KR20080008668A - Method for selective binding, separation or purification of proteins using magnetic nanoparticles - Google Patents

Abstract

A method for selective binding, separation or purification of specific proteins using magnetic nanoparticles is provided to bind the nanoparticles selectively to a specific amino acid-tagged protein and separate the protein effectively. A method for selective binding, separation or purification of specific proteins includes the steps of: binding magnetic nanoparticles of 1-1000nm in size, which consists of Group I transition metals (iron, manganese, nickel, cobalt, zinc, etc) or transition metal ions, to specific amino acid-tagged proteins in In-Vivo blends; separating the proteins bound with the nanoparticles from the In-Vivo blends by an externally applied magnetic field; and separating the specific proteins from the separated nanoparticles.

Description

Method for selective binding, separation or purification of proteins using magnetic nanoparticles}

1 is a diagram illustrating a process of selectively binding, separating or purifying a specific protein using magnetic nanoparticles.

2 is a step-by-step depiction of the preparation of nickel nanoparticles having a nickel oxide coating incorporating imidazole.

FIG. 3 is a transmission electron microscopy photograph showing nanoparticles (right) dispersed in water and immobilized with imidazole and the outer skin / internal structure (left) of the nanoparticles.

FIG. 4 is a fluorescence photograph and fluorescence spectra of histidine-containing Green Fluorescent Protein (left) and histidine-free immunoglobulin (normal mouse IgG) protein (right). 1 is a solution before separation using nanoparticles, 2 is a solution after protein separation using nanoparticles, and 3 is a protein solution separated from the nanoparticle surface.

The present invention relates to protein binders consisting of transition metal magnetic nanoparticles, which selectively bind to specific proteins. More specifically, iron, manganese, nickel, cobalt having the property of selectively binding to proteins including amino acids selected from the group consisting of histidine, asparagine, arjidine, sirtin, glutamine, lysine, methionine, proline and tryptophan And it relates to a protein binder consisting of a transition metal selected from the group consisting of zinc ions or magnetic nanoparticles comprising these transition metal ions.

The present invention also relates to a method for selectively binding, separating or purifying a specific protein using a protein binder consisting of magnetic nanoparticles. The present invention is a method for separating specific proteins in an easy and fast and economical way compared to the conventional method using metal-ion affinity chromatography.

More specifically, the present invention comprises the steps of combining magnetic nanoparticles containing transition metals such as iron, manganese, nickel, cobalt, zinc or ions of these transition metals with a specific protein included in the biological mixture; Using a magnetic field to capture specific proteins bound to the magnetic nanoparticles and to separate them from the biological mixture; The present invention relates to a method for selectively binding, separating or purifying a specific protein using magnetic nanoparticles, the method comprising separating a specific protein from a conjugate between the separated nanoparticle and the specific protein.

The present invention is a method for selectively separating or purifying a protein containing a specific amino acid from a biological mixture by using a specific affinity between nickel oxide including nickel, which is a transition metal, and a specific amino acid, and oxidizing having a diameter in the nanometer range. It is for a novel use of nickel coated magnetic nickel nanoparticles as specific protein binders.

To date, histidine containing proteins with 6 to 10 consecutive histidine amino acids in the terminal structure have been primarily isolated and purified using metal-ion affinity chromatography.

In the prior art for separating proteins comprising contiguous histidine sequences purified metal-ion affinity chromatography is used, utilizes a reversible coupling and separation between the Co ^{2 +} or Ni ^{2 +,} such as transition metal ions and histidine amino acid . Filling materials used in columns of metal-ion affinity chromatography are most often made by coordinating transition metal ions after linking chelating ligands to the filling material.

Magnetic nanoparticles having a size of several nanometers are currently widely used in medical-biological applications such as MRI contrast agents, hyperthermia treatments, drugs or gene delivery. Magnetic nanoparticles with small size and large surface area have excellent dispersibility in water or biological solution, can be easily and quickly combined with biomolecules, and can be easily separated from biological mixture by external magnetic field. Have. Due to these properties, magnetic nanoparticles are expected to be useful for the isolation and purification of biomolecules such as proteins and cells.

Recently, Xu Professor's group at Hong Kong University of Science and Technology produced magnetic nanoparticles in the form of a combination of nickel and nitrilotriacetic acid (Ni-NTA), effectively producing only proteins containing consecutive histidine sequences. Separation techniques have been disclosed (C. Xu et. Al. "Nitrilotriacetic Acid-Modified Magnetic Nanoparticles as a General Agent to Bind Histidine-Tagged Proteins." J. Am. Chem. Soc. 2004, 26, 3392).

