CN105777863B - A method of the isolated polypeptide from glyoxaline ion liquid - Google Patents
A method of the isolated polypeptide from glyoxaline ion liquid Download PDFInfo
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- CN105777863B CN105777863B CN201610171171.XA CN201610171171A CN105777863B CN 105777863 B CN105777863 B CN 105777863B CN 201610171171 A CN201610171171 A CN 201610171171A CN 105777863 B CN105777863 B CN 105777863B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
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- C07K1/16—Extraction; Separation; Purification by chromatography
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
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- C07K14/665—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
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Abstract
The present invention is a kind of method of isolated polypeptide from glyoxaline ion liquid, belongs to the purifies and separates technical field of biomolecule.A kind of method of isolated polypeptide from glyoxaline ion liquid removes free glyoxaline ion liquid using salting-out method back extraction, then will thoroughly be removed with peptide molecule by the imidazole-like ionic of chemical bonds using the reagent of silver ion.The method of isolated polypeptide is simple and effective in the slave glyoxaline ion liquid, and low in cost, low energy consumption, and product purity is high, greatly improves applicability and application range of the glyoxaline ion liquid in chemiluminescent polypeptide.
Description
Technical field:
The present invention relates to the purifies and separates technical fields of biomolecule, more particularly to one kind to divide from glyoxaline ion liquid
Method from polypeptide.
Background technique:
Due to may include in polypeptide various combination and quantity hydrophily and hydrophobic amino acid residue, and it is foldable
Complicated secondary structure is formed, the diversity and changeability of its solubility property are caused.Therefore, in chemiluminescent polypeptide, the choosing of solvent
Select often a major challenge.Currently, common inorganic and organic solvent can not all be compatible with the dissolution of the polypeptide of all kinds.
Ionic liquid at room temperature is often simply called ionic liquid, is a kind of ionic compound for being able to maintain liquid at room temperature.From
Sub- liquid is widely used in material, the energy and chemical field, is famous green industry reagent.In recent years, ionic liquid at room temperature,
Especially imidazole ion liquid is applied initially as novel dissolvent in chemiluminescent polypeptide field.In research so far, miaow
Oxazolinium ion liquid all shows excellent solvability to various types of polypeptides, before having both preferable bio-compatibility and Cabbeen
Body catalytic properties, are widely used in polypeptide fragment connection reaction [1], and amino acid couplings react [2] and peptide carrier synthesis [3].
However, solvability just powerful to polypeptide due to glyoxaline ion liquid, the peptide separation being dissolved in ionic liquid is come out
Also very difficult；Have in document separated from ionic liquid by ether cycling extraction method [2] or liquid chromatography [4] it is more
Peptide, but can not often completely remove the ionic liquid contained in polypeptide sample.This is primarily due to the yin of glyoxaline ion liquid
Ion or cation have stronger chemical bonding energy to the amino acid residue in peptide molecule, such as cysteine, lysine
Power and interaction [4].Since combination of the glyoxaline ion liquid on polypeptide is easily to polypeptide conformation itself and biomembrane ion
Channel etc. impacts, and may cause denaturation or the inactivation of polypeptide drugs, and the application to polypeptide in field of medicaments causes obstacle
[4].In most cases, there are chemical bonds between polypeptide and ionic liquid molecules, and containing polar residues, concentration often exists
MM rank, also uncomfortable spent ion exchange resin and antisolvent crystallization technology [5] method.Therefore, this phenomenon restricts significantly
Application of the ionic liquid in chemiluminescent polypeptide.
For this problem, the method for the present invention provides a kind of from glyoxaline ion liquid isolated polypeptide passes through salt
The physical method of effect and the chemical method of silver ion removing are analysed, efficiently and easily completely removes ionic liquid；Meanwhile most
Make to limits configuration and the activity of polypeptide unaffected.
