TECHNICAL FIELD 

The present invention relates to, for example, a method for immobilizing proteins that can be extensively utilized when immobilizing proteins on the surface of a carrier. 

BACKGROUND ART 

Information concerning protein-protein interactions and protein-compound interactions is very useful in discovering novel drug targets and/or novel candidate compounds for pharmaceutical preparations. Through a comprehensive analysis of protein functions, protein functions can be analyzed with the use of proteins expressed on a minute scale. This is important in terms of cost and throughput. In order to attain the aforementioned information or to implement protein function analysis with the use of proteins expressed on a minute scale, apparatuses that analyze interactions in real time based on the principle of surface plasmon resonance (SPR) without the use of a radioisotope, such as the Biacore 3000 (Biacore), have been used in recent years. 

In such SPR-based interaction analysis, either the proteins or compounds to be analyzed are immobilized on a sensor chip, other proteins or compounds are allowed to react on the sensor chip, and changes in masses resulting from protein-protein or protein-compound interactions are detected as an SPR signal. Since this SPR-based interaction analysis is a technique for highly sensitive function analysis, the amount of proteins required therefor is advantageously small. 

In SPR-based interaction analysis, for example, a method wherein amino groups of proteins are allowed to couple to carboxyl groups on a sensor chip under conditions of low pH and low salt concentration (i.e., amine coupling) is used when immobilizing proteins on the sensor chip. Under such conditions, however, proteins are likely to be deactivated, and acidic proteins cannot be immobilized. When a tag such as a histidine tag is used, a wide variety of histidine tag fusion proteins can be immobilized on the sensor chip. Such immobilization is unstable, and interaction analysis is impossible. In order to overcome such drawbacks, therefore, the present inventors continuously conducted tag-mediated binding and amine coupling of tag fusion proteins, developed a technique whereby almost all types of proteins could be stably immobilized on a sensor chip at physiological pH and physiological salt levels (affinity amine coupling), and already filed for a patent with regard to the same (JP Patent Application No. 2002-335334). 

Protein expression systems that can express proteins regardless of species have been developed (e.g., a wheat germ cell-free system). In such expression systems, however, the expression levels of the target proteins are often very low. When small amounts of target proteins are immobilized on a sensor chip via the aforementioned affinity amine coupling technique, contaminating proteins are simultaneously coupled. This often yields unsatisfactory results in terms of an S/N ratio in the interaction assay. 

DISCLOSURE OF THE INVENTION 

Given the above circumstances, the present invention is directed to providing a method for stably immobilizing various types of target proteins on a carrier regardless of the amounts of the target proteins and without nonspecifically immobilizing contaminating proteins. 

The present inventors have conducted concentrated studies in order to attain the above object and consequently discovered the following. When proteins having first tag portions and second tag portions are purified with the first tag portions and the purified proteins are immobilized on a carrier, the proteins are allowed to react with the carrier for immobilization after reactive groups on the carrier have been activated. This enables the second tag portions of the proteins to interact with and covalently bind to the carrier. Also, various types of target proteins can be stably immobilized on the carrier regardless of the amounts of the target proteins and without nonspecifically immobilizing contaminating proteins. Such discovery has led to the completion of the present invention. 

The present invention includes the following. 

(1) A method for immobilizing proteins comprising: step 1 of purifying target proteins to be immobilized, which have a first tag portion and a second tag portion, with the use of the first tag portion; step 2 of activating reactive groups capable of covalently binding to the proteins on a carrier for immobilization; and step 3 of allowing a solution containing the proteins purified in step 1 to react with the carrier after step 2, wherein, in step 3, the proteins are immobilized on the carrier via interactions between the second tag portion and the site of the carrier to which the second tag portion binds and via covalent binding between the reactive groups and the proteins. 

(2) The method for immobilizing proteins according to (1), wherein step 1 comprises a step of separating and extracting the proteins via a purification means having the site to which the first tag portion binds. 

(3) The method for immobilizing proteins according to (2), wherein the site to which the first tag portion binds is an antibody that reacts with the first tag portion. 

(4) The method for immobilizing proteins according to (3), wherein the first tag portion is a FLAG tag, and the site to which the first tag portion binds is an anti-FLAG tag antibody. 

