[DESCRIPTION] [Invention Tit Ie]

METHOD FOR SELECTIVE BINDING, SEPARATION OR PURIFICATION OF PROTEINS USING MAGNETIC NANOPARTICLES [Technical Field]

<i> 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.

<2> Furthermore, 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.

<3> More specifically, 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.

<4> 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.

[Background Art] <5> Until now, metal-ion affinity chromatography has been mainly used for separation and purification of proteins containing six to ten continuous histidine amino acids at their terminal end. <6> The conventional technique for the separation and purification of proteins containing a continuous histidine uses metal-ion affinity

2+ 2+ chromatography to reversibly bind Co , Ni or a similar transition metal ion with the histidine amino acid. 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.

<7> 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.

<8> Recently, professor Xu of Hong Kong University of Science and Technology disclosed a technique for the effective separation of proteins containing a continuous histidine sequence by producing and using magnetic nanoparticles bound to nickel and nitri lotriacetic acid (Ni-TNA) (C. Xu et . al . "Nitri lotriacetic Acid-Modified Magnetic nanoparticles as a General Agent to Bind Histidine-Tagged Proteins." J. Am. Chem. Soc. 2004, 26, 3392.)

<9> However, the nanoparticles for separating proteins disclosed by Xu et al . are produced through a series of complicated sequential organic reaction steps. <io> The concept that histidine proteins bind well to transition metal oxide surfaces is well known in the art. Recently, using these characteristics, a process where histidine proteins were selectively arranged on the surfaces of nickel oxide and cobalt oxide has also 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 . al. "Global Analysis of Protein Activites Using Proteome Chips." Science 2001, 293, 2101.)

<ii> The conventional method for making the materials used for separation of histidine proteins requires that the column packing materials be bound to ligands such as nitrilotriacet ic acid, which is a problem due to the complicated organic synthesis process that is required. Also, for methods using chromatography, separation of proteins require a relatively long continuous process, which presents a problem due to the difficulty of conducting the next reaction of the protein within a short time after HTS (High Throughput Screening) . [Disclosure] [Technical Problem]

<12> To find a solution for the aforementioned problems to the conventional method, 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.

<i3> Column packing materials used in conventional metal ion affinity chromatography are prepared via a series of complicated organic synthesis processes which include the synthesis of chelate ligands and the bonding of the chelate ligands to packing materials. However, the objective of the present invention is to provide a method for the separation or purification of proteins through a remarkably simple process using the wide surface area of magnetic nanoparticles. Compared to conventional metal ion affinity chromatography method, the method of the present invention allows easier, faster, and more economical separation of proteins. [Technical Solution] ci4> As aforementioned, 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.

<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.

<16> Preferably, 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.

<\i> More preferably, 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.

<n> The magnetic nanoparticles used in the protein binding agents of the present invention have a size range between 1 to 1000 nm. Particularly, 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.

=i9> By using the protein binding agents of the present invention, 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. c20> Preferably 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. More specifically, 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.

<2i> 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.

;23> 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. [Advantageous Effects]

<24> By utilizing selective binding between a specific amino acid and a nanoparticle with a transition metal, or ions thereof , on its surface, and the distinctive characteristic of these complexes which allow them to be collected by magnetic field, 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.

<25> While, the metal ions bound to packing materials are workable in the conventional 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. [Description of Drawings]

<26> Fig. 1 is a graphical illustration of the selective binding, separation, or purification process of a specific protein using magnetic nanoparticles.

<27> Fig. 2 shows a step-by-step synthesis process of nickel coated nickel nanoparticle bound to imidazol.

<28> 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). <29> 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.

2 refers a solution after separation of the protein using the nanoparticles.

3 refers to a solution of the protein separated from the surface of the nanoparticle.

[Best Mode]

<30> Hereinafter, the present invention will be described in greater detail with reference to the following examples. The examples are given only for illustration of the present invention and not to be limiting the present invention.

<31>

<32> [Example 1] Synthesis of nickel oxide coated nickel nanoparticle <33> 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. Thus prepared 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 ⁰C, 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.

<34> After adding an excess of acetone to the solution of dispersed nickel oxide coated nickel nanoparticles, the said nanoparticles were obtained as a black solid form with a nickel core and a nickel oxide shell structure. (Fig. 2)

<35> In order to modify the surface of the prepared nanoparticles to a hydrophilic nature, said nanoparticles were then re-dispersed in chloroform including imadazole (0.5 g/ml , 5 ml) and then stirred for 6 more hours. <36> After allowing the reaction solution to cool to room temperature, an excess of hexane was added, and said solution was separated through centrifugat ion, and then the surface was stabilized with imidazole, whereupon the nickel oxide coated nickel nanoparticles were produced with an average diameter of 13 run. (Fig.3)

<37>

<38> [Example 2] Binding of nickel nanoparticles coated with nickel oxide and proteins containing histidine

<39> The nickel oxide coated nickel nanoparticles (50 μg) were added to histidine tagged Green Flourescent Protein (GFP, 30 μg/ml, 250 μl) and stirred for approximately 30 minutes.

<40> After using magnets to separate the nanoparticles bound to the proteins in the solution, the separated nanoparticles were then re-dispersed in a imidazole aqueous solution (0.1 g/ml, 250 μl) and stirred for 30 minutes to separate proteins bound to the surface of the nanoparticles.

<4i> Again, by using magnetic force to separate/remove the nanoparticles, the histidine tagged Green Fluorescent Proteins (GFP, 30 μg/ml, 250 μl) remained in the solution.

<42> By measuring the fluorescence spectrum of the GFP protein in each step of said process, it was found that approximately 90% of the existing histidine tagged GFP proteins in the initial solution were bound to the nanoparticles, and approximately 70% of histidine tagged GFP proteins were collected inside the imidazol solution. (Fig.4)

<43>

<44> [Comparative Example 1] Reaction of nickel nanoparticles coated with nickel oxide and histidine proteins <45> The same process carried out in Example 2 was performed, except that instead of using histidine tagged green fluorescent protein, immune globulin

(normal mouse IgG, 30 μg/ml, 250 μl) was used without histidine tagging, and was tagged instead with red fluorescence. <46> By measuring the fluorescence spectrum in each step of said process, it was found that only approximately 10% of the existing normal mouse IgG proteins in the initial solution were bound to the nanoparticles.

<47>

<48> [Example 3] Separation of histidine tagged protein using nickel oxide coated nickel nanoparticles

<49> 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 μl) of immune globulin (30 μg/ml) tagged with red fluorescence instead of histidine tag, and green fluorescent protein tagged with histidine (30 ug/ml) was used.

<50> By measuring the fluorescence spectrum in each step of said process, it was found that approximately 90% of the existing histidine tagged GFP proteins in the initial solution were bound to the nanoparticles, and although approximately 50% of histidine tagged GFP proteins were collected inside the imidazol solution, only 18% of the normal mouse IgG proteins were bound to the nanoparticles.