WO2007132998A1

WO2007132998A1 - Linker molecules for substrate surface treatment and specific protein immobilization, and method for preparing the same

Info

Publication number: WO2007132998A1
Application number: PCT/KR2007/002250
Authority: WO
Inventors: Bong Hyun Chung; Tai Hwan Ha; Sun Ok Jung; Sang Jeon Chung; Suh Hyun Lee; Jung-Won Kim
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2006-05-12
Filing date: 2007-05-08
Publication date: 2007-11-22
Also published as: KR100889587B1; KR20070109874A

Abstract

The present invention relates to a linker molecule for immobilizing a protein on a substrate surface and a preparation method thereof. More specifically, relates to a linker molecule, which has a functional group binding to a substrate, and a functional group binding to a protein, at both ends of a protein-immobilizing ligand, respectively, as well as a preparation method thereof and a method for immobilizing a protein through the linker molecule. The disclosed linker molecule can form a self-assembled monolayer on a substrate selected from the group consisting of gold, silver, semiconductor, glass, silicon and polymer, so as to minimize non-specific binding in a process of immobilizing a protein on the substrate. Accordingly, the linker molecule will be useful for biochips, biosensors and other supports for protein immobilization.

Description

Linker Molecules for Substrate Surface Treatment and Specific Protein Immobilization, and Method for Preparing the Same

TECHNICAL FIELD

The present invention relates to a linker molecule for immobilizing a protein on a substrate and a preparation method thereof, and more particularly to a linker molecule which has a functional group binding to a substrate, and a substance binding to a protein at both ends of a protein-immobilizing ligand, a preparation method thereof and a method for immobilizing a protein through the linker molecule.

BACKGROUND ART

One of key technologies of fabricating biochips or biosensors is protein immobilization technology. Existing methods of immobilizing a protein on a solid substrate surface employ either physical adsorption of protein on a membrane without controlling orientation or formation of a covalent bond by a non-selective chemical reaction. Recently, a method of controlling the orientation of antibodies using calixcrown derivatives was reported (Han, M. et al., Proteomics., 3:2289, 2003). In addition, a method of immobilizing a protein using a biotin- streptavidin/avidin complex by linking biotin to a protein and then applying the protein on a solid surface treated with streptavidin or avidin was reported, and a method of reducing non-specific binding using a polymer also appeared. Moreover, when a gold thin film is used, an expensive dextran polymer surface is used, which is typically used in measurement methods which use the surface plasmon resonance phenomenon. However, these methods require much cost and time for surface treatment, and do not immobilize only a specific protein, but rather non-specifically immobilize all proteins, and the orientation of the immobilized proteins is unfavorable. In addition, highly purified proteins should be used in order to prevent non-specific binding.

In order to minimize non-specific binding of proteins which are immobilized on substrates in biosensors, biochips and immobilization supports, modification of a surface with oligo- or polyethylene glycol has been steadily performed since the 1990s (Witesides, G. et al. Chem. Rev., 105:1103, 2005). Since this oligo- or polyethylene glycol is a substance which greatly contributes to blocking the nonspecific binding of proteins, methods of immobilizing proteins or cells using this substance have been frequently used, and studies on the synthesis of linkers using this substance and application thereof have been actively conducted.

Biacore, Inc. developed and sells chips having a surface treated with dextran and chips having a surface treated with NTA bound to dextran. NTA immobilized on a substrate strongly binds to histidine through metal ions, and thus makes it possible to immobilize biomaterials having histidine. However, the biochips must employ the SPR sensor of Biacore, Inc., and the substrate thereof is limited to a gold thin film, thus reducing the utilization of the biochips.

Prior methods for protein immobilization include: a method of immobilizing a protein by integrating an active group (a chemical functional group for immobilizing a protein by chemical binding) onto a substrate using plasma (Korean

Patent No. 448880); a method comprising forming a porous sol-gel thin film having sufficiently increased non-surface area, on a solid substrate surface, using a sol-gel process, and then immobilizing a protein on the porous thin film by physical adsorption (Korean Patent No. 577694); a method of immobilizing antithrombotic protein on a polytetrafluoroethylene (PTFE) surface by a plasma reaction (Korean Patent No. 491700), and a method of immobilizing a protein by providing a protein- immobilizing enzyme, which binds 2 enzymes to which more than 2 cationic amino acid residues are continuously fused (Korean Patent Laid-Open Publication No. 10- 2003-0034136).

However, such prior methods have shortcomings, such as protein denaturation caused by chemical processes, a reduction in protein immobilization ability due to protein immobilization methods which rely only upon physical adsorption, an increase in the cost and time for surface modification, and difficulties in mass production.

In addition, the following methods are known: a method of immobilizing proteins to a hydrophobic polymeric layer attached to a solid support, using a substrate (WO 2003/072752); a method of immobilizing proteins on plastic surfaces using a buffer substance (EP 0916949); and a method of immobilizing a protein by bringing the protein into contact with a hydrophobic solid surface (WO 2005/070968). However, a method of specifically immobilizing a recombinant protein on a substrate surface using a linker molecule is not yet known in the art to which the present invention pertains.

Thus, there is an urgent need to develop a new method, which solves the above- described problems and can immobilize not only highly purified protein, but also recombinant proteins having affinity tags from cell culture broth (complex protein mixture), on a substrate surface.

Accordingly, the present inventors have made many efforts to develop a protein immobilization method which imparts a definite orientation and binding specificity to a target protein. As a result, the present inventors have found that a protein chip having high orientation and specificity can be fabricated using a linker molecule, which has a material forming a self-assembled monolayer on a substrate linked to a material forming an affinity bond with a protein, thereby completing the present invention.

SUMMARY OF THE INVENTION

Therefore, it is a main object of the present invention to provide a linker molecule for immobilizing a protein on a substrate surface, which can minimize the problems occurring in the prior processes of immobilizing proteins on substrate surfaces, for example, lack of orientation, non-specificity, and high cost required for purifying a protein to be immobilized, as well as a preparation method thereof.

Another object of the present invention is to provide a protein chip comprising said linker molecule, and a preparation method thereof.