(5) The method for immobilizing proteins according to (1), wherein the reactive groups are carboxyl groups and step 3 comprises subjecting the carboxyl groups to amine coupling with amino groups of the target proteins to be immobilized. 

(6) The method for immobilizing proteins according to (1), wherein the second tag portion is a histidine tag, and step 3 comprises subjecting the histidine tag to interaction with the carrier. 

(7) The method for immobilizing proteins according to (6), wherein step 3 comprises subjecting the histidine tag to a chelate-mediated interaction with the carrier. 

(8) The method for immobilizing proteins according to (7), wherein step 3 comprises subjecting the histidine tag to an Ni.sup.2+-nitrilotriacetic acid (Ni-NTA)-mediated interaction with the carrier. 

(9) The method for immobilizing proteins according to (7), wherein step 3 comprises subjecting the histidine tag to an Ni.sup.2+-iminodiacetic acid (Ni-IDA)-mediated interaction with the carrier. 

(10) The method for immobilizing proteins according to (1), wherein the site of the carrier to which the second tag portion binds is an antibody that reacts with the second tag portion. 

(11) The method for immobilizing proteins according to (10), wherein the second tag portion is a histidine tag, the antibody is an anti-histidine tag antibody, and step 3 comprises subjecting the histidine tag to the anti-histidine tag antibody-mediated interaction with the carrier. 

(12) A method for detecting the protein-analyte affinity comprising a step of allowing a sample containing a target analyte to be detected to react with the carrier for immobilization having the proteins immobilized thereon via the method for immobilizing proteins according to any one of (1) to (11) and a step of detecting the affinity between the proteins immobilized on the carrier and the analyte contained in the sample. 

(13) The method for detecting the protein-analyte affinity according to (12), wherein the step of detecting the affinity comprises detecting the affinity between the proteins and the analyte based on the principle of surface plasmon resonance. 

(14) A carrier for immobilization having proteins immobilized thereon via the method for immobilizing proteins according to any one of (1) to (11). 

(15) The carrier for immobilization having proteins immobilized thereon according to (14), which comprises a substrate, the polysaccharide molecule chain provided on the substrate and having reactive groups capable of covalent binding to the target proteins to be immobilized, which are introduced therein, and the target proteins to be immobilized, wherein the proteins are covalently bound to the reactive groups and interact with the polysaccharide molecule chain via a chelate. 

Hereafter, the present invention is described in detail. 

The method for immobilizing proteins according to the present invention can be employed when immobilizing proteins on a carrier for immobilization. This method is not restricted to a specific technical scope. For example, the method for immobilizing proteins according to the present invention can be employed when preparing a sensor chip having immobilized thereon proteins for analysis based on the principle of surface plasmon resonance (SPR). This method can also be utilized when preparing a sensor chip based on principles other than SPR. Examples of such principles other than SPR include the principle of quartz crystal microbalance (QCM) and that of dual polarization interferometry (DPI). 

Further, the application of the method for immobilizing proteins according to the present invention is not limited to the preparation of sensor chips based on the principle of SPR or that of QCM. For example, the method of the present invention can be applied to the preparation of a so-called protein chip (a protein array) or an affinity bead (an affinity column). 

Hereafter, a sensor chip used for SPR-based analysis is described by way of example. As shown in FIG. 1 (see Original Patent), this sensor chip comprises an optically transparent substrate 1, a metal membrane 2 provided on a principal surface of the substrate 1, and a carrier for immobilization 3 provided on the metal membrane 2. The carrier for immobilization 3 is prepared by immobilizing a self-assembled monolayer (SAM) having reactive groups such as carboxyl groups or an SAM and carboxymethyl dextran on the metal membrane 2. 

The carrier for immobilization 3 comprises reactive groups that covalently bind to the target proteins to be immobilized. The reactive groups of the carrier for immobilization 3 refer to functional groups that covalently bind to the target proteins to be immobilized. Examples of reactive groups include carboxyl groups and thiol groups. The carrier for immobilization 3 may comprise a polysaccharide molecule chain into which reactive groups that covalently bind to the target proteins to be immobilized have been introduced. When the carrier for immobilization 3 comprises such a polysaccharide molecule chain, the target proteins to be immobilized covalently bind to reactive groups in the polysaccharide molecule chain, and the proteins form chelates with the polysaccharide molecule chain. Thus, the target proteins to be immobilized are immobilized on the carrier for immobilization 3. An example of such a polysaccharide molecule chain is dextran. 

