CN102174077B - Oligopeptide molecules with intramolecular disulfide bonds and application thereof to assisting in protein oxidation renaturation - Google Patents
Oligopeptide molecules with intramolecular disulfide bonds and application thereof to assisting in protein oxidation renaturation Download PDFInfo
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Abstract
The invention relates to oligopeptide molecules with intramolecular disulfide bonds and application thereof to assisting in protein oxidation renaturation. The oligopeptide molecules are the oligopeptide micromolecules which can be subjected to oxidation-reduction with DTT (dithiothreitol) to assist in protein oxidation renaturation. The two oligopeptides have the amino acid sequences shown as the SEQ ID No. 1 and the SEQ ID No. 2, thus the oligopeptide molecules can better assist in protein oxidation renaturation under the condition that the pH value is neutral and can greatly improve the protein oxidation renaturation rate and yield. The oligopeptide micromolecules provided by the invention can be coupled with the DTT which is a strong reducing agent carried by renatured protein to form an oxidation-reduction pair, thus the excessive DTT in the renatured protein does not need to be removed, other reducing agents do not need to be added to renaturation solution, the operation is simple, and the renaturation cost is reduced.
Description
Technical field
The present invention relates to a kind ofly can form with DTT the oligopeptides molecule that contains intramolecular disulfide bond of redox couple auxiliary protein oxidizing and refolding, and the using method in the auxiliary protein oxidizing and refolding, the protein renaturation technical field in biotechnology belonged to.
Background technology
Along with the development of science and technology, gene recombination technology becomes the important channel that produces the protein with medical or industrial value.The protein that much has important medicine and industrial value all contains disulfide linkage, as interleukin-, human growth hormone, human interferon, tumour necrosis factor, Regular Insulin, tissue-type plasminogen activator etc.The applying gene recombination method, in the above-mentioned protein of expression in escherichia coli, tends to form occlusion body.Though the formation of occlusion body is beneficial to the separation and purification of target protein, in occlusion body, target protein does not have activity because higher structure is incorrect, therefore must could obtain bioactive protein by renaturation.The folding process that contains the protein of disulfide linkage is known as oxidizing and refolding or oxidative folding, the correct tertiary structure of Protein formation in this process, and a plurality of cysteine residues accurately match and form correct disulfide linkage simultaneously, recover the natural enzyme activity.
Protein disulfide isomerase (PDI) is an important oxydo-reductase of catalytic proteins oxidizing and refolding in body.It not only can the catalysis disulfide linkage isomery can also promote the formation of disulfide linkage.therefore people have designed the small molecules mercapto alcohol of this enzyme function of a lot of simulations as reductive agent auxiliary protein renaturation, mercapto alcohol as fragrant as the small molecules of simulating the low acid ionization constant (pKa) of PDI, simulate simultaneously the small molecules (±)-trans-1 of its low pKa and intramolecular disulfide bond character, 2-bis-(2-acetyl mercapto) hexanaphthene (BMC) (Method of folding proteins with synthetic dithiol catalysts, US patent, US591043, 1999) and the Cys-Gly-Cys tripeptides mercapto that is formed by halfcystine-glycine-halfcystine alcohol (Small-moleculecatalysts of oxidative protein folding.Current Opinion in Chemical Biology, 2008, 12, 740-745.).The small molecules mercapto alcohol of these simulations PDI function does not all contain disulfide linkage, and it,, as the oxidizing and refolding of reductive agent for protein, is undertaking the isomerized effect of promotion protein disulfide.These molecules are the oxidizing and refolding of auxiliary protein well, accelerates the yield of speed and the raising renaturation of renaturation.But in sex change reduction protein example, excessive reducing agent dithiothreitol (DTT) needs to remove by the method for dialysis or gel filtration chromatography.The removal operation of DTT has not only extended the production cycle but also can improve the production costs such as manpower and equipment loss.In addition, these small molecules mercapto alcohol lost efficacy because being easy to very much oxidation in air, therefore its storage liquid is difficult for preserving.When oxygenant (as Sleep-promoting factor B) commonly used and DTT formed redox couple auxiliary protein oxidizing and refolding, existence can not be near renaturation under condition of neutral pH again, and the relatively low problem of renaturation yield.So for actual production, be necessary to develop can with sex change also the entrained DTT of the crude protein small molecules oxygenant that forms redox couple carry out the oxidizing and refolding of auxiliary protein.