Still another object of the present invention is to provide a protein immobilized using said linker molecule, and a preparation method thereof.

To achieve the above object, the present invention provides a method for preparing an A-B-C-type linker molecule for immobilizing a protein on a substrate, the method comprising the steps of: (a) binding a material (A) which serves to form a self-assembled monolayer on a substrate surface, to one end of a protein- immobilizing ligand (B) which serves to inhibit non-specific binding of a protein; and (b) binding an affinity material (C) which binds to the protein, to the other end of the protein-immobilizing ligand.

The present invention also provides an A-B-C-type linker molecule for immobilizing a protein on a substrate, in which a material (A) forming a self- assembled monolayer on a substrate surface and an affinity material (C) binding to a protein, are bound to both ends of a protein-immobilizing ligand (B) inhibiting the non-specific binding of the protein, respectively. The present invention also provides a method for preparing a protein chip, the method comprising the steps of: (a) forming a self-assembled monolayer of the linker molecule on a substrate through a material (A) for forming the self-assembled monolayer on the substrate surface by introducing the A-B-C-type linker molecule into a substrate, and exposing an affinity material (C) which binds to a protein; and (b) binding a protein to the exposed affinity material (C).

The present invention also provides a protein chip, in which a protein is bound to a substrate through the A-B-C-type linker molecule.

The present invention also provides a method for immobilizing a protein. The method comprises immobilizing the protein on a carrier for protein immobilization through said A-B-C-type linker molecule and an immobilized protein, in which a protein is bound to a support for protein immobilization through said A-B-C-type linker.

The above and other objects, features and embodiments of the present invention will be more clearly understood from the following detailed description and accompanying claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the principle by which a linker is arranged on substrate using the structure of a linker 1 forming a self-assembled monolayer on the surface of a gold thin film. In FIG. 1, A: a functional moiety for attachment to the substrate surface; B: a moiety for inhibiting non-specific binding; and C: a functional moiety for biomolecule immobilization. FIG. 2 shows the structure of a linker molecule into which glutathione was introduced, which serves to form a self-assembled monolayer on a glass surface.

FIG. 3 shows a method for preparing a linker molecule 1 into which glutathione was introduced, which serves to form a self-assembled monolayer.

FIG. 4 shows a method for preparing a linker molecule 2 into which glutathione was introduced, which serves to form a self-assembled monolayer.

FIG. 5 shows a method for preparing a linker molecule 3 into which nitrilotriacetic acid was introduced, which serves to form a self-assembled monolayer.

FIG. 6 shows a method for preparing a linker molecule 4 into which imidodiacetic acid was introduced, which serves to form a self-assembled monolayer.

FIG. 7 shows a method for preparing a linker molecule 5 for the surface treatment of /3-cyclodextrin chips which serves to form a self-assembled monolayer.

FIG. 8 shows the orientation of antigen-antibody binding, measured after a glutathione S-transferase-tagged staphylococcal protein G is immobilized on a self- assembled monolayer bound to a gold thin film. In FIG. 8, EGFP: green fluorescence protein (antigen); GST: glutathione S-transferase; LA-GSH Ligand: a lipoic acid-glutathione self-assembled monolayer; and Carboxylated Dextran: CM-5 (manufactured by Biacore, Inc.); and (a) shows a structure comprising a self- assembled monolayer of the inventive linker, and (b) shows a structure comprising a CM-5 chip (manufactured by Biacore, Inc.).

FIG. 9 is a graphic diagram showing the immobilization of glutathione S- transferase-tagged staphylococcal protein G on a gold thin film surface having the inventive self-assembled monolayer bound thereto, and the immobilization of staphylococcal protein G on the surface of CM-5.

FIG. 10 shows results obtained by immobilizing staphylococcal protein G on a gold thin film surface having a self-assembled monolayer bound thereto, adding an immunoglobulin protein to the immobilized protein, and then measuring antigen- antibody binding in real time using the surface plasmon resonance phenomenon.

FIG. 11 shows the results of a fluorescence experiment conducted using a biochip comprising the inventive linker molecule.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

In one aspect, the present invention relates to a method for preparing an A-B-C-type linker molecule for immobilizing a protein on a substrate, the method comprising the steps of: (a) binding a material (A) which serves to form a self-assembled monolayer on a substrate surface, to one end of a protein-immobilizing ligand (B) which serves to inhibit non-specific binding of a protein; and (b) binding an affinity material (C) which binds to the protein, to the other end of the protein-immobilizing ligand. Also, the present invention relates to an A-B-C-type linker molecule for immobilizing a protein on a substrate, which is prepared by said method and in which a material (A) which serves to form a self-assembled monolayer on a substrate surface and an affinity material (C) binding to a protein, are bound to both ends of a protein-irnmobilizing ligand (B) which serves to inhibit the non-specific binding of the protein, respectively.

In the present invention, the protein-immobilizing ligand is preferably oligoethylene glycol or polyethylene glycol, for both ends of which amine is substituted. Also, the protein is preferably a protein comprising a protein affinity tag. The protein affinity tag is preferably proteins, such as glutathione-S-transferase and maltose- binding proteins, histidine and the like.

Also, the linker molecule of the present invention preferably has a structure of Formula I or Formula II:

[Formula I]

[Formula II]

wherein, n is 2~200; X and Y are substances binding to a protein or a compound containing amine or thiol(SH), and which has a functional group (e.g., maleimide, maleimidopropionic acid or N-hydroxysuccinimido ester) of any one among the following Formula III: [Formula III]

In the present invention, said Y is selected from the group consisting of L-reduction in glutathione, iminodiacetic acid, nitrilotriacetic acid, maltose derivatives, β- cyclodextrin, galactose, calmodulin, biotin, chitin, cellulose, C-myc, thioredoxine, intain, S-peptide and DNA.

Also, the substrate binding to the linker molecule of Formula I is preferably gold or silver, and the substrate binding to the linker molecule of Formula II is preferably silicon, glass or ceramic.

The linker molecule of Formula I is preferably LA-GSH (lipoic acid-glutathione; linker molecule 1 or 2), LA-NTA (lipoic acid-nitrilotriacetic acid; linker molecule 3), LA-IDA (lipoic acid-iminodiacetic acid; linker molecule 4) or LA-/3- cyclodextrin (linker molecule 5).

