WO2008010687A1 - Procédé de liaison, séparation ou purification sélective de protéines utilisant des nanoparticules magnétiques - Google Patents
Procédé de liaison, séparation ou purification sélective de protéines utilisant des nanoparticules magnétiques Download PDFInfo
- Publication number
- WO2008010687A1 WO2008010687A1 PCT/KR2007/003519 KR2007003519W WO2008010687A1 WO 2008010687 A1 WO2008010687 A1 WO 2008010687A1 KR 2007003519 W KR2007003519 W KR 2007003519W WO 2008010687 A1 WO2008010687 A1 WO 2008010687A1
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- Prior art keywords
- row
- protein
- nanoparticle
- magnetic
- transition metal
- Prior art date
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 99
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 98
- 238000000034 method Methods 0.000 title claims abstract description 41
- 239000002122 magnetic nanoparticle Substances 0.000 title claims abstract description 37
- 238000000926 separation method Methods 0.000 title claims abstract description 28
- 238000009739 binding Methods 0.000 title claims abstract description 22
- 238000000746 purification Methods 0.000 title claims abstract description 14
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims abstract description 46
- 239000002105 nanoparticle Substances 0.000 claims abstract description 46
- 229910052723 transition metal Inorganic materials 0.000 claims abstract description 31
- 150000003624 transition metals Chemical class 0.000 claims abstract description 30
- 229910052759 nickel Inorganic materials 0.000 claims abstract description 22
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims abstract description 18
- 150000002500 ions Chemical class 0.000 claims abstract description 15
- 239000000203 mixture Substances 0.000 claims abstract description 11
- 229910017052 cobalt Inorganic materials 0.000 claims abstract description 10
- 239000010941 cobalt Substances 0.000 claims abstract description 10
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims abstract description 10
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims abstract description 10
- 229910052742 iron Inorganic materials 0.000 claims abstract description 9
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000011701 zinc Substances 0.000 claims abstract description 8
- 229910052725 zinc Inorganic materials 0.000 claims abstract description 8
- 235000018102 proteins Nutrition 0.000 claims description 90
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 28
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 25
- 239000011230 binding agent Substances 0.000 claims description 21
- 235000001014 amino acid Nutrition 0.000 claims description 9
- 229940024606 amino acid Drugs 0.000 claims description 9
- 150000001413 amino acids Chemical class 0.000 claims description 9
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 6
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 6
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- 239000004472 Lysine Substances 0.000 claims description 6
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 6
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 6
- 229910045601 alloy Inorganic materials 0.000 claims description 6
- 239000000956 alloy Substances 0.000 claims description 6
- 235000009582 asparagine Nutrition 0.000 claims description 6
- 229960001230 asparagine Drugs 0.000 claims description 6
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 6
- 229930182817 methionine Natural products 0.000 claims description 6
- 150000003568 thioethers Chemical class 0.000 claims description 6
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims description 4
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims description 4
- 229910052804 chromium Inorganic materials 0.000 claims description 4
- 239000011651 chromium Substances 0.000 claims description 4
- 229960003067 cystine Drugs 0.000 claims description 4
- 235000018417 cysteine Nutrition 0.000 claims description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 2
- 125000000539 amino acid group Chemical group 0.000 claims 1
- 229910021645 metal ion Inorganic materials 0.000 abstract description 15
- 238000001042 affinity chromatography Methods 0.000 abstract description 7
- 238000007796 conventional method Methods 0.000 abstract description 5
- 235000014304 histidine Nutrition 0.000 description 27
- 239000000243 solution Substances 0.000 description 23
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 16
- 229910000480 nickel oxide Inorganic materials 0.000 description 13
- GNRSAWUEBMWBQH-UHFFFAOYSA-N oxonickel Chemical compound [Ni]=O GNRSAWUEBMWBQH-UHFFFAOYSA-N 0.000 description 13
- 239000005090 green fluorescent protein Substances 0.000 description 11
- 239000000463 material Substances 0.000 description 11
- -1 histidine amino acids Chemical class 0.000 description 8
- 238000012856 packing Methods 0.000 description 8
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 5
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 239000013522 chelant Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002189 fluorescence spectrum Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000013537 high throughput screening Methods 0.000 description 2