Summary of the invention
The object of the present invention is to provide the oligopeptides molecule that the contains intramolecular disulfide bond auxiliary technological method that contains the efficient oxidation renaturation of disulfide bond protein matter of DTT that is coupled as oxygenant under alkalescence and pH neutral condition.Present method forms redox couple by the designed oligopeptides of direct application as oxygenant and residual reductive agent DTT coupling and under neutrality or alkaline pH condition, realizes the highly efficient renaturation of protein, without denatured protein being carried out to the operation of reductive agent DTT removal.
The present invention is realized by following technical proposals:
The oligopeptides molecule that contains intramolecular disulfide bond of the present invention, intramolecular disulfide bond in the oligopeptides molecule is formed between two cysteine residues, and it can form redox couple as oxygenant and DTT coupling, alkalescence or and the pH neutral condition under the auxiliary oxidizing and refolding that contains disulfide bond protein matter.
Described oligopeptides molecule is: oxidized form tripeptides or oxidized form pentapeptide.The oxidized form tripeptides has SEQ ID No.1 aminoacid sequence: Cys-Gly-Cys, and wherein intramolecular disulfide bond is formed between two Cys, and the carboxyl terminal amidation.The oxidized form pentapeptide, have SEQ ID No.2 aminoacid sequence: Arg-Lys-Cys-Gly-Cys, and wherein intramolecular disulfide bond is formed between two Cys, and the carboxyl terminal amidation.
The application of oligopeptides molecule in the auxiliary protein oxidizing and refolding that contains intramolecular disulfide bond of the present invention, step is as follows:
(1) the oligopeptides small molecules that will contain intramolecular disulfide bond adds in the solution that contains 20-100mmol/L Tutofusin tris, 0-3mmol/L tetrasodium ethylenediamine tetraacetate, 0-2mol/L urea or 0-1mol/L Guanidinium hydrochloride and forms renaturation solution;
(2) protein or occlusion body reduce denaturation with the urea-denatured liquid of 8mol/L that contains DTT;
(3) protein or the occlusion body solution that reduce denaturation of the sex change liquid through containing DTT, directly be diluted in or the sex change liquid dilution 5-25 through not containing DTT doubly after redilution in the renaturation solution of pH7.0-9.0, and the oligopeptides concentration that contains intramolecular disulfide bond after dilution in protein solution be the DTT volumetric molar concentration 2-3.3 doubly.
The method of the auxiliary protein renaturation that the present invention relates to is applied in sex change reduction protein renaturation, deactivation and contain protein example that disulfide linkage is disconnected and use method of the present invention to carry out renaturation can to guarantee higher renaturation yield.
Gordian technique of the present invention has 4 points: at first, the structure of CGC and RKCGC oligopeptides, they all have intramolecular disulfide bond, so just can guarantee in the process of renaturation they not can and the mercapto alcohol of protein molecule between form metastable mixing disulfide linkage, and the further renaturation of the long-time existence meeting arrestin matter of this mixing disulfide linkage; Next is after CGC and RKCGC oligopeptides are reduced by DTT, and mercapto alcohol pK on a halfcystine is arranged in molecule
aValue is lower than redox agent commonly used, thereby make under its condition of pH in neutrality and also can form the sulfydryl negatively charged ion, owing to only having the sulfydryl negatively charged ion, it is the active condition that the reaction of mercapto alcohol disulfide exchange occurs, so CGC and RKCGC oligopeptides small molecules can be accelerated the reaction of mercapto alcohol disulfide exchange, promote protein disulfide to form, thereby accelerate renaturation and improve the renaturation yield; Three of key need to be regulated the molar weight that adds the oligopeptides molecule according to the molar weight of the DTT that contains in the renaturation system, mainly the speed that in the protein molecule of controlled denaturation reduction, disulfide linkage forms, make between disulfide linkage formation and isomery and reach balance preferably, thereby promote the formation of correct disulfide linkage; It is finally the processing of DTT residual in denatured protein solution, while applying other the auxiliary renaturation of small molecules oxygenant, need to by the method that gel filtration chromatography or 24h dialyse, remove by DTT wherein, and refolding method of the present invention does not need to carry out this operation, directly DTT is assisted to renaturation.