The substrate of a biochip or a biosensor, which is formed using the inventive linker molecule forming a self-assembled monolayer, can be used in various applications, including enabling proper orientation of proteins to be immobilized and increasing measurement sensitivity.

The linker molecule according to the present invention can minimize non-specific binding in a protein immobilization process by forming a self-assembled monolayer on a substrate selected from the group consisting of gold, silver, semiconductor, ceramic, glass, silicon and polymer, and thus is useful for biochips, biosensors and other supports for protein immobilization.

In another aspect, the present invention relates to a method for preparing a protein chip, the method comprising the steps of: (a) forming a self-assembled monolayer of a linker molecule on a substrate through a material (A) forming a self-assembled monolayer on a substrate surface by introducing the A-B-C-type linker molecule into the substrate, and exposing an affinity material (C) which binds to a protein; and (b) binding a protein to the exposed affinity material (C). Also, the present invention relates to a protein chip, which is prepared by said method, and in which a protein is bound to a substrate through the A-B-C-type linker molecule.

As used herein, the term "linker molecule" refers to a compound forming a self- assembled monolayer, which is used in a method of immobilizing a recombinant protein having a protein affinity tag on a substrate (FIG. 1). In FIG. 1, moiety (A) is a material serving to bind a self-assembled monolayer to a substrate. Specifically, a linker molecule 1 shown in FIG. 3 contains a disulfide group which is stable and can form a monomolecular layer by being adsorbed on a gold or silver thin film surface. Moiety (B) in FIG. 1 is an efficient spacer group, which serves to maintain the distance between the protein and the solid surface and to inhibit non-specific binding. For this purpose, oligoethylene glycol or polyethylene glycol may be used. Finally, moiety (C) in FIG. 1 comprises a material for immobilizing a recombinant protein having an affinity tag.

Meanwhile, when a multicellular organism-derived protein is produced in the form of a recombinant protein in E. coli, there is a problem in that the target protein is generally produced in the form of an inclusion body which is inactive in cells. To solve this problem, technologies, which stably express a target protein in the form fused with an affinity tag having high water solubility and easily isolate and purify, have been developed (Nilsson, B. et ai, Curr. Opinion. Struct. Biol, 2:569, 1992; Nygren, P. et ah, Trends BiotechnoL, 12:184). A recombinant fusion protein tagged with a protein having an affinity for the moiety C of the linkers used in the present invention can be easily prepared through such methods.

In the present invention, for the synthesis of an affinity ligand for effectively binding an affinity- tagged recombinant protein to a substrate, oligoethylene glycol or polyethylene glycol, for both ends of which amine is substituted, is used as a starting material (protein-immobilizing ligand) for the synthesis of the moiety (B) of FIG. 1.

The moiety (A) of FIG. 1 is a material binding to a substrate, lipoic acid can be used for a gold or silver thin film, a linker having trimethoxysilane at one end and CONH, NCO or C(O)CH₂ at the other end, as shown in FIG. 2, can be used for a ceramic, glass or silicon surface instead of said lipoic acid.

Glutathione forming an affinity bond with a protein tagged with glutathione S- transferase is bound to a material which forms an affinity bond with the protein, the moiety (C) of FIG. 1. Also, to immobilize a maltose binding protein (MBP)- tagged protein, 0-cyclodextrin used, and nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA), which form a complex with metal ions, is bound to a histidine-tagged protein which forms a coordinate bond with nickel ion.

For example, for affinity binding to a glutathione S-transferase (GST)-tagged protein, the moiety (C) is modified with glutathione. For this purpose, as shown in FIG. 3, lipoic acid and maleimidopropionic acid are prepared in the form of N- hydroxysuccinimide ester intermediates 8 and 18, which react specifically with amine, respectively. α,ω-Diamino oligoethylene glycol 11 is allowed to react with compound 8 to synthesize an intermediate 9. Then, the intermediate 9 is allowed to react with the compound 18 to prepare a compound 10. In the reaction between the compound 8 and the a,ω- diamino oligoethylene glycol 11, the α,ω-diamino oligoethylene glycol must be used in an amount of at least 10 times the compound 8 to sufficiently produce a primary intermediate product 9 in which amine is bound to lipoic acid only at one end of the intermediate 9.

The reaction between the primary intermediate product 9, containing amine bound to lipoic acid at one end thereof, and the maleimidopropionic acid N- hydroxysuccinimide ester 18, occurs at a ratio of 1:1, to produce the secondary intermediate product 10. The compound 10 thus prepared is allowed to react with glutathione 19 in a methanol solution, thus preparing a linker molecule 1 as a desired material.

Glutathione can be introduced into a linker molecule 2 shown in FIG. 4 in the same manner used for the linker molecule 1 of FIG. 3. For the synthesis of the linker molecule 2, a compound 20 is first allowed to react with an oligoethylene glycol (or polyethylene glycol) 11 to synthesize a diamine intermediate 12 containing a t- butoxycarbonyl (Boc) protecting group at one end thereof. Then, the amine group at the other end of the intermediate 12 is allowed to react with a maleic anhydride 21 to prepare an intermediate 14. Then, Boc group is removed using trifluoroacetic acid, thus preparing an intermediate 15 containing the regenerated amine group. The intermediate 15 is allowed to react with the intermediate 8 to prepare a linker molecule 6. The linker molecule 6 is treated with glutathione to prepare another linker molecule 2.

A linker molecule 3 in FIG. 5 is used to immobilize a histidine-tagged protein and is synthesized in the following manner. An intermediate 16 formed from the reaction between the intermediate 8 and the intermediate 12, shown in FIG. 3 and FIG. 4, is treated with trifluoroacetic acid to remove the Boc protecting group, thus synthesizing an intermediate 9 containing the regenerated amine group at the end thereof. The intermediate 9 is allowed to react with succinic anhydride 22 to synthesize an intermediate 17 having a carboxyl group at the end thereof, and then an N-hydroxysuccmimide ester intermediate 7 is formed. Then, the intermediate 7 is treated with nitrilotriacetic acid (NTA, compound 23), thus synthesizing the desired linker molecule 3.

