH4.5, centrifugal or membrane filtration removing protein masses, then directly goes up the CM Sepharose FF post that planned balance is good, flow velocity 100cm/h, gradient elution the complete component of Fractional Collections renaturation.Fig. 1 is shown in each step monitoring above, by being oxidized and reducing SDS-PAGE electrophoresis, determines that the elution fraction collected is the Interferon alfacon-1 that renaturation is complete, structure is correct.
Embodiment two
PEG modifies and adopts Mal-mPEG modifier to carry out pointed decoration, and Mal-mPEG modifier molecular weight is 20KD, and modification condition is: damping fluid adopts Tris-HCl, pH8.5, modifier and Interferon alfacon-1 mol ratio are 10: 1, and modifying temperature is 4 degree, and the modification time is 6h.
First use sample-loading buffer (50mM Tris-HCl, pH8.5) to balance chromatography column (about 5 column volumes), get anion-exchange column on the supernatant liquor after modifying after sample filtering, loading linear rate of flow 4ml/min.Use sample-loading buffer (50mMTris-HCl, pH8.5) drip washing after treating loading, drip washing linear rate of flow is 4ml/min, is washed till 280nm and absorbs recurrence baseline.With elution buffer (50mM Tris-HCl, 1M NaCl, pH8.5) from sample after the gradient-purified modification of 0-100%, wash-out linear rate of flow is 5ml/min, rinses 10 column volumes, collects elution peak respectively.Elution peak carries out SDS-PAGE electrophoresis and mass spectrometric detection.Fig. 2 is shown in each step monitoring above, and result shows, collecting component is that PEG modifies Interferon alfacon-1.
Embodiment three
According to Pharmacopoeia of People's Republic of China (version three in 2005) regulation, adopt cytopathic-effect inhibition assay in the activity of cell-based assay Interferon alfacon-1 and PEG modified outcome thereof and activity change.Principle: effect human amniotic cell (WISH) can being protected to destroy from vesicular stomatitis virus (VSV) according to Interferon, rabbit; dye with the human amniotic cell (WISH) of Viola crystallina to survival; its absorbancy is measured in wavelength 570nm place; the protective effect curve of Interferon, rabbit to human amniotic cell (WISH) can be obtained, measure interferon biological activity according to this.When human amniotic cell (WISH) is by the attack of VSV virus, the cell of half can be protected to be not 1 activity unit by the dilution inverse of Interferon, rabbit attacked.Detect the front initial value of denaturation renaturation operation, Interferon alfacon-1 activity is 5.44 × 10
8it is 5.2 × 10 that U/mg, PEG modify Interferon alfacon-1 activity
7u/mg.
Embodiment four
In sex change liquid (50mM Tris-HCl, 1mM EDTA, 6M Guanidinium hydrochloride, pH8.5), finally add the beta-mercaptoethanol of cumulative volume 1%, denaturation time 9h.Room temperature centrifugal (the centrifugal 30min of 8000 × g), gets supernatant liquor.Renaturation solution protein concentration is remained on 0.2mg/ml.Dimly release volume preparation renaturation solution (50mM Tris-HCl, 1mM EDTA, pH8.5), 4 DEG C of precoolings.Renaturation keeps 4 DEG C of operations.Be placed on magnetic stirring apparatus by precooling renaturation solution, produced to stirring bubble-free by magnetic stirring apparatus adjustment of rotational speed, get inclusion body sex change liquid and dropwise add in renaturation solution, rate of addition remains on 2ml/min.Inclusion body sex change liquid is added dropwise to complete, 4 DEG C of standing 48h.4 DEG C centrifugal (the centrifugal 30min of 8000 × g), get supernatant liquor, gained supernatant is renaturation solution.
Carry out denaturation renaturation process monitoring, contrast Interferon alfacon-1, Interferon alfacon-1+PEGylation (molecular chaperones), PEG modify Interferon alfacon-1 three test group, after traditional dilution refolding process, protein recovery and activity recovery, the results are shown in Figure 3.As seen from the figure, covalently bound PEG chain can effectively promote target protein renaturation, and protein recovery and activity recovery are all close to 100%.And by contrast, the PEG protein recovery that non-covalent attachment PEG or interpolation dissociate and activity recovery are only about 30%.
Embodiment five
The correct folding ratio of tradition dilution refolding is generally no more than 30%, the yield of correct folding protein is low normally due to the congregation between polypeptide chain, the concentration of protein is the principal element affecting protein aggregation, and thus, general concentration controls at 0.1-1mg/ml; If metaprotein adds in renaturation solution too fast, easily form flocks.For this reason, the subject matters such as traditional dilution refolding normal plane faces that process volume is large, effective concentration is low, the separating pressure of false folding product and aggregate is large after renaturation.For above problem, the renaturation effect that significantly can promote target protein is combined at covalency PEG, make protein recovery and activity recovery close on the basis of 100%, the upper limit that can be improved by test determination recombinant protein concentration, to reduce the process volume of recycling.The results are shown in Figure 4, visible, after covalency PEG combines, the renaturation concentration of Interferon alfacon-1 not only breaks through the scope of conventional 0.1-1mg/ml, and concentration reach 2mg/ml still can play promote correct folding, suppress the effect assembled.