Summary of the invention:
It is an object of the invention to propose a kind of method of isolated polypeptide from glyoxaline ion liquid, to solve polypeptide hardly possible
The problem of to be isolated and purified from ionic liquid.
Technical solution used by this reality is invented combines physics back extraction separation and chemical removal separation method: step 1,
First mixed according to a certain percentage with preferred polypeptide organic solvent with the polypeptide solution containing glyoxaline ion liquid；Step 2, it drips
Add preferred stripping agent to solution split-phase occur, forms solvent layer and back extraction oxidant layer, separate solvent phase and stripping agent phase；Step 3,
Step 2 mutually is repeated three times or more than three times to separating obtained solvent；Step 4, into separating for several times solvent phase after purification, add
Add the ionic liquid scavenger of silver ion, reaction releases heat, quickly generates heterocycle carbine silver complex [6] or insoluble silver
The white flock precipitate of salt, centrifugal filtration remove precipitating；Step 5, after purification, freeze-drying removes solvent to acquired solution, is free of
The polypeptide sterling of ionic liquid.
In the technical solution, polypeptide organic solvent can be isopropanol, isobutanol, propylene glycol, butanediol, acetonitrile,
Acetone, methyl acetate, ethyl acetate, dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, methylene chloride, chloroform；Its
In, the preferred standard of solvent are as follows: the solvability having to isolated target polypeptides, which is higher than, has ionic liquid used
Solvability.
In the technical solution, glyoxaline ion liquid be can be containing alkyl imidazolium cation or imidazoles anionoid
Ionic liquid.The ionic liquid structural formula containing glyoxaline cation may is that
The ionic liquid structural formula containing imidazole anion may is that
Wherein, the R in structural formula1、R2、R3、R4、R5It can be hydrogen, alkyl, containing halogen, oxygen-containing functional group, nitrogenous function
Group, phosphorous functional group, sulfur-bearing functional group；X-It can be the acid radical anion of inorganic acid or organic acid；Y+Can be choline,
Amine, monokaryon imidazoles, double-core alkyl imidazolium cation.
In the technical solution, polypeptide be can be containing the life for being keyed nature or non-natural amino acid residue by peptide
Object molecule.
In the technical solution, stripping agent can be water (comprising buffered aqueous solution), methanol, ethyl alcohol or normal propyl alcohol；It is excellent
The standard of choosing are as follows: the solvability having to ionic liquid is higher than the solvability having to isolated target polypeptides, and can be with
Make solution system that salting-out effect occur under certain mixed proportion, forms Phase separation.
In the technical solution, the ionic liquid scavenger of silver ion is the dispersed system or energy of silver ion
The dispersed system that silver ion is generated by physical chemistry means, can be, but not limited to be the solution of silver salt, silver salt it is suspended
Liquid, the solution of silver oxide, silver oxide suspension or their mixture；Preferred standard are as follows: polypeptide stability is not influenced, with
Ionic liquid is swift in response completely, and generates the precipitating being easily isolated.
Compared to the prior art the method for the isolated polypeptide provided by the invention from glyoxaline ion liquid has as follows
Innovation and advantage:
1. the method for the isolated polypeptide provided by the invention from glyoxaline ion liquid, using ionic liquid in miscible azeotropic
Salting-out effect in object can easily remove the ionic liquid of most of free state, avoid polypeptide products during the separation process
It is lost；
2. the method for the isolated polypeptide provided by the invention from glyoxaline ion liquid, utilizes the change of glyoxaline ion liquid
Learning characteristic will quickly be taken off by way of adding silver ion scavenger with imidazol ion of the chemical bonds on polypeptid residue
It removes, separation costs are cheap；
3. the method for the isolated polypeptide provided by the invention from glyoxaline ion liquid is compatible with containing variety classes residue
Polypeptide, glyoxaline cation and imidazole anion can be removed simultaneously, polypeptide activity itself is not exposed to influence.