Nanoparticles for protein separation in the method disclosed by Xu et al. Are prepared through a complex series of sequential organic reaction processes.

It has long been known that histidine proteins bind well to the surface of transition metal oxides, and recently, a technique for selectively arranging histidine proteins to only nickel oxide or cobalt oxide surfaces has been disclosed. (Nam, J.-M. et. Al. "Bioactive Protein Nanoarrays on Nickel Oxide Surfaces Formed by Dip-Pen Nanolithography." Angew. Chem. Int. Ed. 2004, 43, 1246. Zhu, H. et. "Global Analysis of Protein Activities Using Proteome Chips." Science 2001, 293, 2101.)

The conventional method for making a histidine protein separation material has a problem of going through a complex multi-step organic synthesis process because it is prepared by connecting a ligand such as nitrilotriacetic acid to the filler. In addition, the chromatographic method is a continuous process that takes a relatively long time to separate the protein, so it is difficult to use the protein for high throughput screening (HTS), etc., where the next reaction must be carried out within a short time. there was.

In order to solve the problems of the above-mentioned prior arts, the present invention is to prepare a metal ion selectively binding to a specific amino acid nanometer-sized magnetic nanoparticles to selectively bind to a protein containing a specific amino acid, magnetic material using a magnetic field It provides a method for capturing specific proteins bound to nanoparticles and effectively separating them from biological mixtures.

 Filling materials used in conventional metal-ion affinity chromatography are prepared through a series of complex, multi-step organic synthesis processes that synthesize chelate ligands and link to the filler material. However, it is an object of the present invention to provide a method for separating or purifying proteins using a large surface area of magnetic nanoparticles prepared through a very simple and economical process. The method of the present invention is a method for separating specific proteins in an easy and fast and economical way compared to the conventional method using metal-ion affinity chromatography.

The primary object of the present invention described above is to have the property of selectively binding to a protein comprising a specific amino acid selected from the group consisting of histidine, asparagine, arzidine, sirtin, glutamine, lysine, methionine, proline and tryptophan It is achieved by providing a protein binder consisting of magnetic nanoparticles comprising a transition metal selected from the group consisting of iron, manganese, nickel, cobalt and zinc or ions of these transition metals.

The binding between the specific protein and the magnetic nanoparticles in the present invention is imidazole, benzopyrrole, amine, thiol and the like contained in amino acids such as histidine, asparagine, arzidine, sirtin, glutamine, lysine, methionine, proline, and tryptophan. By a reversible coordination bond between the functional group and the transition metal or ions of the transition metal present on the surface of the magnetic nanoparticle.

Preferably, the nanoparticles to be used as the protein binder of the present invention, magnetic nanoparticles containing monocyclic transition metals such as iron, manganese, chromium, nickel, cobalt, zinc or ions of these transition metals on the surface thereof and these A composition comprising nanoparticles.

More preferably, the nanoparticles used as the protein binder of the present invention

Selected from the group consisting of oxides, sulfides and phosphides of monocyclic transition metals such as iron, manganese, chromium, nickel, cobalt and zinc or oxides, sulfides, phosphides and alloys of these transition metals or alloys of these transition metals Transition metal compound nanoparticles or a composition comprising them.

Magnetic nanoparticles used as the protein binder of the present invention have a diameter of 1 to 1000 nm. Preferably, the magnetic nanoparticles used as the protein binder of the present invention have a diameter of 1 to 100 nm. More preferably, the magnetic nanoparticles used as the protein binder of the present invention have a diameter of 2 to 50 nm.

Proteins that are selectively separable using the protein binding agents of the present invention are proteins comprising sequences of amino acids such as consecutive histidine, asparagine, arzidine, sirtin, glutamine, lysine, methionine, proline, tryptophan and the like.

Preferably, the protein binding agent, which is a protein binder of the present invention, can be separated by binding to a magnetic nanoparticle comprising a transition metal or a ion of a transition metal, and has 4 to 12 consecutive histidine sequences at the ends of the amino acid sequence of the protein. It is a protein containing.

Another object of the present invention is achieved by providing a new method for selectively binding, separating or purifying specific proteins using magnetic nanoparticles. More specifically, another object of the present invention is to combine magnetic nanoparticles containing transition metals such as iron, manganese, nickel, cobalt, zinc or ions of these transition metals with a specific protein included in the biological mixture; Using a magnetic field applied externally to separate the specific protein associated with the magnetic nanoparticles from the biological mixture; Or by separating specific proteins bound to the separated nanoparticles from the nanoparticles by providing a method for selectively binding, separating or purifying specific proteins using magnetic nanoparticles.