- 150000002411 histidines Chemical class 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 229910001428 transition metal ion Inorganic materials 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- QGLWBTPVKHMVHM-KTKRTIGZSA-N (z)-octadec-9-en-1-amine Chemical compound CCCCCCCC\C=C/CCCCCCCCN QGLWBTPVKHMVHM-KTKRTIGZSA-N 0.000 description 1
- 241000412565 Argentina sphyraena Species 0.000 description 1
- AILDTIZEPVHXBF-UHFFFAOYSA-N Argentine Natural products C1C(C2)C3=CC=CC(=O)N3CC1CN2C(=O)N1CC(C=2N(C(=O)C=CC=2)C2)CC2C1 AILDTIZEPVHXBF-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 206010020843 Hyperthermia Diseases 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- 239000002616 MRI contrast agent Substances 0.000 description 1
- 235000016594 Potentilla anserina Nutrition 0.000 description 1
- 108010026552 Proteome Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 239000012300 argon atmosphere Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229910000428 cobalt oxide Inorganic materials 0.000 description 1
- IVMYJDGYRUAWML-UHFFFAOYSA-N cobalt(ii) oxide Chemical compound [Co]=O IVMYJDGYRUAWML-UHFFFAOYSA-N 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000000979 dip-pen nanolithography Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 125000000487 histidyl group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C([H])=N1 0.000 description 1
- 230000036031 hyperthermia Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- BMGNSKKZFQMGDH-FDGPNNRMSA-L nickel(2+);(z)-4-oxopent-2-en-2-olate Chemical compound [Ni+2].C\C([O-])=C\C(C)=O.C\C([O-])=C\C(C)=O BMGNSKKZFQMGDH-FDGPNNRMSA-L 0.000 description 1
- MGFYIUFZLHCRTH-UHFFFAOYSA-N nitrilotriacetic acid Chemical group OC(=O)CN(CC(O)=O)CC(O)=O MGFYIUFZLHCRTH-UHFFFAOYSA-N 0.000 description 1
- 238000006053 organic reaction Methods 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000005486 sulfidation Methods 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229910000314 transition metal oxide Inorganic materials 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000011282 treatment Methods 0.000 description 1
- RMZAYIKUYWXQPB-UHFFFAOYSA-N trioctylphosphane Chemical compound CCCCCCCCP(CCCCCCCC)CCCCCCCC RMZAYIKUYWXQPB-UHFFFAOYSA-N 0.000 description 1
- ZMBHCYHQLYEYDV-UHFFFAOYSA-N trioctylphosphine oxide Chemical compound CCCCCCCCP(=O)(CCCCCCCC)CCCCCCCC ZMBHCYHQLYEYDV-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82B—NANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
- B82B1/00—Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01F—MAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
- H01F1/00—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
- H01F1/0036—Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties showing low dimensional magnetism, i.e. spin rearrangements due to a restriction of dimensions, e.g. showing giant magnetoresistivity
- H01F1/0045—Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
- H01F1/0054—Coated nanoparticles, e.g. nanoparticles coated with organic surfactant
Definitions
- the present invention relates to a protein binding agent which selectively binds to specific proteins comprising transition metal magnetic nanopart icles. More specifically, the present invention is directed to a protein binding agent comprising magnetic nanoparticles selected from the group consisting of iron, manganese, nickel, cobalt or ions thereof, which bind selectively to proteins comprising an amino acid selected from the group consisting of histidine, asparagine, argedine (argenine), cyrtine (cystine), glutamine, lysine, methionine, proline, or tryptophan.
- the present invention relates to a method for the selective binding, separation, or purification of specific proteins using the protein binding agent comprising the magnetic nanoparticles.
- the present invention provides an easier, faster, and more economical method for the separation of specific proteins compared to the conventional method for metal-ion affinity chromatography.
- the present invention relates to a method for selective binding, separation, or purification of a specific protein using magnetic nanoparticle, which comprises: binding a magnetic nanoparticle which includes a transition metal such as iron, manganese, nickel, cobalt, zinc, etc. or ions thereof, to a specific protein contained in a biological mixture! separating said specific protein bound with said nanoparticle from said biological mixture by means of magnetic field; and separating said specific protein from the nanopart icle-protein complex.
- a magnetic nanoparticle which includes a transition metal such as iron, manganese, nickel, cobalt, zinc, etc. or ions thereof
- the present invention relates to a method for selective separation or purification of a protein containing a specific amino acid from a biological mixture by using the specific affinity between nickel oxide and a specific amino acid, and a novel use of magnetic nickel nanopart icles coated with nickel oxide and measured in nanometers.
- the packing materials used in the columns of metal-ion chromatography are mostly prepared by binding chelate ligands to materials used for packing and then binding the chelate ligand bound packing material with transition metal ions via coordinate covalent bonding.