The carrier for immobilization 3 comprises the site to which the second tag portion of the target protein to be immobilized binds. Such site is adequately selected in accordance with the second tag portion. For example, such site may be nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) when the second tag portion is a histidine tag, it may be glutathione when the second tag portion is a glutathione S-transferase tag, and it may be maltose when the second tag portion is a maltose-binding protein tag. When the second tag portion is an antigenic peptide, the site of the carrier to which the second tag portion binds may be an antibody that undergoes an antigen-antibody reaction with the antigenic peptide. 

In the method for immobilizing proteins according to the present invention, any protein can be immobilized without particular limitation, as long as such protein has 2 tag portions, i.e., the first and the second tag portions. 

The first tag portion refers to a tag portion that is used when purifying the target proteins to be immobilized, which interacts with the site to which the first tag portion binds in the process of purification. The site to which the first tag portion binds is adequately selected in accordance with the first tag portion. For example, such site may be nitrilotriacetic acid (NTA) or iminodiacetic acid (IDA) when the first tag portion of the protein is a histidine tag, it may be glutathione when the first tag portion is a glutathione S-transferase tag, and it may be maltose when the first tag portion is a maltose-binding protein tag. When the first tag portion is an antigenic peptide, the site to which the first tag portion binds may be an antibody that undergoes an antigen-antibody reaction with the antigenic peptide. Examples of the first tag portion include a histidine tag (hereafter referred to as a "His tag"), a glutathione S-transferase tag (hereafter referred to as a "GST tag"), a maltose binding protein tag (hereafter referred to as an "MBP tag"), and an antigenic peptide tag. The "antigenic peptide tag" employs a peptide in which an antibody is present. Examples thereof include a His tag, a His G tag, an HA tag, a C-myc tag, a myc tag, a BPV-1 tag, a cl tag, a Cre recombinase tag, a FLAG tag, an NS1 (81) tag, a green fluorescent protein (GFP) tag, an IRS tag, a LexA tag, a Thioredoxin tag, a Polyoma virus medium T antigen epitope tag, an SV40 Large T Antigen tag, a Paramoxyvirus SV5 tag, a Xpress tag, a GST tag, and an MBP tag. A FLAG tag, an MBP tag, and a GST tag are particularly preferable as first tag portions. 

The second tag portion interacts with the site of the carrier for immobilization 3 to which the second tag portion binds and plays a key role in the binding that takes place between proteins and the carrier for immobilization 3. Any site that differs from the first tag portion can serve as the second tag portion. Examples of the first tag portion listed above can be employed as the second tag portion. Particularly preferable examples of the second tag portion include a His tag and a GST tag. 

Proteins having any characteristics or properties can be employed without limitation. For example, proteins may be basic or acidic, and they may be hydrophobic or hydrophilic. 

A protein having a first tag portion and a second tag portion can be prepared using a recombinant vector comprising the first tag portion and the second tag portion in frame with a protein-encoding gene and allowing the expression of a fusion protein comprising the first tag portion, the second tag portion, and the protein. The first tag portion and the second tag portion of the fusion protein may be located on the N-terminal side or the C-terminal side of the protein, as long as they each independently function as a tag; i.e., as long as they can interact with the site to which the first or second tag portion binds. The first tag portion and the second tag portion of the fusion protein may be located adjacent to or separate from each other. 

A target protein to be immobilized, which has a first tag portion and a second tag portion, can be prepared via any method without particular limitation. Examples of such method include: (1) a method wherein the protein-encoding gene is introduced into a vector, the resulting recombinant vector is introduced into a host cell, and the protein is then allowed to express in the host cell; and (2) a method wherein the protein is allowed to express in a cell-free system, such as a wheat germ cell-free system. 