A linker molecule 4 in FIG. 6 can be used to immobilize a histidine-tagged protein, like the linker molecule 3, and is prepared by allowing the intermediate 7 in FIG. 4 to react with an IDA 24 instead of the NTA 23.

The linker molecule 5 shown in FIG. 7 is used to immobilize a maltose-binding protein (MBP)-tagged protein and is prepared by allowing the intermediate 9 in FIG. 3 to react with monotosyl β-cyclodextrin.

The number of ethylene glycol in the diamine-substituted oligoethylene glycol, a starting material, may be in the range of two to the desired number (a few tens to a few hundreds).

Using the linker molecules obtained through said reactions, surfaces for the immobilization of recombinant proteins tagged with a protein affinity material can be formed within 4 hours. As used herein, the term "affinity tag" refers to a tag specifically binding to a specific material, and examples thereof may include glutathione-S-transferase tag, maltose-binding protein tag and histidine tag. The linker molecules thus prepared will be useful for the immobilization of recombinant proteins on biosensors for the measurement of antigen-antibody binding, protein- protein binding and protein-small molecule binding, on biochips, and supports for protein immobilization.

In still another aspect, the present invention relates to a method for immobilizing a protein, the method comprises immobilizing the protein on a support for protein immobilization through said A-B-C-type linker molecule. Also, the present invention relates to an immobilized protein prepared by the method, in which a protein is bound to a support for protein immobilization through said A-B-C-type linker.

In the present invention, examples of the support for protein immobilization may include membranes and beads.

In the present invention, in order to examine whether a protein was immobilized on a substrate through formation of a self-assembled monolayer, an experiment using a surface plasmon resonance sensor was performed.

For this purpose, the above-synthesized linker molecule was dissolved in dimethylsulfoxide (DMSO), and a gold thin film-coated surface plasmon resonance sensor chip was immersed in the solution for 3 hours to form a self-assembled monolayer.

As used herein, the term "self-assembled" refers to a phenomenon in which specific nanostructures showing new physical properties are spontaneously formed due to interatomic covalent bonds or intermolecular attractive forces. Typically, this phenomenon appears in self-assembled monolayers, biomaterials containing DNA, nano- and micro-particles, etc.

As shown in FIG. 9, the surface plasmon resonance sensorgram upon the immersion of glutathione S-transferase-tagged staphylococcal protein G was 1300 RU, suggesting that an effective protein monolayer was formed in a short time.

In many cases, even though a protein layer is formed, a protein loses its activity in an immobilization process for various reasons, including immobilization of active sites on a surface. The activity of the glutathione S-transferase-tagged staphylococcal protein G used in the present invention can be determined by examining whether it specifically binds to an antibody in a solution.

FIG. 8 is a schematic diagram showing a process in which the glutathione S- transferase-tagged staphylococcal protein G formed on a surface plasmon resonance sensor chip surface having the inventive self-assembled linker bound thereto adsorbs antibodies present in a solution. FIG. 10 shows that the linker molecule developed according to the present invention immobilizes a significantly large amount of antibodies compared to commercialized CM-5.

In many cases, an immobilized protein (herein, glutathione S-transferase-tagged staphylococcal protein G) loses its activity, and one reason therefor is that the three- dimensional structure of the protein is modified or degraded due to continuous contact with surfaces (non-specific interaction).

Oligoethylene glycol has been known to inhibit non-specific interaction of proteins, and the moiety (B) of FIG. 1 , which is used in the present invention to minimize non-specific binding, comprises oligoethylene glycol. Thus, it is considered that the linker molecule, which contains oligoethylene glycol in addition to the moiety (C) of FIG. 1, which specifically immobilizes a protein, contributes to the maintenance of the activity of the glutathione S-transferase-tagged staphylococcal protein G immobilized on a substrate).

The surface plasmon resonance experiment results shown in FIG. 9 indicate the efficiency of the glutathione linker molecule according to the present invention. As shown in FIG. 9, the linker molecule according to the present invention immobilizes glutathione S-transferase in an amount similar to or slightly smaller than that of the prior commercialized surface plasmon resonance sensor chip, but the protein linked through the inventive linker molecule has excellent activity. Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It will however be obvious to one skilled in the art that these examples are illustrative only and the scope of the present invention is not limited thereto.

Example 1 : Synthesis of glutathione linker molecule 1 which forms self-assembled monolayer (see FIG. 3)

(1) Synthesis of N-lipoyl-2,2'-(ethylene-l,2-dioxy)bisethylamide (intermediate 9)

375 mg of lipoic acid N-hydroxysuccinimide (NHS) ester 8 was dissolved in 20 ml of methylene chloride, and then 1.9 g of 2,2 '-(ethylene- l,2-dioxy)bis(ethylamine) 11 and 0.35 g of triethylamine were added thereto. The mixture was stirred at room temperature for 6 hours.

The precipitated NHS was removed by filtration, the solvent was evaporated under reduced pressure, and the residue was dissolved in 20 ml of chloroform and washed three times with distilled water. The organic layer was washed with a saturated saline solution and then dried with anhydrous magnesium sulfate (anhydrous MgSO₄). The resulting material was concentrated in a vacuum, and the residue was purified through silica gel chromatography (chloroform: methanol =2:1), thus 185 mg of a compound 9 was prepared. The structure of the product was analyzed by ¹H-NMR. ^rlH-NMR (300 MHz, CDCl₃): δ 1.5 (m, 2H: -CH-CH₂-CH₂-CH₂-CH₂- COOH), 1.6-1.7 (m, 4H: -CH-CH₂-CH₂-CH₂-COOH), 1.9 (sextet, J= 6Hz, IH: -S- CH₂-CH₂-CH-S-), 2.2 (t, J= 9Hz, 2H: -CH₂-CH₂-CH₂-COOH), 2.5 (sextet, J- 6Hz, IH: -S-CH₂-CH₂-CH-S-), 2.9 (t, J= 5Hz, 2H: -0-CH₂-CH₂-NH₂), 3.1-3.2 (m, 2H: - S-CH₂-CH₂-CH-S-), 3.5 (m, 2H: -CONH-CH₂-CH₂-O-), 3.5-3.6 (m, 5H: IH for - S-CH₂-CH₂-CH-S- and 4H for (-CH₂-O-CH₂-CH₂-NH₂ or -NHCO-)₂), 3.6 (s, 4H: (- CH₂-O-CH₂-CH₂-NH₂ or -NHCO-)₂J .