Binding and separation of specific proteins of the methods of the present invention may comprise mixing magnetic nanoparticles in a solution comprising the specific protein; Capturing nanoparticles that bind to the specific protein using a magnetic field; And removing the material not captured by the magnetic field.

Separation and recovery of the specific protein selectively bound to the binder consisting of magnetic metal ion nanoparticles used in the method of the present invention, the binder between the binder of the present invention and the specific protein is imidazole, pyridine, amine, pyrrole, benzopyrrole, etc. It is made through the process of separating the specific protein and the binder of the present invention by mixing with a solution or acidic aqueous solution containing a material that coordinates the metal ion as shown.

Hereinafter, the components and technical features of the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention in detail, and the scope of the present invention is not limited to the contents of the following examples.

EXAMPLE 1

Synthesis of Nickel Nanoparticles Coated with Nickel Oxide

Nickel acetoacetonate (Ni (acac) ₂ (0.2 g)) and oleylamine (oleylamine, 2.0 ml) were heated in an argon atmosphere to prepare a nickel-oleylamine complex. The nickel-oleylamine compound thus prepared was added to a mixed solution of trioctylphosphine oxide (TOPO, 5.0 g) and trioctylphosphine (TOP, 0.3 ml), and then slowly heated up to 250 ° C. The mixture solution was reacted for 30 minutes while maintaining the temperature of 250 ° C, and then slowly cooled to room temperature. Excess ethanol was added to the solution, and the precipitated nanoparticles were centrifuged to obtain a solid form, which was then dispersed in hexane and oxidized in air for several days to form nickel oxide on the skin of the nanoparticles.

Excess acetone was added to the solution in which nickel nanoparticles coated with nickel oxide were dispersed and centrifuged to obtain nanoparticles having a nickel nucleus and a nickel oxide shell structure as a black solid (FIG. 2).

To hydrophilically modify the surface of the prepared nanoparticles, the nanoparticles were dispersed again in chloroform containing imidazole (0,5 g / ml, 5 ml) and stirred for 6 hours.

After the reaction solution was cooled to room temperature, excess hexane was added and centrifuged to prepare nickel nanoparticles coated with a nickel oxide layer stabilized with imidazole and having an average diameter of 13 nm.

EXAMPLE 2

Coupling Nickel Nanoparticles Coated with Nickel Oxide and Proteins Containing Histidine

Nickel nanoparticles coated with nickel oxide (50 μg) were added to histidine-labeled green fluorescent protein (Green Fluorescent Protein, GFP, 30 μg / ml, 250 μl) and then stirred for about 30 min.

After the protein-bound nanoparticles were separated from the solution using a magnet, the separated nanoparticles were again dispersed in an aqueous imidazole solution (0.1 g / ml, 250 μl) and stirred for 30 minutes to remove the proteins attached to the nanoparticle surface. Separated.

Again, the nanoparticles were separated and removed using a magnet, leaving histidine-labeled Green Fluorescent Protein (GFP, 30 μg / ml, 250 μl) separated from the nanoparticles.

By measuring the fluorescence spectrum of the GFP protein at each step of the process, about 90% of the histidine labeled GFP protein present in the initial solution binds to the nanoparticles, and about 70% of the histidine labeled GFP protein is an aqueous solution of imidazole. It can be seen that it is recovered into the stomach (FIG. 4).

[Comparative Example 1]

Reaction of Nickel Nanoparticles Coated with Nickel Oxide with Histidine Unlabeled Protein

Example 2 except for using immunoglobulins (normal mouse IgG, 30 μg / ml, 250 μl) labeled with histidine unlabeled and red fluorescent instead of using histidine-labeled green fluorescent protein The reaction was carried out in the same manner.

By measuring the red fluorescence spectrum at each step of the process, it can be seen that only about 10% of the normal mouse IgG protein present in the initial solution binds to the nanoparticles. (Figure 4)

EXAMPLE 3

Isolation of Histidine Labeled Protein Using Nickel Oxide-coated Nickel Nanoparticles

A mixture of histidine-labeled immunoglobulin (30 μg / ml) and histidine-labeled green fluorescent protein (30 μg / ml) instead of using a solution of histidine-labeled green fluorescent protein The reaction was carried out in the same manner as in Example 2 except that a solution (250 μl) was used.

By measuring the green and red fluorescence spectra at each step of the process, about 90% of the histidine labeled GFP protein present in the initial solution binds to the nanoparticles, and about 50% of the histidine labeled GFP protein is an aqueous solution of imidazole. While recovered into the genus, only 18% of the immunoglobulin (normal mouse IgG) protein present in the initial solution was found to bind to the nanoparticles.