- Magnetic nanoparticles of nanoparticle size are currently being used widely for biomedical purposes such as MRI contrast agents, hyperthermia treatments, drugs or gene transfer, etc..
- Nanoparticles with small sizes and large surface areas have distinctive properties in that they have superior dispersability in water or biological solutions, bind quickly and easily with biological molecules, and separate easily from biological mixtures using external force applied by a magnetic field. Due to these distinctive properties, there are many promising applications for these nanoparticles in biomedical use relating to proteins, cells and other similar biomolecular structures.
- the present invention provides a method for producing nanometer sized metal ion magnetic nanoparticles for selective binding with specific proteins, and the effective separation of said specific proteins bound to said magnetic nanoparticles from a biological mixture using a magnetic field.
- the first objective of the present invention can be achieved by providing a protein binding agent comprising a of transition metal selected from the group consisting of iron, manganese, nickel, cobalt, and zinc, or ions thereof, which bind selectively to proteins comprising an amino acid selected from the group consisting of histidine, asparagine, argedine(argenine), cyrtine(cysteine), glutamine, lysine, methionine, proline, and tryptophan.
- a protein binding agent comprising a of transition metal selected from the group consisting of iron, manganese, nickel, cobalt, and zinc, or ions thereof, which bind selectively to proteins comprising an amino acid selected from the group consisting of histidine, asparagine, argedine(argenine), cyrtine(cysteine), glutamine, lysine, methionine, proline, and tryptophan.
- ⁇ 15> The binding of a specific protein and magnetic nanoparticle of the present invention is achieved through reversible coordinate covalent bonding of the functional group such as, imidazole, benzopyrrol, amine, thiol, etc., contained in the amino acid, which can be selected from a group consisting of histidine, asparagine, argentine, cystine, glutamine, lysine, methionine, proline, tryptophan etc., and said transition metal or ion thereof existing on the surface of the magnetic nanoparticle.
- the functional group such as, imidazole, benzopyrrol, amine, thiol, etc.
- the nanopaticles used in the protein binding agents of the present are selected from the group consisting of magnetic nanoparticles comprising, at the surface of said nanoparticle, a transisiton metal or ions thereof from the first row of transition metals, consisting of iron, manganese, chromium, nickel, cobalt, zinc etc., or ions thereof.
- the nanoparticles used in the protein binding agents of the present invention are selected from the group consisting of nanoparticles composed of elements from the first row of transition metals including iron, manganese, chromium, nickel, cobalt, zinc, etc, or transition metal chemical compounds such as oxides, sulfides, phosphides of said transition metals, and also the alloys of said transition metals, or the oxides, sulfides, phosphides of said alloys of said transition metals.
- the magnetic nanoparticles used in the protein binding agents of the present invention have a size range between 1 to 1000 nm.
- the magnetic nanoparticles comprised in the protein binding agents of the present invention have a size range between 1 to 100 nm. More preferably, the magnetic nanoparticles used in the protein binding agents of the present invention have a size range between 2 to 50 nm.
- proteins including an amino acid sequence of histidines in a continuous sequence, asparagine, arginine, cystine, glutamine, lysine, methionine, proline, tryptophan etc. can be selectively separated.
- the protein, which can be separated by binding to the nanoparticles comprising transition metals or ions thereof includes a continuous sequence of 4 to 12 histidines at the end of the amino acid sequence.
- c2i> Another objective of the present invention can be achieved by providing a novel method for the selective binding, separation, or purification of specific proteins.
- another objective of the present invention is to offer a method for the selective binding, separation, or purification of specific proteins using magnetic nanoparticles, which comprises: binding a magnetic nanoparticle which includes a transition metal such as manganese, nickel, cobalt, zinc, etc., or ions thereof to a specific protein contained in a biological mixture; separating said specific protein bound with said nanoparticles from said biological mixture by means of magnetic field; or separating said specific protein from the bound nanoparticle-protein complex.
- the method for binding and separation of the specific protein according to the present invention can be achieved by providing the method which comprises: mixing magnetic nanoparticles with a solution containing specific proteins; collecting nanoparticles that are being bound to said specific proteins by means of magnetic field; and removing materials which are not collected by means of magnetic field.
- the step of the separation and recovery of the specific protein bound selectively to the protein binding agent comprised of magnetic metal ion nanoparticles of the present invention is carried out via separating the specific protein from the protein binding agent of the present invention by mixing the protein binding agent complex and the specific protein in the solution containing materials which form coordinate covalent bonds with metal ions, such as imidazole, pyridine, amine, pyrrole, benzopyrrole, etc., or in an aqueous acidic solution.