(2) Synthesis of N-lipoyl-N'-(3-maleimido)propionyl-2,2'-(ethylene-l,2-dioxy) bisethylamide (intermediate 10)

80 mg of the compound 9 was dissolved in 10 ml of methylene chloride, and 70 mg of the compound 18 and 50 μi of triethylamine were added thereto. The mixture was stirred at room temperature for 6 hours. The precipitated NHS was removed by filtration, the solvent was evaporated under reduced pressure, and the residue was dissolved in 20 ml of chloroform and washed three times with distilled water. The organic layer was washed with a saturated saline solution and dried with anhydrous magnesium sulfate (MgSO₄). The resulting material was concentrated in a vacuum, and the residue was purified through silica gel chromatography (chloroform: methanol =1:2), thus 73 mg of a compound 10 was prepared. The structure of the product was analyzed by ¹H-NMR. ^rlH-NMR (300 MHz, CDCl₃): 1.5 (m, 2H: -CH-CH₂-CH₂-CH₂-CH₂-CONH-), 1.6-1.7 (m, 4H: -CH-CH₂-CH₂-CH₂-CONH-), 1.9 (sextet, J= 6Hz, IH: -S-CH₂- CH₂-CH-S-), 2.2 (t, J= 9Hz, 2H: -CH₂-CH₂-CH₂-CONH), 2.5 (sextet, J = 6 Hz, IH: -S-CH₂-CH₂-CH-S-), 2.6 (t, J=7Hz, 2H: (-CH-CO-)₂N-CH₂-CH₂- NHCO-, MPA part), 3.1-3.2 (m, 2H: -S-CH₂-CH₂-CH-S-), 3.5 (m, 4H: -(CONH-CH₂-CH₂-O-)^, 3.5-3.6 (m, 5H: IH for -S-CH₂-CH₂-CH-S- and 4H for (-CH₂-O-CH₂-CH₂-NHCO- )₂, MPA part), 3.6 (s, 4H: (-CH₂-O-CH₂-CH₂- NHCO-)₂), 3.8 (t, J= 7Hz, 2H: (-CH- CO-)₂N-CH₂-CH₂-NHCO-, MPA part), 6.7 (s, 2H: (-CH-CO-)₂N-CH₂-CH₂-NHCO-, MPA part) j .

(3) Synthesis of N-lipoyl-N'-(3^t-beta-(glutathione S)-succinimido)propionyl-2,2^t- (ethylene- 1 ,2-dioxy)bisethylamide ( 1 ) 50 mg of the intermediate 10 was dissolved in 10 ml of methanol. While the solution was stirred at room temperature, a solution of 30 mg of glutathione 19 dissolved in 1 ml of distilled water was added dropwise thereto at a rate of 0.01 ml/min for 1 hour and 40 minutes, followed by further stirring for 1.5 hours. The solvent was removed under reduced pressure, and the residue was washed several times with methanol and ethyl ether and dried in a vacuum, thus a linker molecule 1 as a final product was prepared. The structure of the product was analyzed by H- NMR. ^r1H-NMR (300 MHz, D₂O): 1.5 (m, 2H: -CH-CH₂-CH₂-CH₂-CH₂-CONH-), 1.6-1.7 (m, 4H: -CH-CH₂-CH₂-CH₂-CONH-), 1.9 (sextet, J= 6Hz, IH: -S-CH₂- CH₂-CH-S-), 2.1 (q, J=I Λ, 2H in GSH), 2.2 (t, J= 9Hz, 2H: -CH₂-CH₂-CH₂- COOH), 2.4-2.6 (IH for -S-CH₂-CH₂-CH-S-, 2H for (-CH-CO-)₂N-CH₂-CH₂- NHCO-) and 2H in GSH), 2.7 (m, IH in MPA), 3.0 (m, 2H in GSH and IH in MPA) 3.1-3.2 (m, 2H: -S-CH₂-CH₂-CH-S-), 3.5 (m, 4H: -(-CH₂-O-CH₂-CH₂- NHCO-)₂), 3.5-3.6 (m, 5H: IH for -S-CH₂-CH₂-CH-S- and 4H for (-CH₂-O-CH₂- CH₂-NHCO-)₂), 3.6 (s, 4H: (-CH₂-O-CH₂-CH₂-NHCO-)₂), 3.8 (m, 2H for (CH-CO- )₂N-CH₂-CH₂-NHCO- and IH in GSH), 3.9 (s, 2H in GSH), 4.0 (m, IH in MPA), 4.5 (t, J=IO Hz, IH in GSH) j

Example 2: Synthesis of linker molecule 2 which forms self-assembled monolayer 2 (see FIG. 4)

(1) Synthesis of N-Boc-2,2' -(ethylene- l,2-dioxy)bisethylamine (intermediate 12)

1 g of 2,2' -(ethylene- l,2-dioxy)bis(ethylamine) 11 was dissolved in 50 ml of methanol, and a solution of 0.736 g of di-tert-butyl dicarbonate 20 and 0.75g of triethylamine dissolved in 30 ml of methanol was added dropwise slowly thereto. Then, the solution was stirred at room temperature for 10 hours. The solvent was removed under reduced pressure, and the residue was dissolved in 50 ml of chloroform, washed two times with an aqueous solution of sodium hydrogen carbonate, and then dried with anhydrous magnesium sulfate. The resulting solution was concentrated under reduced pressure, thus 1.03 g of a compound 12 was prepared. The structure of the product was analyzed by ¹H-NMR. ^rlH NMR (300MHZ, CDC13): δ 1.4 (s, 9H: -CO-O-(CH₃)₃), 2.86 (t, 2H: -O- CH₂-CH₂-NH₂, J= 5.1 Hz), 3.27-3.32 (m, 2H: -0-CH_2-CH_2-NHCO-), 3.48-3.58 (m, 8H: NH₂-CH₂-(CH₂OCH₂)₂-CH₂-), 5.16 (broad s, IH: -0-CH₂-CH₂-NHCO-O-