The oligopeptides that contains disulfide linkage that the present invention introduces is compared and is had the following advantages with oxygenant auxiliary protein oxidizing and refolding method commonly used at present as the method for oxygenant auxiliary protein oxidizing and refolding: first, oxygenant that the present invention adopts can with the DTT auxiliary protein oxidizing and refolding that is coupled, so can save the operation steps from removing the protein soln that reduces denaturation by strong reductant DTT, thereby save renaturation time and cost; The second, oligopeptides small molecules of the present invention can improve speed and the yield of oxidative folding of protein greatly, and renaturation speed and yield are all higher than the effect of Sleep-promoting factor B under same condition; The 3rd, the oligopeptides small molecules that the present invention introduces, the also renaturation of auxiliary protein well under neutral pH, and the general only auxiliary protein oxidizing and refolding preferably under alkaline condition of oxygenant commonly used; The 4th, the oligopeptides small molecules that the present invention adopts can with sex change also the entrained strong reductant DTT coupling of crude protein form redox couple, thereby need in renaturation solution, not add reductive agent, simple to operate, and reduced the renaturation cost.
The accompanying drawing explanation
The activity yield temporal evolution figure of 1.4mmol/L CGC and GSSG assisted Reduction sex change lysozyme renaturation under the pH neutral condition in Fig. 1: embodiment 1.
The activity yield temporal evolution figure of 0.84mmol/L RKCGC and GSSG assisted Reduction sex change lysozyme renaturation under condition of neutral pH in Fig. 2: embodiment 2.
The activity yield temporal evolution figure of the lysozyme renaturation of 1.4mmol/LRKCGC and GSSG assisted Reduction sex change under condition of neutral pH in Fig. 3: embodiment 3.
The activity yield temporal evolution figure of 1.2mmol/L CGC and GSSG assisted Reduction sex change lysozyme renaturation under condition of neutral pH in Fig. 4: embodiment 4.
The activity yield comparison diagram of 0.84mmol/L CGC and GSSG assisted Reduction sex change lysozyme renaturation under the alkaline pH condition in Fig. 5: embodiment 5.
The activity yield temporal evolution figure of 0.84mmol/L RKCGC and GSSG assisted Reduction sex change lysozyme renaturation under the alkaline pH condition in Fig. 6: embodiment 6.
The activity yield comparison diagram of the 1.0mmol/L RKCGC in Fig. 7: embodiment 7 and the GSSG ribonuclease A renaturation of assisted Reduction sex change under the alkaline pH condition.
Embodiment
Following example will be further described method provided by the invention.As oxygenant can with DTT coupling form redox couple and pH 7.0 and more than the oligopeptides molecule that contains intramolecular disulfide bond of auxiliary protein oxidizing and refolding well, intramolecular disulfide bond is formed between two cysteine residues.
