CN103626831A - Method used for selective modification of protein carbon terminal carboxyl groups with polyethylene glycol - Google Patents
Method used for selective modification of protein carbon terminal carboxyl groups with polyethylene glycol Download PDFInfo
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- CN103626831A CN103626831A CN201210300769.6A CN201210300769A CN103626831A CN 103626831 A CN103626831 A CN 103626831A CN 201210300769 A CN201210300769 A CN 201210300769A CN 103626831 A CN103626831 A CN 103626831A
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- staphylokinase
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Abstract
The invention relates to a polyethylene glycol (PEG) modification method. The method is capable of realizing selective modification of carbon terminal carboxyl groups of proteins and polypeptides, and each protein molecule and polypeptide molecule is connected with one PEG molecule. According to the method, staphylokinase is used as a model protein. The polyethylene glycol (PEG) modification method comprises following main steps: (1) under slightly acidic conditions (pH 5.0), carboxyl groups of staphylokinase and amino groups of cystamine are connected; and (2) reduction of disulfide bonds of cystamine is realized using a reducing agent, and generated sulfhydryl groups are reacted with methoxy polyethylene glycol-maleinimide (mPEG-Mal). pKa value of the carbon terminal carboxyl groups of staphylokinase is 2.1 to 2.4, pKa value of side chain carboxyl groups of aspartic acid is 3.7 to 4.0, and pKa value of side chain carboxyl groups of glutamic acid is 4.2 to 4.5, so that under the conditions with a pH value of 5.0, PEG is capable of realizing selective modification of the carbon terminal carboxyl groups of staphylokinase. The method is mainly used for PEG selective modification of the carbon terminal carboxyl groups of the proteins and the polypeptides; a novel PEG modification method is provided; and an advantage of the method is that uniformity of modification sites of the PEG modification product is excellent.
Description
Technical field
The invention belongs to protein chemistry, biomedical sector, relates to the modifying method of a kind of polyoxyethylene glycol (PEG) selective modification c-terminal of protein carboxyl.
Background technology
The bio-pharmaceutical that utilizes the technological development such as molecular biology, structure biology, molecular immunology, genetically engineered, protein engineering, monoclonal antibody, can make the treatment of various diseases become possibility.Wherein, gene recombinant protein and polypeptide drugs are class bio-pharmaceuticals of giving prominence to the most.From the first genetically engineered drug recombinant human insulin in nineteen eighty-two listing so far, lots of genes recombinant protein and polypeptide drugs are for clinical study, and have obtained huge success.Compare with traditional small molecules chemicals, bio-pharmaceutical identical with endogenous protein aspect molecular structure and/or biological function or approach identical, therefore have biological effect significantly, the advantage such as determined curative effect, side effect be little.Yet the problems such as bio-pharmaceutical ubiquity administration difficulty, antigen-antibody reaction, the body-internal-circulation transformation period is short, bioavailability is low.At present, developed the performance that multiple technologies are improved bio-pharmaceutical, as slow control-release microsphere technology, implants technology, original position Microspheres Technique, liposome embedded technology, nanometer ball technology and chemical modification technology.Wherein polyoxyethylene glycol (PEG) modification technique is the most successfully one of technology of application at present.
Uncharged polymkeric substance that PEG is a kind of linearity, can be freely curling in solution, has nontoxic, faint antigenicity and good biocompatibility.PEG covalent modification can increase protein the body-internal-circulation transformation period, reduce its antigenicity, increase its solvability, change its biodistribution in vivo.1977, people (J.Biol.Chem.1977, the 252:3578-3581) reported first such as Davis PEG modifying protein.Subsequently, PEG modification technique is widely used at biomedical and biological technical field.PEG modification technique has become one of effective means of pharmacokinetics/pharmacodynamic property improving pharmaceutical grade protein.At present, the pharmaceutical grade protein that existing multiple PEG modifies, by U.S. FDA approval listing, as PEG modified human growth hormone receptor antagonist (U.S. Pfizer company, commodity are called Somavert), is used for the treatment of acromegaly and hyperplasia disease; PEG modifies alpha-interferon (U.S. Schering-Plough company, commodity are called PEG-Intron), is used for the treatment of hepatitis C.