- metal ions such as imidazole, pyridine, amine, pyrrole, benzopyrrole, etc.
- the present invention provides a faster, easier, and more economical method for the separation of specific proteins compared to existing methods for metal-ion affinity chromatography.
- the preparation process of the present invention is very simple and economical since the method uses the affinity between ions formed by the oxidation, sulfidation, phosphation, etc., and specific proteins. Also, the preparation process of the present invention can be applied to a commercially large scale protein separation process, since the preparation method of the present invention makes it faster to separate and recover the proteins than the conventional metal ion chromatography which uses the packing material bound to metal ions.
- Fig. 1 is a graphical illustration of the selective binding, separation, or purification process of a specific protein using magnetic nanoparticles.
- Fig. 2 shows a step-by-step synthesis process of nickel coated nickel nanoparticle bound to imidazol.
- Fig. 3 shows a Transmission Electron Microscopy picture of the attained forms of imidazol stabilized nanoparticles dispersed in water(right) and the exterior/interior structure of the nanoparticle (left).
- Fig. 4 is a fluorescence image and fluorescence spectrum of Green Fluorescent Protein tagged with histidine (left) and a normal mouse IgG protein untagged with histidine, and instead tagged with red fluorescence (right). 1 refers to a solution before separation using the nanoparticles.
- Nickel-oleyl amine complex was prepared by reacting nickel acetoacetonate (Ni(acac) 2 (0.2 g)) and oleyl amine (2.0 ml) with heating under an argon atmosphere.
- nickel-oleyl amine complex solution was injected into a mixture solution of trioctylphosphine oxide(T0P0, 5.0 g) and trioctylphosphine (TOP, 0.3 ml) and was heated slowly up to 250 ° C .
- the resulting solution was aged for 30 minutes at 250 0 C, and then cooled slowly to room temperature.
- the nanoparticles were precipitated by adding excess ethanol to the said solution and separated through centrifugation and obtained as a solid state.
- the nanoparticles were re-dispersed with hexane, and after several days under the air for oxidation, the nickel oxide was formed on the outer skin of the nanoparticle.
- the nickel oxide coated nickel nanoparticles (50 ⁇ g) were added to histidine tagged Green Flourescent Protein (GFP, 30 ⁇ g/ml, 250 ⁇ l) and stirred for approximately 30 minutes.
- GFP Green Flourescent Protein
- the separated nanoparticles were then re-dispersed in a imidazole aqueous solution (0.1 g/ml, 250 ⁇ l) and stirred for 30 minutes to separate proteins bound to the surface of the nanoparticles.
- Example 2 The same process used in Example 2 was carried out for the reaction, except that instead of using the histidine tagged green fluorescent protein solution, a mixed solution (250 ⁇ l) of immune globulin (30 ⁇ g/ml) tagged with red fluorescence instead of histidine tag, and green fluorescent protein tagged with histidine (30 ug/ml) was used.
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nanotechnology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Molecular Biology (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Medical Informatics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Inorganic Chemistry (AREA)
- Biomedical Technology (AREA)
- Power Engineering (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Peptides Or Proteins (AREA)
Abstract
La présente invention concerne un procédé de liaison, séparation ou purification sélective de protéines utilisant des nanoparticules magnétiques. Cette invention concerne plus spécifiquement un procédé de séparation de protéines spécifiques plus simple, plus rapide et plus économique comparé au procédé conventionnel de chromatographie d'affinité pour les ions métalliques. Cette invention concerne également un procédé de liaison, séparation ou purification sélective d'une protéine spécifique utilisant une nanoparticule magnétique, lequel procédé consiste : à lier une nanoparticule magnétique, qui comprend un métal de transition tel que le fer, le manganèse, le nickel, le cobalt, le zinc etc. ou des ions de ceux-ci, à une protéine spécifique contenue dans un mélange biologique; à séparer ladite protéine spécifique liée à la nanoparticule du mélange biologique au moyen d'un champ magnétique; puis à séparer cette protéine spécifique du complexe nanoparticule-protéine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2006-0068178 | 2006-07-20 | ||