The oligopeptides molecule that contains intramolecular disulfide bond as oxygenant is a kind of three peptide molecules, and have SEQ ID No:1 aminoacid sequence: Cys-Gly-Cys, intramolecular disulfide bond are formed between two Cys, and the carboxyl terminal amidation;
The oligopeptides molecule that contains intramolecular disulfide bond as oxygenant is a kind of pentapeptide molecule, and have SEQ ID No:2 aminoacid sequence: Arg-Lys-Cys-Gly-Cys, intramolecular disulfide bond are formed between two Cys, and the carboxyl terminal amidation;
the method that contains the oligopeptides auxiliary protein oxidizing and refolding of intramolecular disulfide bond with above-mentioned any one, the protein that sex change liquid through containing DTT reduces denaturation or occlusion body solution, directly be diluted in or the sex change liquid dilution 5-25 through not containing DTT doubly after redilution in the renaturation solution of pH7.0-9.0, renaturation solution is by 20-100mmol/L Tutofusin tris (Tris), 0-3mmol/L tetrasodium ethylenediamine tetraacetate (EDTA), 0-2mol/L urea or 0-1mol/L Guanidinium hydrochloride and the oligopeptides small molecules that contains intramolecular disulfide bond form, after dilution in protein solution oligopeptides concentration be the DTT volumetric molar concentration 2-3.3 doubly.
Embodiment 1:1.4mmol/L CGC oligopeptides is applied to the renaturation of assisted Reduction sex change N,O-Diacetylmuramidase under condition of neutral pH
14.3mg being dissolved in 1.0mL, N,O-Diacetylmuramidase contains 8mol/L urea, 100mmol/L DTT, 100mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) and 1mmol/L EDTA, in the sex change buffered soln of pH 8.5, mix and reduced denaturation 3 hours in 40 ℃; After sex change completes, by C18 reverse-phase chromatography mensuration, obtaining wherein residual DTT content is 84mmol/L; The sex change N,O-Diacetylmuramidase that obtains is carried out to 20 times of dilutions with the sex change damping fluid that does not contain DTT and obtain the metaprotein quality sample.Renaturation buffer is comprised of 20mmol/L Tris-HCl, 1.0mol/L urea, 1mmol/L EDTA and 1.4mmol/L CGC oligopeptides or Sleep-promoting factor B (GSSG), and pH value of solution is 7.0.To the metaprotein quality sample, do not remove the operation of DTT, and directly with renaturation solution by 10 times of denatured protein diluted samples, after dilution, the concentration of Lysozyme in Solution is 5 μ mol/L, DTT is 0.42mmol/L.Solution after dilution is placed in the water bath with thermostatic control of 28 ℃ and starts renaturation.The activity of N,O-Diacetylmuramidase detects take micrococci as substrate, and the protein renaturation liquid after the phosphate buffered saline buffer of the pH 6.2 of 1.30mL 0.25mg/mL substrate and 0.10mL dilution fully mixes, the absorbancy variation of assaying reaction liquid under 450nm.Determination of activity temperature: 28 ℃.Ratio by the changing down under light absorption value changing down between 0-90s and natural enzyme catalytic condition calculates the lysozyme activity yield.Activity at renaturation different time sampling and measuring protein.Other renaturation condition is identical, and the auxiliary renaturation effect comparison of different small molecules oxygenants as shown in Figure 1.By the single index match, can obtain under the CGC oligopeptides is auxiliary, the rate constant of the lysozyme renaturation that reduces denaturation is 0.0345min
-1And the rate constant of its renaturation is 0.0255min under the auxiliary condition of Sleep-promoting factor B (GSSG)
-1, with GSSG, comparing as seen, CGC can improve the speed of the lysozyme renaturation that reduces denaturation as oxygenant.As seen from the figure, under condition of neutral pH, the renaturation speed of the N,O-Diacetylmuramidase that the CGC oligopeptides of usining reduces denaturation during as oxygenant and ultimate yield are all higher than usining the renaturation of GSSG as oxygenant.