(2) Synthesis of N-Boc-N'-maleyl-2,2^t -(ethylene- l,2-dioxy)bisethylamide (intermediate 13)

The intermediate 12 (1 g )syrithesized in Example 2-(l) above was dissolved in 20 ml of methylene chloride, and then 0.2 g of maleic anhydride 21 and 50 mg of dimethylaminopyridine (DMAP) were added thereto. The mixture solution was stirred at room temperature for 2 hours and then evaporated under reduced pressure. The residue was dissolved in 50 ml of methylene chloride and extracted with 30 ml of an aqueous sodium solution of hydrogen carbonate, and the aqueous layer was acidified to pH 3 by the addition of dilute hydrochloric acid. The aqueous solution was extracted again with methylene chloride, and the organic layer was dried with anhydrous magnesium sulfate. The resulting material was concentrated under reduced pressure, thus 0.65 g of an intermediate 13 as a final product was prepared. The structure of the product was analyzed by ¹H-NMR. ^r1H NMR (300MHz, CDCl₃): δ 1.47 (s, 9H: -COO-(CH₃)₃), 3.32 (t, 2H: -O- CH₂-CH₂-NHCO-, J= 5.1 Hz), 3.55-3.68 (m, 1OH: -N-CH₂-(CH₂OCH₂)₂- CH₂-), 4.89 (broad s, IH: CH-CONH-CH₂), 6.26-6.44 (dd, 2H: CO-CH-CH- CONH-CH₂, J= 13.1 Hz), 5.76 (broad s, IH: -O-CH₂-CH₂-NHCO-O-(CH₃)₃), 9.78 (broad s, IH: - CH-CH-COOH)J (3) Synthesis of N,N-maleimidoyl-N'-Boc-2 X -(ethylene- l,2-dioxy)bisethylamide (intermediate 14)

The intermediate 13 (0.5 g) synthesized in Example 2-(2) above was dissolved in 30 ml of acetic anhydride, 0.59 g of sodium acetate was added thereto, and the solution was then stirred at 120 ^°C for 45 minutes. The solvent was removed under reduced pressure, and the residue was dissolved in methylene chloride, washed three times with water and phosphate buffer (pH 7.2), and then dried with anhydrous magnesium sulfate. The resulting solution was concentrated under reduced pressure, thus 0.38 g of an intermediate 14 was prepared. The structure of the product was analyzed by ¹H-NMR. ^r1H NMR (300MHz, CDCl₃): δ 1.44 (s, 9H: -COO-(CH₃)₃), 3.25-3.31 (m, 2H: - 0-CH₂-CH₂-NHCO-), 3.47-3.75 (m, 1OH: -N-CH₂-(CH₂OCH₂)₂-CH₂), 5.02 (broad s, IH: CH₂-NHCO-O-), 6.7 (s, 2H: -O-CH₂-CH₂-N(-CH-CO-)₂)j

(4) Synthesis of N,N-maleimidoyl-N'-liρoyl-2,2 '-(ethylene- 1 ,2-dioxy)bisethylamide (linker molecule 6)

The compound 14 (0.3 g) synthesized in Example 2-(3) above was dissolved in a mixture of 10 ml of trifluoroacetic acid and methylene chloride (1:1), and the solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure, and a process of dissolving the remaining material in methylene chloride and removing the solvent under reduced pressure was repeated three times to completely remove trifluoroacetic acid. The residue was dried in a vacuum, thus preparing 140 mg of an intermediate 15.

The intermediate 15 (75 mg) was dissolved in 10 ml of methylene chloride. Then, 100 mg of lipoic acid NHS ester 8, 140 μJL of triethylamine and 20 mg of dimethylaminopyridine (DMAP) were added thereto, and the solution was stirred at room temperature for 10 hours. The solvent was removed under reduced pressure, and the residue was dissolved in 20 ml of methylene chloride. The organic layer was washed three times with distilled water and dried with anhydrous magnesium sulfate. The resulting residue was purified through silica gel chromatography (acetone: methylene chloride = 1:3), thus 60 mg of a linker molecule 6 was prepared. The structure of the product was analyzed by ¹H-NMR. ^rlH-NMR (300 MHz, CDCl₃): 1.47 (m, 2H: -CH-CH₂-CH₂-CH₂-CH₂-CONH-), 1.59-1.76 (m, 4H: -CH-CH₂-CH₂-CH₂-CONH-), 1.90 (sextet, J= 6.6Hz, IH: -S- CH₂-CH₂-CH-S-), 2.2 (t, J= 7.5Hz, 2H: -CH₂-CH₂-CH₂-CONH), 2.45 (sextet, J= 6.6Hz, IH: -S-CH₂-CH₂-CH-S-), 3.06-3.21 (m, 2H: -S-CH₂-CH₂-CH-S-), 3.39-3.45 (m, 2H: -(CONH-CH₂-CH₂-O-), 3.49-3.76 (m, 8H: -(CONH-CH₂-CH₂- O-CH₂)₂, 2H: -0-012-CH₂-NCO-, IH: -S-CH₂-CH₂-CH-S-), 6.18 (broad s, IH: CH₂-CONH-CH₂-CH₂-O-), 6.71 (s, 2H: -O-CH₂-CH₂-N(-CH-CO-)₂)j

(5) Synthesis of N,N-(T-beta-(glutathione S)-succinimido)-N'-liρoyl-2.2^>- (ethylene- 1 ,2-dioxy)bisethylamide (linker molecule 2)