Examples of plasmid DNAs into which the genes encoding the target proteins to be immobilized, which have the first and the second tag portions, have been introduced that are employed in method (1) for preparing proteins include: E. coli-derived plasmids (e.g., pET-based plasmids such as pET30b, pBR-based plasmids such as pBR322 and pBR325, pUC-based plasmids such as pUC118, pUC119, pUC18, and pUC19, and pBluescript); Bacillus sutbtilis-derived plasmids (e.g., pUB110 and pTP5); and yeast-derived plasmids (e.g., YEp-based plasmids such as YEp13 and YCp-based plasmids such as YCp50). Examples of phage DNAs include .lamda. phages (e.g., Charon 4A, Charon 21A, EMBL3, EMBL4, .lamda.gt10, .lamda.gt11, and .lamda.ZAP). Further, vectors derived from animal viruses such as a retrovirus or a vaccinia virus, vectors derived from plant viruses such as cauliflower mosaic virus, or vectors derived from insect viruses such as a baculovirus can also be employed. 

In order to insert genes encoding target proteins to be immobilized, which have a first tag portion and a second tag portion, into a vector, cDNAs of such genes are first cleaved with an adequate restriction enzyme, and the cleaved cDNAs are inserted into the restriction site or multicloning site of an adequate vector DNA to ligate the cDNAs to the vector. Alternatively, part of a vector and part of the cDNA of the gene may be provided with homologous regions to ligate them via an in vitro method using PCR or other means or via an in vivo method using yeast or the like. 

Subsequently, a recombinant vector containing the gene encoding the target protein to be immobilized, which has the first tag portion and the second tag portion, is introduced into the host. Thus, a transformant that expresses the protein of interest can be obtained. The host is not particularly limited as long as the gene can be expressed therein. Examples of host include: plants of the Gramineae, Brassicaceae, Solanaceae, and Leguminosae; bacteria of Escherichia such as Escherichia coli, Bacillus such as Bacillus subtilis, or Pseudomonas such as Pseudomonas putida; yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; animal cells such as COS cells and CHO cells; and insect cells such as Sf9 cells. 

The recombinant vector can be introduced into plants via conventional transformation techniques, such as electroporation, the agrobacterium method, the particle gun method, or the PEG method. 

The recombinant vector can be introduced into bacteria via any method of introducing DNA into bacteria. Examples thereof include the calcium ion-based method and electroporation. 

The recombinant vector can be introduced into yeast via any method of introducing DNA into yeast. Examples thereof include electroporation, the spheroplast method, and the lithium acetate method. 

Examples of animal host cells include simian COS7 cells, Vero cells, Chinese hamster ovarian (CHO) cells, and murine L cells. The recombinant vector can be introduced into animal cells via any method of introducing DNA into animal cells. Examples thereof include electroporation, the calcium phosphate method, and lipofection. 

An example of an insect host cell is the Sf9 insect cell. The recombinant vector can be introduced into insect cells via any method of introducing DNA into insect cells. Examples thereof include the calcium phosphate method, lipofection, and electroporation. 

In method (2) for preparing proteins, Proteios (Toyobo Co., Ltd.) can be employed as the wheat germ cell-free system, for example. When Proteios (Toyobo Co., Ltd.) is employed, the mRNA is first synthesized in a reaction system that comprises, as a template, a recombinant vector for the wheat germ cell-free system (e.g., a pEU3-NII plasmid (Toyobo Co., Ltd.)) containing a gene encoding a target protein to be immobilized, which has a first tag portion and a second tag portion, and thermo T7 RNA polymerase (Toyobo Co., Ltd.). The synthesized mRNA is then subjected to protein removal via phenol/chloroform treatment and is then buffer-exchanged with the Proteios buffer via ethanol precipitation. In accordance with the protocol of Proteios, the target proteins to be immobilized, which have a first tag portion and a second tag portion, are synthesized using the synthesized mRNA. 

In the method for immobilizing proteins according to the present invention, the target protein to be immobilized is first purified with the first tag portion. The term "purified with the first tag portion" refers to the interaction between the first tag portion of the target protein to be immobilized and the site to which the first tag portion binds, which is used to separate and extract the target protein to be immobilized from biological material containing contaminating proteins and the like. Examples of techniques for protein purification include affinity chromatography and a method involving the use of affinity resins and magnetic beads. Examples of a means for purification that has the site to which the first tag portion binds include a carrier having such site immobilized thereon and a column filled with such carrier. Examples of carriers include agarose and sepharose. 