Detailed description of the invention:
Fig. 1 is the high performance liquid chromatography of the polypeptide LYRAGCRANK in ionic liquid 1- ethyl-3-methylimidazole acetate
Figure.
Fig. 2 is the efficient liquid that polypeptide LYRAGCRANK is isolated from ionic liquid 1- ethyl-3-methylimidazole acetate
Phase chromatogram.
Fig. 3 is the ESI mass spectrum that polypeptide LYRAGCRANK is isolated from ionic liquid 1- ethyl-3-methylimidazole acetate
Figure.
Fig. 4 is the high-efficient liquid phase color of the leucine enkephalin in ionic liquid 1-butyl-3-methyl imidazolium hexafluorophosphate
Spectrogram.
Fig. 5 is leucine enkephalin is isolated from ionic liquid 1-butyl-3-methyl imidazolium hexafluorophosphate efficient
Liquid chromatogram.
Fig. 6 is the ESI for the leucine enkephalin isolated from ionic liquid 1-butyl-3-methyl imidazolium hexafluorophosphate
Mass spectrogram.
Fig. 7 is the mu-conotoxin SIIIA high performance liquid chromatography in ionic liquid 1- ethyl-3-methylimidazole acetate
Figure.
Fig. 8 is to isolate the efficient of mu-conotoxin SIIIA from ionic liquid 1- ethyl-3-methylimidazole acetate
Liquid chromatogram.
Fig. 9 is the ESI that mu-conotoxin SIIIA is isolated from ionic liquid 1- ethyl-3-methylimidazole acetate
Mass spectrogram.
Figure 10 is the high-efficient liquid phase chromatogram of the mu-conotoxin SIIIA in ionic liquid choline imidazole salts.
Figure 11 is the high-efficient liquid phase chromatogram that mu-conotoxin SIIIA is isolated from ionic liquid choline imidazole salts.
Figure 12 is the ESI mass spectrogram that mu-conotoxin SIIIA is isolated from ionic liquid choline imidazole salts
Specific embodiment:
Experimental method used in following embodiments is conventional method unless otherwise specified.
Experimental material as used in the following examples, reagent etc. can be obtained by commercial sources or known experimental method
?.
Embodiment 1
In document [1], polypeptide LYRAGCRANK was once closed using ionic liquid 1- ethyl-3-methylimidazole acetate as solvent
At.In the present embodiment, polypeptide LYRAGCRANK is carried out in ionic liquid 1- ethyl -3- methyl using method provided by the invention
Separation in imidazoleacetic acid salt.
Experimental provision is mainly 50 milliliters of point bottom with a scale test tubes, filters, pipette, centrifuge, water bath device, concussion
Blender and low-temperature vacuum drying device.Step 1, polypeptide LYRAGCRANK is dissolved in ionic liquid 1- ethyl -3- methyl miaow
In zole acetic acid salt, the polypeptide ionic liquid solution that 1mL concentration is 3nmol/L is formed.Step 2, the propylene glycol of 10mL is added, shakes
Stirring.Step 3,200 μ L pure water are added, misty phenomenon is generated, gained mixed liquor is made point for 30 minutes with most high speed centrifuge separation
Layer.Step 4, the water layer containing ionic liquid of test tube bottom is carefully removed.Step 5, step 3 and 4 is repeated to resulting solution, directly
To no longer there is misty phenomenon when pure water is added dropwise.Step 6, under conditions of water-bath, the third two containing 20% silver nitrate are slowly added dropwise
Alcoholic solution；Stop being added dropwise immediately when not continuing to and generate to white precipitate；The reaction equation of step 6 are as follows:
Step 7, it is centrifugated 20 minutes, and is filtered to remove precipitating with quartz fibre filter paper；Step 8, acquired solution is led to
It crosses and is purified using the gel filtration method of dilute hydrochloric acid eluent, is lyophilized under cryogenic vacuum, obtain the polypeptide crystal powder of white
End.