The compound 6 (50 mg) synthesized in Example 2-(4) above was dissolved in 10 ml of methanol, and a solution of 20 mg of glutathione in 1 ml of distilled water was added dropwise with stirring at room temperature. Then, the mixture solution was stirred at room temperature for 5 hours. The solvent was removed under reduced pressure, and the residue was washed several times with methylene chloride and methanol, and then dried in a vacuum, thus a linker molecule 2 as a final product was prepared. The structure of the product was analyzed by ¹H-NMR. ^rlH-NMR (300 MHz, D₂O): 1.46 (m, 2H: -CH-CH₂-CH₂-CH₂-CH₂-CONH-), 1.57-1.74 (m, 4H: -CH-CH₂-CH₂-CH₂-CONH-), 1.90 (sextet, J= 6Hz, IH: --CH₂- CH₂-CH-S-), 2.11-2.26 (m, 2H: NHCO-CH₂-CH₂- in GSH & 2H: -CH₂-CH₂-CH₂- COOH), 2.40-2.59 (m, IH: -S-CH₂-CH₂-CH-S- & 2H: -NHCO-CH₂-CH₂-InGSH), 3.1 (m, IH: -S-CH₂-CH₂-CH-S-), 3.15-3.26 (m, 4H: -NCO-CH₂-CH-S-CH₂- and 2H: -S-CH₂-CH₂-CH-S-), 3.32-3.47 (m, 2H: -CONH-CH₂-CH₂-O-), 3.49-3.77 (m, 8H: -CONH-CH₂-(CH₂-O-CH₂)₁, 2H: -O-CH2-CH₂-NCO-, 1H:-NCO-CH₂-CH-S- CH₂, IH in GSH & IH in GSH), 4.7 (m, IH for GSH) j

Example 3: Preparation of a chip with glutathione linker molecule

In order to immobilize the linker molecule 1 synthesized in Example 1 on a substrate surface, a gold thin film surface was treated with a mixture solution of 95% sulfuric acid and 30% hydrogen peroxide (3:1 v/v) at 60 ^°C for 30 minutes, and then immersed in a dimethylsulfoxide (DMSO) solution containing 1 mM of the linker molecule 1, at room temperature for 3 hours or longer, thus forming a self-assembled monolayer on the gold thin film surface. The chip having the self- assembled monolayer formed thereon was washed with dimethylformamide (DMF) and triple distilled water, thus preparing a chip with glutathione linker molecule.

Example 4: Preparation of glutathione S-transferase-tagged staphylococcal protein G

To construct a staphylococcal protein G gene tagged with glutathione S-transferase at the N-terminal end, two primers containing a portion of the staphylococcal protein G gene were constructed.

In order to insert the staphylococcal protein G gene into glutathione S-transferase tag expression vector pGEX 4T- 1 (Amersham Pharmasia Biotech Inc., USA), Ndel was introduced into an N-terminal primer (SEQ ID NO: 1), and a Xhol restriction enzyme cleavage site was introduced into a C-terminal primer (SEQ ID NO: 2). Using the primer pair of SEQ ID NO: 1 and SEQ ID NO: 2, a Streptococcus sp. KCCM 41566 genomic gene was amplified by PCR. The resulting fragment was cleaved with the corresponding restriction enzymes (Ndel and Xhoϊ), and then inserted into the pGEX 4T- 1 vector cut with restriction enzymes, thus constructing a pGST-protein G vector. SEQ ID NO: 1 (sense primer): 5'-CATATGCACCACCACCACCACCACA

AAGGCGAAACAACTACTGAAGCT-3' SEQ ID NO: 2 (antisense primer): S'-CTCGAGTTATTCAGTTACCGTAA

AGGTCTTAGTC-3'

E. coli BL21 transformed with the constructed pGST-protein G vector was shake- cultured at 37 °C . When the culture medium reached an absorbance of 0.6 at OD 600 nm, IPTG was added to a final concentration of 1 mM to induce protein expression. After 4 hours, E. coli pellets collected by centrifugation were disrupted with ultrasonic waves (Branson, Sonifier 450, 3 kHz, 3 W, 5 min) to obtain a recombinant protein solution. The obtained protein solution was added to a buffer solution (12 mM Tris-HCl, pH 6.8, 5% glycerol, 2.88 mM mercaptoethanol, 0.4% SDS, 0.02% phenol bromide blue), and the mixture was heated at 100 ^°C for 5 minutes, loaded on polyacrylamide gel and electrophoresed for 1 hour to resolve a recombinant protein.

Example 5: Measurement of antigen-antibody binding in gold thin film chip having glutathione S-transferase-tagged staphylococcal protein G immobilized thereon

The gold thin film surface having the GSH self-assembled monolayer formed thereon, prepared in Example 3, was immersed in 0.1 mg/ml of the glutathione S- transferase-tagged staphylococcal protein G solution prepared in Example 4 and was allowed to react for 30 minutes.

The chip having the protein immobilized thereon was mounted on a surface plasmon resonance sensor (Biacore 3000), 0.1 mg/ml of an antibody and 0.1 mg/ml of an antigen were allowed to react with each other on the chip surface at a rate of 5 βllvnm, and the antibody-antigen binding on the surface was measured in real time. Also, the glutathione S-transferase-tagged staphylococcal protein G was immobilized on a commercially available CM-5 sensor chip (Biacore), and the antibody-antigen reaction thereon was also measured.

Specifically, for immobilization, the CM-5 surface having a carboxyl group was allowed to react with a mixture of 0.1 M N-hydroxysuccinimide (NHS) and 0.4 M l-ethyl-3-dimethylaminopropyl carbodiimide (EDC) at a rate of 7 μi/min for 7 minutes, so that it was activated with N-hydroxysuccinimidyl ester. The glutathione S-transferase-tagged staphylococcal protein G (GST-protein G fusion protein) was allowed to react with the activated chip surface to immobilize it on the chip surface, and the remaining active group was allowed to react with 1 M ethanolamine solution on the chip surface at a rate of 7 μH/mm for 7 minutes so as to inactivate it. Then, the immobilized surface was mounted on the surface plasmon resonance sensor (Biacore 3000), and then an antigen and an antibody were allowed to react with each other on the chip surface in the same manner as described above. The antibody-antigen binding on the chip surface was measured.

FIG. 8 schematically shows the above method of measuring the antibody-antigen method. The surface plasmon resonance sensor was used to compare the effects of the immobilization methods on protein orientation and protein-protein binding. As a result, the chip fabricated in Example 3 had correct protein orientation and showed rapid and simple protein immobilization and high sensitivity, compared to the commercially available CM-5 chip (FIG. 10).