It is mainly amino (comprising that N holds alpha-amino acylations to modify with the acylations of alkylation modification, lysine side-chain epsilon-amino and modifies), carboxyl and the sulfydryl etc. of modifying protein and polypeptide that PEG modifies.The modification reaction wherein carrying out on the epsilon-amino of lysine side-chain is random modification reaction.Because protein contains a plurality of lysine residues conventionally, and be usually located at protein surface, therefore easy and PEG modifier reacts, and is swift in response, and modification rate is high.But owing to being random modification, so the composition more complicated of PEG modified outcome, the site of modified outcome is heterogeneity also.The biological activity of these isomer is often not quite similar, and the stability between being difficult to while causing mass-producing to be prepared to realize batch, does not meet the requirement that drug manufacture and new drug are declared.
Another kind of reaction is PEG pointed decoration, mainly comprises the modification of the alpha-amino modification of N-terminal and cysteine residues.The alpha-amino modification of N-terminal is normally carried out reductive alkylation reaction by reductive agent (as sodium cyanoborohydride) and the modifier of this class of PEG-aldehyde.This modification reaction is also not exclusively specific, and the lysine residue of protein side chain also can participate in reaction.In mono-modified product, the ratio that is modified at N-terminal accounts for more than 85% conventionally.And cysteine residues modification is to utilize modifier, as mono methoxy polyethylene glycol maleimide (mPEG-Mal) and the free sulfhydryl groups of protein molecule react.The specificity of this modification reaction is very high.But most of albumen is not contain free sulfhydryl group, need on protein molecule, introduce halfcystine by engineered method.Yet the halfcystine of introducing easily causes the mispairing of the natural disulfide linkage of protein, form intermolecular disulfide linkage simultaneously, thereby affect the biological activity of protein.
Also has at present a kind of method that can modifying protein carboxyl.The method is utilized the carboxyl reaction of adipic dihydrazide and carbodiimide (EDC) and protein, introduces after hydrazides group and PEG aldehyde generation modification reaction.Yet, this modifying method need to add a large amount of adipic dihydrazide and carbodiimide, the poor selectivity of modification reaction, easily and the carboxyl reaction of aspartic acid and L-glutamic acid side chain, also can occur in protein molecular and intermolecular cross-linking (J.Mol.Recognit.1996,9:644-651).
The present invention be take staphylokinase as model proteins.Staphylokinase (staphylokinase, SAK) be a kind of non-enzyme albumen of being secreted by some streptococcus aureus, molecular weight is 15.5kDa, itself does not have enzymic activity, when it and Profibrinolysin (Plg) in conjunction with after can plasminogen activation, make it to become activated plasmin and bring into play thrombolytic effect (Fig. 1).Staphylokinase is first class national new drug, is used for the treatment of cardiovascular and cerebrovascular thrombotic diseases, has become one of focus of the development of novel thrombolytic agent in recent years.PEG modifies can extend staphylokinase circulating half-life in vivo, because hold at N in its active centre, therefore selects to hold carboxyl as decorating site away from the C in active centre.
The present invention utilizes EDC and cystamine to introduce mercapto groups at the C-terminal of staphylokinase, then reacts with mPEG-Mal, realizes C end pointed decoration (Fig. 2).The cystamine that present method is used and the mol ratio of albumen test little (being about 2-3), can effectively avoid in the molecule of protein or the problem of intermolecular cross-linking; This reaction has utilized the pKa value (2.1-2.4) of C-terminal carboxyl lower than the pKa value of aspartic acid (3.7-4.0) and L-glutamic acid (4.2-4.5) side chain carboxyl group.When the mol ratio of EDC and cystamine and staphylokinase is lower, cystamine can be preferentially combined with the carboxyl of C-terminal, thereby reaches the object of selective modification c-terminal of protein carboxyl.
Summary of the invention
The object of the present invention is to provide a kind of method of PEG selective modification c-terminal of protein carboxyl.The method has overcome the poor selectivity existing in traditional protein carboxyl modified, easily occurs in protein molecule and the problem of intermolecular cross-linking.