Embodiment 2:0.84mmol/LRKCGC oligopeptides is applied to the renaturation of assisted Reduction sex change N,O-Diacetylmuramidase under condition of neutral pH
14.3mg being dissolved in 1.0mL, N,O-Diacetylmuramidase contains 8mol/L urea, 100mmol/L DTT, 100mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) and 1mmol/L EDTA, in the sex change buffered soln of pH 8.5, mix and reduced denaturation 3 hours in 40 ℃; After sex change completes, by C18 reverse-phase chromatography mensuration, obtaining wherein residual DTT content is 84mmol/L; The sex change N,O-Diacetylmuramidase that obtains is carried out to 5 times of dilutions with the sex change damping fluid that does not contain DTT and obtain the metaprotein quality sample.Renaturation buffer is comprised of 100mmol/L Tris-HCl, 2mol/L urea, 3mmol/L EDTA and 0.84mmol/LRKCGC oligopeptides or Sleep-promoting factor B (GSSG), and pH value of solution is 7.0.To the metaprotein quality sample, do not remove the operation of DTT, directly with renaturation solution by 40 times of denatured protein diluted samples, after dilution, the concentration of Lysozyme in Solution is 5 μ mol/L, DTT is 0.42mmol/L.Solution after dilution is placed in the water bath with thermostatic control of 28 ℃ and starts renaturation.The activity of N,O-Diacetylmuramidase detects take micrococci as substrate, and the protein renaturation liquid after the phosphate buffered saline buffer of the pH 6.2 of 1.30mL 0.25mg/mL substrate and 0.10mL dilution fully mixes, the absorbancy variation of assaying reaction liquid under 450nm.Determination of activity temperature: 28 ℃.Ratio by the changing down under light absorption value changing down between 0-90s and natural enzyme catalytic condition calculates the lysozyme activity yield.Activity at renaturation different time sampling and measuring protein.Other renaturation condition is identical, and the auxiliary renaturation effect comparison of RKCGC oligopeptides and Sleep-promoting factor B as shown in Figure 2.As seen from the figure, the renaturation yield of N,O-Diacetylmuramidase and the speed renaturation under all auxiliary higher than the sweet peptide of oxidized form Guang (GSSG) that reduces denaturation under RKCGC auxiliary.
Embodiment 3:1.4mmol/L RKCGC oligopeptides is applied to the renaturation of assisted Reduction sex change N,O-Diacetylmuramidase
14.3mg being dissolved in 1.0mL, N,O-Diacetylmuramidase contains 8mol/L urea, 100mmol/L DTT, 100mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) and 1mmol/L EDTA, in the sex change buffered soln of pH 8.5, mix and reduced denaturation 3 hours in 40 ℃; After sex change completes, by C18 reverse-phase chromatography mensuration, obtaining wherein residual DTT content is 84mmol/L; The sex change N,O-Diacetylmuramidase that obtains is carried out to 25 times of dilutions with the sex change damping fluid that does not contain DTT and obtain the metaprotein quality sample.Renaturation buffer is comprised of 50mmol/L Tris-HCl, 0.5mol/L Guanidinium hydrochloride, 1mmol/L EDTA and 1.4mmol/L RKCGC oligopeptides or Sleep-promoting factor B (GSSG), and pH value of solution is 7.0.By 8 times of denatured protein diluted samples, after dilution, the concentration of Lysozyme in Solution is 5 μ mol/L with renaturation solution, and DTT is 0.42mmol/L.Solution after dilution is placed in the water bath with thermostatic control of 28 ℃ and starts renaturation.The activity of N,O-Diacetylmuramidase detects take micrococci as substrate, and the protein renaturation liquid after the phosphate buffered saline buffer of the pH 6.2 of 1.30mL0.25mg/mL substrate and 0.10mL dilution fully mixes, the absorbancy variation of assaying reaction liquid under 450nm.Determination of activity temperature: 28 ℃.Ratio by the changing down under light absorption value changing down between 0-90s and natural enzyme catalytic condition calculates the lysozyme activity yield.Activity at renaturation different time sampling and measuring protein.Other renaturation condition is identical, and the auxiliary renaturation effect comparison of RKCGC oligopeptides and Sleep-promoting factor B as shown in Figure 3.As seen from the figure, the renaturation yield of N,O-Diacetylmuramidase and the speed of reducing denaturation under RKCGC auxiliary is the renaturation of sweet peptide GSSG under auxiliary far above the oxidized form Guang all, under RKCGC is auxiliary, after 2 hours, yield can reach 90%, and under similarity condition, even GSSG still can only make yield reach 60% after 4 hours in renaturation.