Products obtained therefrom is polypeptide LYRAGCRANK, and yield 95.4% (is not included in oxidized form polypeptide)；High-efficient liquid phase chromatogram
In do not find the characteristic peak of ionic liquid, the polypeptide products peak compared with the polypeptide products peak (Fig. 1) in ionic liquid, after separation
(Fig. 2) is not subjected to displacement or divided；ESI mass spectrum does not detect ionic liquid residual (Fig. 3).
Embodiment 2
In document [3], polypeptide leucine enkephalin was once synthesized by carrier of glyoxaline ion liquid.In the present embodiment
In, leucine enkephalin is carried out in ionic liquid 1-butyl-3-methyl imidazolium hexafluorophosphate using method provided by the invention
In separation.
Experimental provision is mainly 50 milliliters of point bottom with a scale test tubes, filters, pipette, centrifuge, water bath device, concussion
Blender and low-temperature vacuum drying device.Step 1, leucine enkephalin is dissolved in ionic liquid 1-butyl-3-methyl imidazolium
In hexafluorophosphate, the polypeptide ionic liquid solution that 1mL concentration is 3nmol/L is formed.Step 2, the methyl acetate of 10mL is added,
Concussion stirring.Step 3,150 μ L methylene chloride are added dropwise, generate misty phenomenon；The centrifuge separation of gained mixed liquor is made point for 30 minutes
Layer.Step 4, the dichloromethane layer containing ionic liquid of test tube bottom is carefully removed.Step 5, step 3 is repeated to resulting solution
With 4, until no longer occurring misty phenomenon when pure water is added dropwise.Step 6, under conditions of water-bath, the silver oxide containing saturation is slowly added dropwise
Methanol solution；Stop being added dropwise immediately when not continuing to and generate to white precipitate；The reaction equation of step 6 are as follows:
Step 7, it is centrifugated 20 minutes, and is filtered to remove precipitating with quartz fibre filter paper；Step 8, acquired solution is led to
It crosses and is purified using the gel filtration method of dilute hydrochloric acid eluent, is lyophilized under cryogenic vacuum, obtain the polypeptide crystal powder of white
End.
Products obtained therefrom is leucine enkephalin, yield 99.2%；The spy of ionic liquid is not found in high-efficient liquid phase chromatogram
Peak is levied, compared with the polypeptide products peak (Fig. 4) in ionic liquid, the polypeptide products peak after separation is not subjected to displacement or divides (figure
5)；ESI mass spectrum does not detect ionic liquid residual (Fig. 6).
Embodiment 3
In document [4], polypeptide mu-conotoxin SIIIA is once molten with ionic liquid 1- ethyl-3-methylimidazole acetate
Agent is oxidized folding.In the present embodiment, polypeptide mu-conotoxin SIIIA is carried out in ionic liquid using method provided by the invention
Separation in body 1- ethyl-3-methylimidazole acetate.
Experimental provision is mainly 50 milliliters of point bottom with a scale test tubes, filters, pipette, centrifuge, water bath device, concussion
Blender and low-temperature vacuum drying device.Step 1, mu-conotoxin SIIIA is dissolved in ionic liquid 1- ethyl -3- methyl miaow
In zole acetic acid salt, the polypeptide ionic liquid solution that 1mL concentration is 3nmol/L is formed.Step 2, the isopropanol of 10mL is added, shakes
Stirring.Step 3,100 μ L pure water are added dropwise, generate misty phenomenon；Gained mixed liquor, which is centrifugated 30 minutes, to be made to be layered.Step 4,
Carefully remove the layer containing ionic liquid aqueous phase of test tube bottom.Step 5, step 3 and 4 is repeated to resulting solution, until being added dropwise pure
No longer occurs misty phenomenon when water.Step 6, under conditions of water-bath, the isopropanol that the silver acetate containing 2.4mg/mL is slowly added dropwise is molten
Liquid；Stop being added dropwise immediately when no longer generating to white precipitate；The reaction equation of step 6 are as follows:
Step 7, it is centrifugated 20 minutes, and is filtered to remove precipitating with quartz fibre filter paper；Step 8, acquired solution is led to
It crosses and is purified using the gel filtration method of dilute hydrochloric acid eluent, is lyophilized under cryogenic vacuum, obtain the polypeptide crystal powder of white
End.