Technical scheme of the present invention is as follows:
A kind of modifying method of PEG selective modification PROTEIN C end carboxyl, key step is: in 2-(N-morpholine) ethyl sulfonic acid (MES) damping fluid (pH 5.0) system, the carboxyl of staphylokinase is connected to staphylokinase: cystamine: the ratio of carbodiimide is 1:2:2 with the carbodiimide for amino (EDC) of cystamine.The entrained disulfide linkage of cystamine is after three [2-propyloic] phosphines (TCEP) reduction, and the sulfydryl of generation reacts with PEG-maleimide (PEG-Mal), can realize the selective modification of staphylokinase C-terminal carboxyl.Because Ion Exchange Medium can adsorb PEG modified protein and former albumen, and can not adsorb PEG molecule, therefore with SP cationic exchange coloum or Q anion-exchange column, remove unreacted PEG.Because PEG modified protein and former albumen have larger difference on molecular size, can collect PEG modified outcome with gel-filtration column, remove the former albumen of a small amount of unmodified.
Described staphylokinase carboxyl and the crosslinking reaction of cystamine, is characterized in that described cystamine contains uncle's ammonia and disulfide linkage, and wherein cystamine can replace with other compound that contains uncle's ammonia and disulfide linkage, but first-selection is cystamine.
Described MES buffer solution system, is characterized in that not containing carboxyl and amino in buffer solution system, avoids interference the crosslinking reaction of staphylokinase carboxyl and cystamine.
Described staphylokinase carboxyl and the crosslinking reaction of cystamine, is characterized in that used linking agent is carbodiimide, also can connect carboxyl and amino with other linking agent, but first-selection is carbodiimide.
Described PEG modifier, is characterized in that described modifier is to modify the straight chain type of sulfydryl and the mPEG-Mal of Y type.In this two classes modifier, the molecular weight ranges of polyoxyethylene glycol is 1000-40000 dalton.
The modifying method of the carboxyl of described PEG selective modification PROTEIN C end, described method is applicable to the selective modification of the C end carboxyl of range protein and polypeptide.
With respect to traditional carboxyl modified method, the present invention has overcome the poor selectivity existing in traditional protein carboxyl modified, easily occurs in protein molecular and the problem of intermolecular cross-linking, can realize the selective modification of C-terminal carboxyl.
Accompanying drawing explanation:
The molecular structure of Fig. 1 staphylokinase
The terminal modified schema of Fig. 2 C
Fig. 3 is staphylokinase and cystamine reaction in embodiment 1, and the gel-filtration analysis of spectra that adds modified outcome after PEG modifier; Use Superdex 200 (1.0cm * 30cm) gel-filtration column to identify.With 20mM phosphoric acid buffer (pH 7.4) balance and wash-out gel-filtration column containing 0.1M sodium sulfate, flow velocity is 0.5mL/min, and room temperature detects, and detection wavelength is 280nm, and sample size is 100 μ L.
Fig. 4 is the SDS-PAGE electrophorogram of embodiment 1 Central Plains albumen and mono-modified product.Swimming lane 1-4 represents respectively the mono-modified product after the reacted product of staphylokinase, staphylokinase and cystamine, the PEG of standard protein, unmodified modify.
Fig. 5 is the former albumen in embodiment 2, the mono-modified product peptide figure collection of illustrative plates after tryptic digestion.
Fig. 6 is the biological activity comparison of embodiment 3 Central Plains albumen, staphylokinase and the reacted product of cystamine and mono-modified product.
Embodiment
The C end carboxyl selective modification of embodiment 1 recombinant staphylokinase
Implementation method
Staphylokinase is replaced in the MES damping fluid (pH 5.5) of 0.05mo1/L, then add EDC and cystamine in 4 ℃ of reactions 3 hours, wherein the mol ratio of EDC and cystamine and staphylokinase is 2:2:1, then add TCEP to carry out reduction reaction, 4 ℃ are reacted 2 hours, the PEG-mal that is finally 20kDa with molecular weight reaction, staphylokinase and polyethyleneglycol modified dose of mol ratio 1:4, in 4 ℃ of reactions 8 hours.Above-mentioned PEG modified outcome, after separation and purification, is analyzed with Superdex 200 gel-filtration columns and SDS-PAGE electrophoresis.
Result
As shown in Figure 1, there is 1 single feature elution peak in staphylokinase (SAK) on the overanxious post of gel, and introduce sulfydryl on staphylokinase molecule after, the feature elution peak position of the staphylokinase of sulfhydrylation (SAK-SH) does not change substantially.And in conjunction with after PEG molecule, the position of elution peak drifts about left.After this explanation PEG modifies, the molecular weight of SAK increases to some extent.