Embodiment 4:1.2mmol/L CGC oligopeptides is applied to the renaturation of assisted Reduction sex change N,O-Diacetylmuramidase
14.3mg being dissolved in 1.0mL, N,O-Diacetylmuramidase contains 8mol/L urea, 100mmol/L DTT, 100mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) and 1mmol/L EDTA, in the sex change buffered soln of pH 8.5, mix and reduced denaturation 3 hours in 40 ℃; After sex change completes, by C18 reverse-phase chromatography mensuration, obtaining wherein residual DTT content is 84mmol/L; The sex change N,O-Diacetylmuramidase that obtains is carried out to 5 times of dilutions with the sex change damping fluid that does not contain DTT and obtain the metaprotein quality sample.Renaturation buffer is comprised of 50mmol/L Tris-HCl, 1.0mol/L Guanidinium hydrochloride and 1.2mmol/L CGC oligopeptides or Sleep-promoting factor B (GSSG), and pH value of solution is 7.0.By 40 times of denatured protein diluted samples, after dilution, the concentration of Lysozyme in Solution is 5 μ mol/L with renaturation solution, and DTT is 0.42mmol/L.Solution after dilution is placed in the water bath with thermostatic control of 28 ℃ and starts renaturation.The activity of N,O-Diacetylmuramidase detects take micrococci as substrate, and the protein renaturation liquid after the phosphate buffered saline buffer of the pH 6.2 of 1.30mL 0.25mg/mL substrate and 0.10mL dilution fully mixes, the absorbancy variation of assaying reaction liquid under 450nm.Determination of activity temperature: 28 ℃.Ratio by the changing down under light absorption value changing down between 0-90s and natural enzyme catalytic condition calculates the lysozyme activity yield.Activity at renaturation different time sampling and measuring protein.Other renaturation condition is identical, and the auxiliary renaturation effect comparison of different small molecules oxygenants as shown in Figure 4.As seen from the figure, under condition of neutral pH, the DTT that the sex change of usining is also carried in crude protein is as reductive agent, using 1.2mmol/L CGC oligopeptides as the renaturation speed of oxygenant time variation reduction N,O-Diacetylmuramidase and ultimate yield all higher than usining the renaturation of 1.2mmol/L Sleep-promoting factor B (GSSG) during as oxygenant.
The renaturation of embodiment 5:0.84mmol/L CGC assisted Reduction sex change N,O-Diacetylmuramidase under alkaline condition
14.3mg being dissolved in 1.0mL, N,O-Diacetylmuramidase contains 8mol/L urea, 100mmol/L DTT, 100mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) and 1mmol/L EDTA, in the sex change buffered soln of pH 8.5, mix and reduced denaturation 3 hours in 40 ℃; After sex change completes, by C18 reverse-phase chromatography mensuration, obtaining wherein residual DTT content is 84mmol/L; The sex change N,O-Diacetylmuramidase that obtains is carried out to 20 times of dilutions with the sex change damping fluid that does not contain DTT and obtain the metaprotein quality sample.Renaturation buffer is comprised of 20mmol/L Tris-HCl, 1.0mol/L urea, 1mmol/L EDTA and 0.84mmol/L CGC oligopeptides or Sleep-promoting factor B (GSSG), and pH value of solution is 8.5.By 10 times of denatured protein diluted samples, after dilution, the concentration of Lysozyme in Solution is 5 μ mol/L with renaturation solution, and DTT is 0.42mmol/L.Solution after dilution is placed in the water bath with thermostatic control of 28 ℃ and starts renaturation.The activity of N,O-Diacetylmuramidase detects take micrococci as substrate, and the protein renaturation liquid after the phosphate buffered saline buffer of the pH 6.2 of 1.30mL0.25mg/mL substrate and 0.10mL dilution fully mixes, the absorbancy variation of assaying reaction liquid under 450nm.Determination of activity temperature: 28 ℃.Ratio by the changing down under light absorption value changing down between 0-90s and natural enzyme catalytic condition calculates the lysozyme activity yield.The activity of sampling and measuring protein when renaturation 90min.Other renaturation condition is identical, and the auxiliary renaturation effect comparison of different small molecules oxygenants as shown in Figure 5.As seen from the figure, under the condition of pH=8.5, using the renaturation yield of CGC oligopeptides N,O-Diacetylmuramidase during as oxygenant higher than usining the renaturation of Sleep-promoting factor B (GSSG) during as oxygenant.