Products obtained therefrom is mu-conotoxin SIIIA, yield 97.5%；Ionic liquid is not found in high-efficient liquid phase chromatogram
Characteristic peak, compared with the polypeptide products peak (Fig. 7) in ionic liquid, the polypeptide products peak after separation is not subjected to displacement or divides
(Fig. 8)；ESI mass spectrum does not detect ionic liquid residual (Fig. 9).
Embodiment 4
In the present embodiment, method provided by the invention carries out polypeptide mu-conotoxin SIIIA in ionic liquid choline miaow
Separation in azoles salt.
Experimental provision be mainly 50 milliliters of sharp bottom test tubes with a scale, filter, pipette, centrifuge, concussion blender and
Low-temperature vacuum drying device.Step 1, mu-conotoxin SIIIA is dissolved in ionic liquid choline imidazole salts, it is dense forms 1mL
Degree is the polypeptide ionic liquid solution of 3nmol/L.Step 2, the methylene chloride of 15mL, concussion stirring is added.Step 3,100 are added dropwise
μ L water generates misty phenomenon；Gained mixed liquor, which is centrifugated 30 minutes, to be made to be layered.Step 4, containing for test tube bottom is carefully removed
Ionic liquid aqueous phase layer.Step 5, step 3 and 4 is repeated to resulting solution, until no longer occurring misty phenomenon when pure water is added dropwise.
Step 6, the water and methylene chloride mixed solution (volume ratio 1: 1) of the silver oxide containing 1.5mg/mL is added dropwise；It is no longer produced to white precipitate
Stop being added dropwise immediately when raw；The reaction equation of step 6 are as follows:
Step 7, it is centrifugated 20 minutes, and is filtered to remove precipitating with quartz fibre filter paper；Step 8, acquired solution is led to
It crosses and is purified using the gel filtration method of dilute hydrochloric acid eluent, is lyophilized under cryogenic vacuum, obtain the polypeptide crystal powder of white
End.
Products obtained therefrom is mu-conotoxin SIIIA, yield 98.1%；Ionic liquid is not found in high-efficient liquid phase chromatogram
Characteristic peak, compared with the polypeptide products peak (Figure 10) in ionic liquid, the polypeptide products peak after separation is not subjected to displacement or divides
(Figure 11)；ESI mass spectrum does not detect ionic liquid residual (Figure 12).
Embodiment 5
Electrobiophysics method (sodium-ion channel blocking) evaluates the activity of following polypeptide in reference literature [4]: using high
Mu-conotoxin SIIIA that effect liquid phase chromatogram method is separated from ionic liquid 1- ethyl-3-methylimidazole acetate, using efficient
Mu-conotoxin SIIIA that liquid chromatography is separated from ionic liquid choline imidazole salts, using method provided by the invention from
The mu-conotoxin SIIIA separated in ionic liquid 1- ethyl-3-methylimidazole acetate, with use method provided by the invention
The mu-conotoxin SIIIA separated from ionic liquid choline imidazole salts.It the results are shown in Table one:
Table one
The results show that activity is lost using the mu-conotoxin SIIIA that the high performance liquid chromatography of document [4] separates,
And the mu-conotoxin SIIIA of method provided by the invention separation is used to maintain very high activity.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office
Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field
Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention
Addition, selection of concrete mode etc., are included within protection scope of the present invention and the open scope.