As shown in Figure 2, staphylokinase is corresponding to 1 single protein electrophoresis band (the 2nd swimming lane), and corresponding molecular weight is about 14kDa.The staphylokinase of sulfhydrylation (SAK-SH) corresponding protein electrophoresis band position identical with staphylokinase (the 3rd swimming lane).PEG modifies staphylokinase also corresponding to 1 single protein electrophoresis band (the 4th swimming lane), and corresponding molecular weight is about 45kDa.This is due to the PEG molecular energy bound water molecule of modifying, and the apparent molecular weight that makes PEG modify staphylokinase is greater than the protein of same molecular amount.Due to combination be that molecular weight is the PEG molecule of 20kDa, can determine that each staphylokinase molecule combines 1 PEG molecule in PEG modifies staphylokinase.
The PEG decorating site of the mono-modified product of embodiment 2 staphylokinase is identified
Embodiment
The evaluation of PEG decorating site is that the tryptic digestion peptide mapping by more mono-modified product and former albumen carries out.Specific as follows:
With the 0.05M NH containing 2.0M urea
4hCO
3solution (pH 8.2) is fully dialysed staphylokinase and PEG modification staphylokinase, through ultrafiltration and concentration to concentration, is then 1.0mg/ml.Get trypsinase, the liquid storage that is 1.0mg/ml with ultrapure water compound concentration.The ratio that is 1:50 according to proteolytic enzyme and staphylokinase mass ratio adds trypsinase liquid storage, and mixture is hatched 11 hours at 37 ℃, clears up product and carries out the separation of peptide section by Shiseido Proteonavi post (4.6mm * 250mm).95% solvent orange 2 A for pillar (containing the ultrapure water of 0.1% trifluoroacetic acid) and 5% the abundant balance of solvent B (containing the acetonitrile of 0.1% trifluoroacetic acid), flow velocity is 0.5ml/min.Gradient is for to rise to 50% at 100 minutes internal solvent B from 5%.
Result
Fig. 3 is that the enzyme of former albumen and modified outcome is cut peptide figure.As shown in Figure 3, there are a plurality of feature elution peaks in staphylokinase after trypsin hydrolyzing in chromatographic column, the 16th and the 17th peptide section (T16+17) that wherein the peptide section at C terminal amino acid place is tryptic digestion.Staphylokinase is after PEG modifies, and more than 80% T16+17 peptide section disappears.After this shows that T16+17 peptide section combines PEG molecule, because hydrophobicity strengthens, the original position of this peptide section disappears.By contrast, other peptide section of staphylokinase is almost unchanged after PEG modifies.Therefore the C end that, we can infer staphylokinase is by the modification of PEG molecular selectivity.
The biological activity comparison of embodiment 3 modified outcomes and former albumen
Embodiment
The biological activity of staphylokinase is tested to detect by lyase fibrous loop, wherein with the staphylokinase of unmodified in contrast.Obtain after the fibrous loop diameter that staphylokinase (SAK), sulfhydrylation staphylokinase (SAK-SH) and PEG modify staphylokinase (SAK-PEG), the diameter of staphylokinase of unmodified of take is 100%, the relative biological activity of calculating SAK-SH and SAK-PEG.Survey the step of living as follows
(1) take 100mg recombinant human Fibrinogen, be dissolved in the Tris-HCl damping fluid (pH 7.4) of 25ml;
(2) take the agarose of 250mg, join in the Tris-HCl damping fluid (pH 7.4) of 25ml, concentration is 1% (w/v), heating for dissolving in the water-bath of 60 ℃;
(3) agarose of dissolving is at room temperature cooled to 45 ℃;
(4) get the thrombin solution of 20 μ l, join in the recombinant human fibrinogen solution of the fresh configuration of 25ml, mix rapidly;
(5) zymoplasm/recombinant human fibrinogen solution is joined rapidly in agarose solution, mix, pour in 2 flat boards, room temperature is placed 30min, after agarose solidifies, and punching application of sample;
(6) with Tris-HCl damping fluid (pH 7.4), urokinase weaker concn is respectively to 1,5,10,20,40,80IU/ml, every hole application of sample 15 μ l, as typical curve;
(7) with the protein concn of Bradford method working sample, make 2 times of serial dilutions, join respectively Zhong，Mei hole, agar hole application of sample 15 μ l;
(8) flat board is upside down in incubator and is cultivated at 37 ℃, until there is solusphere clearly to occur, with vernier callipers, measure each solusphere diameter, make typical curve, calculate the relative reactivity of SAK-SH and SAK-PEG.