The renaturation of embodiment 6:0.84mmol/L RKCGC assisted Reduction sex change N,O-Diacetylmuramidase under alkaline condition
14.3mg being dissolved in 1.0mL, N,O-Diacetylmuramidase contains 8mol/L urea, 100mmol/L DTT, 100mmol/L Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl) and 1mmol/L EDTA, in the sex change buffered soln of pH 8.5, mix and reduced denaturation 3 hours in 40 ℃; After sex change completes, by C18 reverse-phase chromatography mensuration, obtaining wherein residual DTT content is 84mmol/L; The sex change N,O-Diacetylmuramidase that obtains is carried out to 20 times of dilutions with the sex change damping fluid that does not contain DTT and obtain the metaprotein quality sample.Renaturation buffer is comprised of 20mmol/L Tris-HCl, 1.0mol/L urea, 1mmol/L EDTA and 1.4mmol/L RKCGC oligopeptides or Sleep-promoting factor B (GSSG), and pH value of solution is 9.0.To the metaprotein quality sample, do not remove the operation of DTT, and directly with renaturation solution by 10 times of denatured protein diluted samples, after dilution, the concentration of Lysozyme in Solution is 5 μ mol/L, DTT is 0.42mmol/L.Solution after dilution is placed in the water bath with thermostatic control of 28 ℃ and starts renaturation.The activity of N,O-Diacetylmuramidase detects take micrococci as substrate, and the protein renaturation liquid after the phosphate buffered saline buffer of the pH 6.2 of 1.30mL 0.25mg/mL substrate and 0.10mL dilution fully mixes, the absorbancy variation of assaying reaction liquid under 450nm.Determination of activity temperature: 28 ℃.Ratio by the changing down under light absorption value changing down between 0-90s and natural enzyme catalytic condition calculates the lysozyme activity yield.Activity at renaturation different time sampling and measuring protein.Other renaturation condition is identical, and the auxiliary renaturation effect comparison of different small molecules oxygenants as shown in Figure 6.As seen from the figure, under the alkaline pH condition, the RKCGC oligopeptides of usining reduces denaturation the renaturation speed of N,O-Diacetylmuramidase and ultimate yield during as oxygenant all higher than usining the protein renaturation of Sleep-promoting factor B as oxygenant.