The present invention takes advantage of the selective binding between transition metals or ions of the transition metal or specific amino acids present on the surface of the magnetic nanoparticles and the nature of the binding of the conjugates in the magnetic field. In comparison, specific proteins can be isolated in an easier, faster and more economical way.

In conventional metal ion affinity chromatography, metal ions connected to the filler material act, but the protein binder of the present invention shows that ions formed by oxidation, sulfidation, ignition reaction, etc. on the surface of magnetic nanoparticles show affinity with specific amino acids. In addition to the technical effect that the manufacturing process is very simple and economical, as well as the separation and recovery of the protein is faster than using a metal ion-filled filler, the commercial process must be continued quickly to the next process. It can be applied to large-scale protein separation purification process.

Claims (18)

Iron, manganese, nickel, cobalt and zinc ions that have the ability to selectively bind proteins containing amino acids selected from the group consisting of histidine, asparagine, arzidine, sirtin, glutamine, lysine, methionine, proline and tryptophan A protein binder, comprising a magnetic nanoparticle comprising a transition metal or a transition metal ion selected from the group consisting of: The protein binder comprising the transition metal of claim 1 or magnetic nanoparticles containing ions of the transition metal, wherein the transition metal is a one-cycle transition metal. The protein binder according to claim 2, wherein the one-cycle transition metal is selected from the group consisting of iron, manganese, chromium, nickel, cobalt, zinc and the like. A protein binder comprising magnetic nanoparticles comprising the transition metal or the ion of transition metal according to claim 1, wherein the magnetic nanoparticles are selected from the group consisting of oxides, sulfides, and phosphides of monocyclic transition metals or monocyclic transition metals. Protein binder, characterized in that the. The protein binder comprising magnetic nanoparticles comprising the transition metal or the ion of the transition metal of claim 1, wherein the magnetic nanoparticles are selected from the group consisting of an alloy of one cycle transition metals or oxides, sulfides and phosphides of one cycle transition metal alloy. Protein binder, characterized in that the compound selected. The protein binder consisting of magnetic nanoparticles comprising the transition metal or the ion of the transition metal of claim 1, wherein the nanoparticles have a diameter of 1 to 1000 nm. The protein binder comprising magnetic nanoparticles comprising the transition metal or the ion of the transition metal of claim 1, wherein the nanoparticles have a diameter of 1 to 100 nm. The protein binder consisting of magnetic nanoparticles comprising the transition metal of claim 1 or the ion of the transition metal, characterized in that the diameter of the nanoparticles is 2 to 50nm. Combining magnetic nanoparticles containing transition metals such as iron, manganese, nickel, cobalt, zinc, or ions of the transition metals with a specific protein included in the biological mixture; Using an externally applied magnetic field to separate the specific protein associated with the magnetic nanoparticles from the biological mixture; Or separating the specific proteins bound to the separated nanoparticles from the nanoparticles, selectively binding, separating or purifying the specific proteins using the magnetic nanoparticles. The method of claim 9, wherein the magnetic nanoparticles comprise one cycle transition metal or ions of one cycle transition metal, and selectively bind, separate, or purify a specific protein using magnetic nanoparticles. The method of claim 10, wherein the one-phase transition metal is selected from the group consisting of iron, manganese, chromium, nickel, cobalt, zinc, etc., selectively binding, separating or separating specific proteins using magnetic nanoparticles How to purify. 10. The method according to claim 9, wherein the magnetic nanoparticles are compounds selected from the group consisting of oxides, sulfides, and phosphides of monocyclic transition metals or monocyclic transition metals. Methods of binding, separation or purification. 10. The method according to claim 9, wherein the magnetic nanoparticles are compounds selected from the group consisting of alloys of one-cycle transition metals or oxides, sulfides, and phosphides of one-cycle transition metal alloys. Selectively binding, separating or purifying. 10. The method of claim 9, wherein the nanoparticles have a diameter of 1 to 1000 nm, selectively binding, separating or purifying specific proteins using magnetic nanoparticles. 10. The method of claim 9, wherein the nanoparticles have a diameter of 1 to 100 nm, selectively binding, separating or purifying specific proteins using magnetic nanoparticles. The method of claim 9, wherein the nanoparticles have a diameter of 2 to 50 nm, and selectively bind, separate, or purify specific proteins using magnetic nanoparticles. 10. The magnetic nanoparticle of claim 9, wherein the protein is a protein including those selected from the group consisting of amino acids consisting of histidine, asparagine, arzidine, sirtin, glutamine, lysine, methionine, proline and tryptophan. Selectively binding, isolating or purifying a particular protein. 10. The method of claim 9, wherein the specific protein is a protein containing histidine, selectively binding, separating or purifying the specific protein using magnetic nanoparticles.

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