The glutathione S-transferase-tagged staphylococcal protein G (GST-protein G fusion protein) bound to the self-assembled monolayer of the glutathione linker molecule through affinity binding, and the glutathione S-transferase-tagged staphylococcal protein G immobilized on the CM-5 chip through chemical binding, showed 1300 RU and 3350 RU, respectively, which are about 2.5 fold different from each other. However, as shown in FIG. 10, the amounts of antibodies bound thereto were 2300 RU and 720 RU, respectivedly, suggesting that the glutathione S- transferase-tagged staphylococcal protein G immobilized on the glutathione self- assembled monolayer was immobilized such that it had more excellent orientation characteristics.

Also, in order to measure the activity of a fluorescence biochip to which a protein was bound using the glutathione self-assembled monolayer, the glutathione S- transferase-tagged staphylococcal protein G was immobilized on a substrate surface, and 0.1 mg/ml of 20 anti-biotin spots and 0.1 mg/ml of 20 anti-EGFP spots were arrayed on the substrate surface. Then, 0.1 mg/ml of protein EGFP showing green fluorescence was allowed to react with the antibodies. After completion of the reaction, the chip was measured with GenePix 4200 (Axon, USA) using a 488-nm laser GenePix 4200 (Axon, USA).

As a result, as shown in FIG. 11 , it could be observed that, in the anti-biotin array on the left side, no fluorescent signal appeared, and thus no non-specific binding occurred, and in the array on the right side, signals appeared saturated, and thus specific protein binding predominantly occurred.

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention provides the linker molecule for immobilizing a protein on a substrate surface, and a preparation method thereof. The linker molecule according to the present invention forms a self-assembled monolayer through contact with a substrate, and only a protein having a specific tag is immobilized on the self-assembled monolayer. The immobilized protein can be bound such that it has excellent orientation and, at the same time, does not lose its activity. A chip fabricated using the linker molecule developed according to the present invention has an advantage in that non-specific binding does not occur on a surface having no target protein bound thereto. Thus, according to the present invention, the time and cost required for purifying a protein in the preparation of the protein chip are reduced and the activity of the protein is increased. Therefore, the protein chip according to the present invention is very economical.

While the present invention has been described with reference to the particular illustrative embodiment, it is not to be restricted by the embodiment but only by the appended claims. It is to be appreciated that those skilled in the art can change or modify the embodiment without departing from the scope and spirit of the present invention.

Claims

THE CLAIMS What is claimed is::

1. A method for preparing an A-B-C-type linker molecule for immobilizing a protein on a substrate, the method comprising the steps of:

(a) binding a material (A) which serves to form a self-assembled monolayer on a substrate surface, to one end of a protein- immobilizing ligand (B) which serves to inhibit non-specific binding of a protein; and

(b) binding an affinity material (C) which binds to the protein, to the other end of the protein-immobilizing ligand.

2. The method for preparing an A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 1 , wherein the protein-immobilizing ligand (B) is oligoethylene glycol or polyethylene glycol, for both ends of which amine is substituted.

3. The method for preparing an A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 1, wherein the protein is tagged with a peptide or proteins having an affinity for known ligands such as GSH, maltose, metal-NTA, metal-IDA etc.

4. The method for preparing an A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 3, wherein the target protein tagged with a peptide or protein having an affinity for affinity ligands mentioned in claim 3 is selected from the group consisting of a protein tagged with glutathione-S- transferase, a protein tagged with maltose-binding protein, and a protein tagged with histidine.

5. An A-B-C-type linker molecule for immobilizing a protein on a substrate, in which a material (A) serving to form a self-assembled monolayer on a substrate surface and an affinity material (C) binding to a protein, are bound to both ends of an oligoethyleneglycol linker or a polyethyleneglycol linker for both ends of which amine is substituted (B) serving to inhibit non-specific binding of the protein, respectively.

6. The A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 5, wherein the protein-immobilizing ligand (B) is oligoethylene glycol or polyethylene glycol, for both ends of which amine is substituted.

7. The A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 5, which has a structure of Formula I: [Formula I]

wherein X and Y are substances binding to a protein or a compound containing amine or thiol(SH), and which has a functional group of any one among the following Formula III, n is 2~100: [Formula III]

8. The A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 6, wherein said Y is selected from the group consisting of L- reduced glutathione, iminodiacetic acid, nitrilotriacetic acid, maltose derivatives, galactose, calmodulin, biotin, chitin, cellulose, C-myc, thioredoxine, intain, S- peptide and DNA.

9. The A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 6, wherein the linker molecule of Formula I is selected from the group consisting of linker molecules 1-5.

10. The A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 5, which has a structure of Formula II: [Formula II]

wherein, said triethoxysilane moiety is a functional group to be bound on a glass surface, X and Y has a functional group of any one among the following Formula III, n is 2-100: [Formula III]

11. The A-B-C-type linker molecule for immobilizing a protein on a substrate according to claim 9, wherein said Y is selected from the group consisting of L- reduced glutathione, iminodiacetic acid, nitrilotriacetic acid, maltose derivatives, galactose, calmodulin, biotin, chitin, cellulose, C-myc, thioredoxine, intain, S- peptide and DNA.

12. A method for preparing a protein chip, the method comprising the steps of:

(a) forming a self-assembled monolayer of the linker molecule on a substrate through a material (A) for forming a self-assembled monolayer on a substrate surface by introducing the A-B-C-type linker molecule of any one claim among claims 5-10 into a substrate, and exposing an affinity material (C) which binds to a protein; and

(b) binding a protein to the exposed affinity material (C).

13. A protein chip, in which a protein is bound to a substrate through the A-B-C- type linker molecule of any one claim among claims 5-10.

14. A method for immobilizing a protein, the method comprises immobilizing the protein on a support for protein immobilization through said A-B-C-type linker molecule of any one claim among claims 5-10.

15. An immobilized protein, in which a protein is bound to a support for protein immobilization through said A-B-C-type linker molecule of any one claim among claims 5-10.

16. The immobilized protein, wherein the support for protein immobilization is membrane or bead.