When a protein has a FLAG tag as the first tag portion, for example, a target protein to be immobilized, which has a FLAG tag as the first tag portion, is prepared via the aforementioned method. Subsequently, a solution containing the target protein to be immobilized is brought into contact with a carrier, such as agarose, carrying an anti-FLAG tag antibody. A complex of the target protein to be immobilized and the carrier, which have been bound via an antigen-antibody reaction, is then separated. For example, a FLAG peptide can then be added to the complex to competitively elute the target proteins to be immobilized. Further, the eluted target proteins to be immobilized can be separated from the carrier or the FLAG peptide via, for example, low-speed centrifugation (e.g., at 2,000 g). 

In the method for immobilizing proteins according to the present invention, reactive groups on the carrier for immobilization 3 are then activated. The term "activation" refers to transition of the reactive groups so that they become capable of covalent binding with the target proteins to be immobilized, which are located in the vicinity thereof. If the carrier for immobilization 3 comprises carboxyl groups as reactive groups, for example, a mixed solution of N-ethyl-N'-(dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) may be allowed to react therewith to activate the carboxyl groups. 

In the method for immobilizing proteins according to the present invention, the target protein to be immobilized is allowed to react with the carrier for immobilization 3 to realize an interaction between the second tag portion of the target protein to be immobilized and the carrier for immobilization 3. The term "interaction" used herein refers to the binding of the second tag portion and the site to which the second tag portion binds, which results in the binding of the protein and the carrier for immobilization 3 at a relatively mild rate. In the case of a protein having a His tag as the second tag portion, for example, a metal such as nickel is trapped in NTA or IDA that has been introduced into the carrier for immobilization 3 to form a complex of the His tag with NTA or IDA via nickel. Nickel may be trapped in NTA or IDA before or after activation of the carrier for immobilization 3. Thus, a protein having a His tag can be subjected to interaction with the carrier for immobilization 3 into which NTA or IDA has been introduced. 

When a protein has a GST tag as the second tag portion, a carrier for immobilization 3 into which glutathione has been introduced can be made to interact with the proteins by allowing them to be present together in a physiological phosphate buffer (e.g., PBS) or a physiological Hepes buffer (e.g., HBS). When proteins comprising antigenic peptides and a carrier for immobilization 3 into which antibodies have been introduced are used, they can also be made to interact with each other by allowing them to be present together in a physiological phosphate buffer (e.g., PBS) or a physiological Hepes buffer (e.g., HBS). 

In the method for immobilizing proteins according to the present invention, the second tag portion of the target protein to be immobilized is allowed to interact with the carrier for immobilization 3 as mentioned above. Thus, the target proteins to be immobilized are located in the vicinity of the carrier for immobilization 3 at a relatively high density. This facilitates the covalent binding between the activated reactive groups and the proteins, and the covalent binding can easily take place between them. 

When reactive groups are carboxyl groups, for example, the reactive groups covalently bind to amino groups that are present in the target proteins to be immobilized (i.e., amine coupling). When reactive groups are carboxyl groups, the carboxyl groups are modified with PDEA to form covalent bonds between free thiol groups in the target proteins to be immobilized and the reactive groups (i.e., ligand-thiol coupling). When the target proteins to be immobilized have carboxyl groups, such proteins are allowed to react with PDEA (2-(2-pyridinyldithio) ethaneamine hydrochloride) previously to modify the carboxyl groups with PDEA. The carboxyl groups on the carrier for immobilization 3 are first activated, the carboxyl groups are allowed to react with cystamine dihydrochloride, and the reaction product is then subjected to reduction with dithiothreitol (DTT) for conversion into thiol groups. Covalent bonds are formed between the PDEA-modified carboxyl groups and the thiol groups on the carrier for immobilization 3 (i.e., disulfide coupling). More specifically, surface thiol coupling takes place. 

