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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 PDF

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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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WO
WIPO (PCT)
Prior art keywords
row
protein
nanoparticle
magnetic
transition metal
Prior art date
Application number
PCT/KR2007/003519
Other languages
English (en)
Inventor
Taeg Hwan Hyeon
In Su Lee
Original Assignee
Seoul National University Industry Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Seoul National University Industry Foundation filed Critical Seoul National University Industry Foundation
Publication of WO2008010687A1 publication Critical patent/WO2008010687A1/fr

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Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/16Extraction; Separation; Purification by chromatography
    • C07K1/22Affinity chromatography or related techniques based upon selective absorption processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82BNANOSTRUCTURES FORMED BY MANIPULATION OF INDIVIDUAL ATOMS, MOLECULES, OR LIMITED COLLECTIONS OF ATOMS OR MOLECULES AS DISCRETE UNITS; MANUFACTURE OR TREATMENT THEREOF
    • B82B1/00Nanostructures formed by manipulation of individual atoms or molecules, or limited collections of atoms or molecules as discrete units
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y25/00Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • HELECTRICITY
    • H01ELECTRIC ELEMENTS
    • H01FMAGNETS; INDUCTANCES; TRANSFORMERS; SELECTION OF MATERIALS FOR THEIR MAGNETIC PROPERTIES
    • H01F1/00Magnets or magnetic bodies characterised by the magnetic materials therefor; Selection of materials for their magnetic properties
    • H01F1/0036Magnets 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
    • H01F1/0045Zero dimensional, e.g. nanoparticles, soft nanoparticles for medical/biological use
    • H01F1/0054Coated 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.
PCT/KR2007/003519 2006-07-20 2007-07-20 Procédé de liaison, séparation ou purification sélective de protéines utilisant des nanoparticules magnétiques WO2008010687A1 (fr)

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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Cited By (5)

* Cited by examiner, † Cited by third party
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)

* Cited by examiner, † Cited by third party
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

Patent Citations (3)

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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)

* Cited by examiner, † Cited by third party
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

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Publication number Publication date
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