Result
Determination of activity result shows that the relative reactivity of SAK-SH is 93%, and the relative reactivity of SAK-PEG is 76%.This explanation is carried out PEG modification at the C end away from staphylokinase active centre, can keep to a great extent the biological activity of staphylokinase.
Claims (5)
1. the method for the C-terminal carboxyl of a polyoxyethylene glycol selective modification proteins and peptides, key step is: in the buffer solution system of the 2-of 2-200mM (N-morpholine) ethyl sulfonic acid pH 4.5-8.0, the amino of the carboxyl of protein or polypeptide and cystamine is carried out to crosslinking reaction with carbodiimide, wherein protein or polypeptide: cystamine: the ratio of carbodiimide is 1:5-10:2-50, the entrained disulfide linkage of cystamine is after the reduction of three (2-propyloic) phosphine, the sulfydryl generating reacts with mono methoxy polyethylene glycol maleimide, thereby realize the PEG selective modification of proteins and peptides C-terminal carboxyl.
2. the method for the C-terminal carboxyl of polyoxyethylene glycol selective modification proteins and peptides according to claim 1, described protein or polypeptide are staphylokinase, but are not limited to staphylokinase, are also applicable to the selective modification of other oroteins and peptide C terminal carboxyl(group).
3. the method for the C-terminal carboxyl of polyoxyethylene glycol selective modification proteins and peptides according to claim 1, in the carboxyl of protein or polypeptide and the crosslinking reaction of cystamine, cystamine contains 2 uncle's ammonia and 1 pair of disulfide linkage.
4. the method for the C-terminal carboxyl of polyoxyethylene glycol selective modification proteins and peptides according to claim 1, not containing carboxyl and amino, avoids the crosslinking reaction of carboxyl and cystamine to impact in described MES buffer solution system.
5. the method for the C-terminal carboxyl of polyoxyethylene glycol selective modification proteins and peptides according to claim 1, described polyethyleneglycol modified dose is to modify the straight chain type of sulfydryl or the mono methoxy polyethylene glycol maleimide of Y type, and in this two classes modifier, the molecular weight ranges of polyoxyethylene glycol is 1000-40000 dalton.
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Cited By (4)
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| CN103923161A (en) * | 2014-04-25 | 2014-07-16 | 南开大学 | Novel protein site-specific modification method |
| CN104497097A (en) * | 2014-06-18 | 2015-04-08 | 深圳市第二人民医院 | Protein or polypeptide-polymer grafted copolymer with reduction responsiveness, synthesis method and protein or polypeptide drug thereof |
| CN108395531A (en) * | 2018-01-18 | 2018-08-14 | 东华大学 | It is a kind of package nanogold particle amphoteric ion and morpholine modification Polyamidoamine Dendrimers preparation method |
| CN117088963A (en) * | 2023-07-21 | 2023-11-21 | 博汇美萃生物工程技术(广东)有限公司 | PEG modified long-acting recombinant collagen for scalp repair and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923161A (en) * | 2014-04-25 | 2014-07-16 | 南开大学 | Novel protein site-specific modification method |
| CN103923161B (en) * | 2014-04-25 | 2016-04-06 | 南开大学 | A kind of method of protein pointed decoration |
| CN104497097A (en) * | 2014-06-18 | 2015-04-08 | 深圳市第二人民医院 | Protein or polypeptide-polymer grafted copolymer with reduction responsiveness, synthesis method and protein or polypeptide drug thereof |
| CN108395531A (en) * | 2018-01-18 | 2018-08-14 | 东华大学 | It is a kind of package nanogold particle amphoteric ion and morpholine modification Polyamidoamine Dendrimers preparation method |
| CN108395531B (en) * | 2018-01-18 | 2021-02-26 | 东华大学 | Preparation method of zwitterion and morpholine modified polyamide-amine dendrimer wrapping gold nanoparticles |
| CN117088963A (en) * | 2023-07-21 | 2023-11-21 | 博汇美萃生物工程技术(广东)有限公司 | PEG modified long-acting recombinant collagen for scalp repair and preparation method thereof |
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