The target proteins to be immobilized are purified with the first tag portion, the second tag portion is allowed to interact with the site to which the second tag portion binds, and a covalent bond is formed between the reactive groups and the proteins. This enables immobilization of the target proteins to be immobilized on the carrier for immobilization. According to the method for immobilizing proteins of the present invention, the target proteins to be immobilized are purified with the first tag portion. Thus, biological materials containing contaminating proteins are removed, and the target proteins to be immobilized can be reacted in a dense concentration with the carrier for immobilization 3. In the method for immobilizing proteins of the present invention, the second tag portion is allowed to interact with the site to which the second tag portion binds. Thus, proteins can be present in a dense concentration in the vicinity of the carrier for immobilization 3. According to the method for immobilizing proteins of the present invention, therefore, very small amounts of target proteins to be immobilized can be located in a dense concentration in the vicinity of the carrier for immobilization 3 and can be covalently bound to the carrier for immobilization 3 without nonspecifically immobilizing contaminating proteins, which had been difficult via conventional techniques. 

A sensor chip prepared via the method for immobilizing proteins according to the present invention can be applied to a system that detects an analyte having affinity to the immobilized proteins. 

The term "analyte" refers to a substance having affinity to proteins that have been immobilized. Any known compound or novel compound may be an analyte. Examples thereof include nucleic acids, carbohydrates, lipids, proteins, peptides, amino acids, organic low-molecular weight compounds, a compound library prepared via combinatorial chemistry, a random peptide library prepared via solid-phase synthesis or phage display, and naturally occurring substances derived from microorganisms, plants and animals, or marine organisms. 

As shown in FIG. 2 (see Original Patent), for example, an analyzer based on the principle of SPR comprises: a prism 4 located on the opposite principal surface relative to the principal surface of a substrate 1 on which a carrier for immobilization 3 is located; a light source 6 that permits a polarized light 5 to come into contact with the sensor chip through the prism 4; a detection unit 8 into which a reflected light 7 generated upon reflection of the polarized light 5 enters through the prism 4 via a metal membrane 2; and a flow cell 9 located in contact with the carrier for immobilization 3 having proteins immobilized thereon. 

When the polarized light 5 is applied from the light source 6 to the metal membrane 2 in a manner such that the light is totally reflected, the intensity of the reflected light 7 is partially weakened, according to the principle of SPR. The angle at which a dark portion appears (i.e., a change in the refractive index) depends on the mass on the sensor chip. When the analyte is bound to the protein that has been immobilized on the carrier for immobilization 3, the mass changes (i.e., the mass increases), and the dark portion is shifted from I to II (FIG. 2). It is known that such shift takes place by 0.1 degree from I to II upon binding of 1 ng of substance per mm.sup.2. If the mass decreases upon dissociation, however, the dark portion is shifted back by such degree from II to I. 

The analyzer shown in FIG. 2 flushes a solution comprising a sample containing the analyte into the flow cell 9 and detects the amount of the darkened portion created by the reflected light 7 shifted from I to II with the detection unit 8. This analyzer displays, as the result of detection, changes in the mass on the sensor chip surface on the vertical axis and changes in the mass with the elapse of time as measured data (a sensorgram). The vertical axis is expressed in terms of resonance units (RU), and 1 RU is equivalent to 1 pg/mm.sup.2. Such changes in the refractive index are substantially the same among all biomolecules (i.e., proteins, nucleic acids, and lipids), and interaction can be monitored in real time without labeling such biomolecules. 

With the use of such SPR-based analyzer, interaction between a protein and an analyte can be analyzed, and a novel drug target or a novel candidate compound for a pharmaceutical preparation can be particularly effectively discovered. The sensor chip prepared via the method for immobilizing proteins according to the present invention can immobilize any proteins thereon regardless of the protein species. Also, such sensor chip can securely immobilize proteins for a long period of time. This enables screening of a novel drug target or a novel candidate compound for a pharmaceutical preparation using various species of proteins. 
 

Claim 1 of 11 Claims

1. A method for immobilizing fusion proteins comprising: step 1 of purifying target proteins to be immobilized, which have a first tag portion and a second tag portion by separating and extracting the fusion protein via purification means having the site to which the first tag portion binds; step 2 of activating reactive groups capable of covalently binding to the fusion proteins on a carrier for immobilization, wherein the carrier has the site to which the second tag portion binds; and step 3 of allowing a solution containing the proteins purified in step 1 to react with the carrier after step 2, wherein, in step 3, the fusion proteins are immobilized on the carrier by covalent binding reaction between the reactive groups and the fusion proteins while being concentrated on the carrier by interactions between the second tag portion and the site of the carrier to which the second tag portion binds.

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