Analysis principle is as follows, and as shown in Figure 5, traditional dilution refolding or add molecular chaperones is that random playing with protein contacts promotes correctly to fold, suppresses the effect assembled in renaturation process.But once mispairing body or aggregate are formed, these chance mechanisms do not have error correction.By comparison, covalent attachment PEG is then on molecular scale, and protection target protein unit molecule, show following three aspects, (1) PEG hydration can occur in aqueous, each ethyl oxide unit (-CH
2-CH
2-O-) can tightly in conjunction with 2-3 water molecules.This hydration of PEG also can raise along with the increase of its molecular weight, when the continuation of molecular weight raises, can autofolding be there is in the solution in PEG chain, form the folding segment of more analogous protein or polypeptide secondary structure, these PEG chain segment by interact also can loosely in conjunction with water molecules free in bulk solution.Thus, make PEG modified protein in renaturation process, fast development becomes " independent individual "; (2) this hydration of PEG also shows larger spatial stability effect in the solution.This stereoeffect is in particular in elastic contribution aspect.When the protein surface that another macromolecular substance is modified close to PEG, because the motion of PEG chain is fettered, the loss of conformational entropy is caused to produce elastic repulsion force thus pushed open by macromole.The macromole that these power make other molecules in solution be difficult to modify with PEG fully collides and contacts, thus effectively suppresses the formation of aggregate in renaturation process.(3) kindliness of PEG molecular chain is another important physical basis of its good nature, makes it constantly change conformation to adapt to the surface topology structure of protein, can promote the correct folding of target protein.Thus, PEG is modified to be coupled with renaturation to become a kind of new way that proteinaceous product reuses.
Claims (5)
1. a method for proteinaceous product recycling, comprises and being modified by protein PEG, treats active reduction, after product exceedes validity period, it is thoroughly done denaturing treatment, and the step of refolding.
2. the method for proteinaceous product recycling according to claim 1, wherein:
Non-specific amido modified: Methyl Parathion Hydrolase to be added damping fluid I and dissolve, regulate pH to 7-9, add polyethyleneglycol modified dose, albumen and polyethyleneglycol modified dose of mol ratio are 1: 5-1: 50.Described damping fluid I is 0.02-0.2mol/LTris-Cl or phosphate buffered saline buffer (PB), and pH7-9, under temperature constant state, modifies for 4 DEG C-37 DEG C;
N Amino End Group is modified: Methyl Parathion Hydrolase is added damping fluid II and dissolve, and regulate pH to 4-6, add polyethyleneglycol modified dose, albumen and modifier mol ratio are 1: 1-1: 10; Described damping fluid II is 0.02-0.2mol/LNaAc-HAc or phosphate buffered saline buffer, pH4-6, under temperature constant state, modifies for 4 DEG C-37 DEG C;
Or, sulfydryl modification: Methyl Parathion Hydrolase is added damping fluid III and dissolve, regulate pH to 7-9, add polyethyleneglycol modified dose, albumen and modifier mol ratio are 1: 1-1: 10; Described damping fluid III is 0.02-0.2mol/LTris-Cl or boric acid-borate buffer solution, pH7-9, under temperature constant state, modifies for 4 DEG C-37 DEG C.
Described PEG modifies in the method for Methyl Parathion Hydrolase, and non-specific amido modified polyethyleneglycol modified dose used is mPEG-SC5000, mPEG-SC10000, mPEG-SC20000; It is mPEG-ALD5000, mPEG-ALD10000, mPEG-ALD20000 that N Amino End Group modifies polyethyleneglycol modified dose used; Or used polyethyleneglycol modified dose of sulfydryl modification is mPEG-MAL5000, mPEG-MAL10000, mPEG-MAL20000.
3. the method for proteinaceous product recycling according to claim 1, wherein, the modification time is 3-24 hour.
4. the method for proteinaceous product recycling according to claim 1, wherein, albumen after modification adopts ion exchange chromatography DEAE Sepharose6FF or Q Sepharose6FF purifying to modify component, moving phase is 0.05-0.2mol/L Tris-HCl or phosphate buffered saline buffer pH6-9.5, adds 0-1.5M sodium-chlor.
5. the method for proteinaceous product recycling according to claim 1, wherein, denaturation method, uses denaturing agent 6M Guanidinium hydrochloride or 8M urea and excessive reductive agent process, makes protein molecule become unordered loose stretch-like structure from originally orderly curling imporosity.Refolding method, mixed with dilution refolding damping fluid by metaprotein, placement is spent the night, by reducing the content of denaturing agent and reductive agent, progressively recoverin matter molecular activity.
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Cited By (4)
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| CN106243186A (en) * | 2015-06-15 | 2016-12-21 | 张鹏 | Protein renaturation or the circulation How It Works as the leading operation of protein renaturation can be independently used for |
| CN108743417A (en) * | 2018-07-18 | 2018-11-06 | 山东利特纳米技术有限公司 | A kind of graphene-protein method for synthesizing composite material |
| CN113929731A (en) * | 2021-12-16 | 2022-01-14 | 北京春雷杰创生物科技有限公司 | Method for promoting low molecular weight protein in vitro renaturation and improving immunogenicity |
| CN116174044A (en) * | 2023-02-21 | 2023-05-30 | 集美大学 | New preparation method and application of artificial metalloenzyme with protein framework |
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| CN106243186A (en) * | 2015-06-15 | 2016-12-21 | 张鹏 | Protein renaturation or the circulation How It Works as the leading operation of protein renaturation can be independently used for |
| CN108743417A (en) * | 2018-07-18 | 2018-11-06 | 山东利特纳米技术有限公司 | A kind of graphene-protein method for synthesizing composite material |
| CN113929731A (en) * | 2021-12-16 | 2022-01-14 | 北京春雷杰创生物科技有限公司 | Method for promoting low molecular weight protein in vitro renaturation and improving immunogenicity |
| CN116174044A (en) * | 2023-02-21 | 2023-05-30 | 集美大学 | New preparation method and application of artificial metalloenzyme with protein framework |
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