[1] K ü hl, T.；Chen, M.；Teichmann, K.；Stark, A.；Imhof, D.lonic Liquid 1-
Ethyl-3-Methylimidazolium Acetate:An Attractive Solvent for Native Chemical
Ligation of Peptides.Tetrahedron Lett.2014,55 (27), 3658-3662.
[2] Vallette, H.Room Temperature lonic Liquids (RTI L ' s) Are Convenient
Solvents for Peptide Synthesis！Arkivoc 2006,2006 (4), 200.
[3] Miao, W.；Chan, T.H.lonic-Liquid-Supported Synthesis:A Novel Liquid-
Phase Strategy for Organic Synthesis.Acc.Chem.Res.2006,39 (12), 897-908.
[4] Heime r, P.；Tietze, A.A.；M.；Giernoth, R.；Kuchenbuch, A.；Stark,
A.；Leipold, E.；Heinemann, S.H.；Kandt, C.；Imhof, D.Application of Room-Temperature
Aprotic and Protic lonic Liquids for Oxidative Folding of Cysteine-Rich
Peptides.ChemBioChem 2014,15 (18), 2754-2765.
[5] a kind of 102559943 B of CN: process of separating glucose and ionic liquid.
[6] Hindi, K.M.；Panzner, M.J.；Tessier, C.A.；Cannon, C.L.；Youngs, W.J.The
Medicinal Applications of Imidazolium Carbene-Metal Complexes.Chem.Rev.2009,
109 (8), 3859-3884.
Claims (1)
1. a kind of method of the isolated polypeptide from glyoxaline ion liquid, which is characterized in that combine physics back extraction separation and
Chemical removal separation method includes the following steps: that (1) is first molten with polypeptide organic solvent and the polypeptide containing glyoxaline ion liquid
Liquid mixes according to a certain percentage, used in the solvability that has to isolated target polypeptides of polypeptide organic solvent should be higher than that
The solvability having to glyoxaline ion liquid used；(2) principle for utilizing salting-out effect, adds strippant, to more
Glyoxaline ion liquid in peptide solution is stripped, and the combination of the polypeptide organic solvent and strippant of use has respectively:
Propylene glycol and water, methyl acetate and methylene chloride, isopropanol and water, methylene chloride and water；(3) in separating obtained polypeptide solution
The ionic liquid scavenger of middle addition silver ion, until precipitating stops being added dropwise immediately when no longer generating, the ionic liquid of silver ion
Body scavenger includes: the propylene glycol solution of 20% silver nitrate, the methanol solution of the silver oxide containing saturation, the silver acetate containing 2.4mg/mL
Aqueous isopropanol, the solution that the water of the silver oxide containing 1.5mg/mL is mixed with methylene chloride with volume ratio 1:1；(4) it is filtered to remove
The precipitating of generation；(5) after acquired solution purifying freeze-drying, polypeptide products are obtained.
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| PCT/CN2016/079862 WO2017161619A1 (en) | 2016-03-24 | 2016-04-21 | Method for separating polypeptide from imidazolium ionic liquid |
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| CN1807446A (en) * | 2006-02-27 | 2006-07-26 | 南京财经大学 | Method for separating and extracting protein and/or enzyme from ion liquid |
| CN105203372A (en) * | 2014-05-26 | 2015-12-30 | 中国科学院大连化学物理研究所 | Method for removing ionic liquid in sample solution |
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| CN102559943B (en) * | 2010-12-31 | 2014-02-12 | 北京化工大学 | Process method for separating glucose from ionic liquid |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1807446A (en) * | 2006-02-27 | 2006-07-26 | 南京财经大学 | Method for separating and extracting protein and/or enzyme from ion liquid |
| CN105203372A (en) * | 2014-05-26 | 2015-12-30 | 中国科学院大连化学物理研究所 | Method for removing ionic liquid in sample solution |
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| 功能化离子液体为载体液相合成RGD 三肽;徐中义;《化学研究与应用》;20121031;第24卷(第10期);第1613-1616页 |
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