The ribonuclease A sample renaturation of embodiment 7:1.0mmol/L RKCGC assisted Reduction sex change under alkaline condition
7.0mg being dissolved in 1.0mL, ribonuclease A contains 8mol/L urea, 50mmol/L DTT, and 100mmol/LTris-HCl and 1mmol/L EDTA, reduced denaturation 1 hour in 40 ℃ in the sex change buffered soln of pH 8.5.After sex change completes, by C18 reverse-phase chromatography mensuration, obtaining wherein residual DTT content is 47mmol/L.Renaturation buffer is comprised of 100mmol/L Tris-HCl, 1.0mmol/L RKCGC or Sleep-promoting factor B (GSSG) and 1mmol/L EDTA, and pH value of solution is 8.0.By 100 times of denatured protein diluted samples, in the rear solution of dilution, the concentration of ribonuclease A is 5 μ mol/L with renaturation solution, and DTT is 0.47mmol/L.Solution after dilution is placed in the water bath with thermostatic control of 25 ℃ and starts renaturation.The activity of ribonuclease A detect with cytidine-2 ', 3 '-cyclic phosphoric acid (cCMP) is substrate, 1.00mL the 100mmol/L Tris-HCl damping fluid of the pH 6.0 of 0.25mg/mL substrate fully mixes with 0.10mL protein renaturation sample, the absorbancy of assaying reaction liquid under 284nm changes.Determination of activity temperature: 25 ℃.Ratio by the changing down under light absorption value changing down between 0-90s and natural enzyme catalytic condition carrys out the calculated activity yield.Adopt the described method renaturation of this patent, do not remove the also DTT in crude protein of sex change, and direct dilution refolding.Activity at renaturation sampling and measuring protein after 5 hours.Other renaturation condition is identical, and RKCGC oligopeptides and Sleep-promoting factor B (GSSG) assisted Reduction denatured rnase enzyme A renaturation effect comparison as shown in Figure 7.As seen from the figure, under same renaturation condition, the renaturation yield when when the RKCGC oligopeptides was oxygenant, the yield of ribonuclease A renaturation was oxygenant higher than GSSG.
The oligopeptides of the effective renaturation of coupling DTT auxiliary protein that the present invention proposes, and use it for the renaturation of metaprotein quality sample, by on-the-spot preferred embodiment, be described, person skilled obviously can be changed method as herein described or suitably change and combination within not breaking away from content of the present invention, spirit and scope, realize the technology of the present invention.Special needs to be pointed out is, the replacement that all are similar and change apparent to those skilled in the artly, they are deemed to be included in spirit of the present invention, scope and content.
Claims (2)
1. the oligopeptides molecule that contains intramolecular disulfide bond, it is characterized in that the oligopeptides molecule is oxidized form tripeptides or oxidized form pentapeptide, intramolecular disulfide bond wherein is formed between two cysteine residues, and it can form redox couple as oxygenant and DTT coupling, alkalescence or and the pH neutral condition under the auxiliary oxidizing and refolding that contains disulfide bond protein matter; The sequence of described oxidized form tripeptides is SEQ ID No.1 aminoacid sequence: Cys-Gly-Cys, and wherein intramolecular disulfide bond is formed between two Cys and the carboxyl terminal amidation; The sequence of described oxidized form pentapeptide is SEQ ID No.2 aminoacid sequence: Arg-Lys-Cys-Gly-Cys, and wherein intramolecular disulfide bond is formed between two Cys and the carboxyl terminal amidation.
2. contain the application of oligopeptides molecule in the auxiliary protein oxidizing and refolding of intramolecular disulfide bond, it is characterized in that step is as follows:
(1) will contain the oligopeptides small molecules Cys-Gly-Cys of intramolecular disulfide bond or Arg-Lys-Cys-Gly-Cys adds in the solution that contains 20-100mmol/L Tutofusin tris, 0-3mmol/L tetrasodium ethylenediamine tetraacetate, 0-2mol/L urea or 0-1mol/L Guanidinium hydrochloride and forms renaturation solution;
(2) protein or inclusion body reduce denaturation with the urea-denatured liquid of 8mol/L that contains DTT;
(3) protein or the inclusion body solution that reduce denaturation of the sex change liquid through containing DTT, directly be diluted in or the sex change liquid dilution 5-25 through not containing DTT doubly after redilution in the renaturation solution of pH7.0-9.0, and the concentration that contains the oligopeptides Cys-Gly-Cys of intramolecular disulfide bond or Arg-Lys-Cys-Gly-Cys after dilution in protein solution be the DTT volumetric molar concentration 2-3.3 doubly.
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