| KR1020060068178A KR20080008668A (ko) | 2006-07-20 | 2006-07-20 | 자성체 나노입자를 이용하여 단백질을 선택적으로 결합,분리 또는 정제하는 방법 |
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| Publication Number | Publication Date |
|---|---|
| WO2008010687A1 true WO2008010687A1 (fr) | 2008-01-24 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2007/003519 WO2008010687A1 (fr) | 2006-07-20 | 2007-07-20 | Procédé de liaison, séparation ou purification sélective de protéines utilisant des nanoparticules magnétiques |
Country Status (2)
| Country | Link |
|---|---|
| KR (1) | KR20080008668A (fr) |
| WO (1) | WO2008010687A1 (fr) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013032174A3 (fr) * | 2011-08-26 | 2013-04-25 | (주)바이오니아 | Nécessaire de synthèse protéique, et procédé d'expression et d'extraction de protéines à l'aide d'équipement d'extraction automatique |
| WO2015166415A1 (fr) | 2014-04-28 | 2015-11-05 | Universidade De Aveiro | Nanoparticules chélatrices de silice magnétique modifiée, leur utilisation et leur préparation |
| US9616122B2 (en) | 2008-07-03 | 2017-04-11 | Postech Academy-Industry Foundation | PH sensitive metal nanoparticle and preparation method |
| NO20181290A1 (en) * | 2018-10-05 | 2020-04-06 | Combipro As | A method and a system for purifying a fluid |
| CN111474336A (zh) * | 2020-03-21 | 2020-07-31 | 南昌大学 | 一种铁氰化镍纳米粒化学发光适体传感器的制备方法及基于其检测8-OhdG的方法 |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101313180B1 (ko) * | 2008-09-12 | 2013-09-30 | (주)바이오니아 | 핵산 세포전달용 자성 나노입자 및 이의 제조방법 |
| KR101135054B1 (ko) * | 2008-12-17 | 2012-04-13 | 고려대학교 산학협력단 | 단백질 분리용 나노입자, 그 제조 방법 및 이를 이용한 단백질 분리 정제 방법 |
Citations (3)
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|---|---|---|---|---|
| US6162926A (en) * | 1995-07-31 | 2000-12-19 | Sphere Biosystems, Inc. | Multi-substituted fullerenes and methods for their preparation and characterization |
| US6316153B1 (en) * | 1998-04-21 | 2001-11-13 | The University Of Connecticut | Free-form fabricaton using multi-photon excitation |
| US6830694B2 (en) * | 2000-03-20 | 2004-12-14 | Institut Fuer Neue Materialien Gemeinnuetzige Gmbh | Method for separating components from liquid and gaseous media with nanocomposites |
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- 2006-07-20 KR KR1020060068178A patent/KR20080008668A/ko not_active Ceased
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- 2007-07-20 WO PCT/KR2007/003519 patent/WO2008010687A1/fr active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6162926A (en) * | 1995-07-31 | 2000-12-19 | Sphere Biosystems, Inc. | Multi-substituted fullerenes and methods for their preparation and characterization |
| US6316153B1 (en) * | 1998-04-21 | 2001-11-13 | The University Of Connecticut | Free-form fabricaton using multi-photon excitation |
| US6830694B2 (en) * | 2000-03-20 | 2004-12-14 | Institut Fuer Neue Materialien Gemeinnuetzige Gmbh | Method for separating components from liquid and gaseous media with nanocomposites |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9616122B2 (en) | 2008-07-03 | 2017-04-11 | Postech Academy-Industry Foundation | PH sensitive metal nanoparticle and preparation method |
| EP2308799B1 (fr) * | 2008-07-03 | 2017-11-08 | POSTECH Academy-Industry Foundation | Nanoparticule métallique sensible au ph et procédé de préparation correspondant |
| WO2013032174A3 (fr) * | 2011-08-26 | 2013-04-25 | (주)바이오니아 | Nécessaire de synthèse protéique, et procédé d'expression et d'extraction de protéines à l'aide d'équipement d'extraction automatique |
| US9163272B2 (en) | 2011-08-26 | 2015-10-20 | Bioneer Corporation | Protein synthesis kit, and method for expressing and extracting proteins using automatic extraction equipment |
| WO2015166415A1 (fr) | 2014-04-28 | 2015-11-05 | Universidade De Aveiro | Nanoparticules chélatrices de silice magnétique modifiée, leur utilisation et leur préparation |
| NO20181290A1 (en) * | 2018-10-05 | 2020-04-06 | Combipro As | A method and a system for purifying a fluid |
| NO346022B1 (en) * | 2018-10-05 | 2021-12-27 | Combipro As | A method and a system for purifying a fluid |
| CN111474336A (zh) * | 2020-03-21 | 2020-07-31 | 南昌大学 | 一种铁氰化镍纳米粒化学发光适体传感器的制备方法及基于其检测8-OhdG的方法 |
Also Published As
| Publication number | Publication date |
|---|---|
| KR20080008668A (ko) | 2008-01-24 |
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