JP2006132980A - Method for fixing protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for fixing proteins at an arbitrary position. <P>SOLUTION: According to this method, one or more kinds of proteins made by together bonding a single-chain nucleic acid are mixed with each other to fix the proteins at an arbitrary position of the nucleic-acid by utilizing nucleic acid hybridization. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

    The present invention relates to a technique for immobilizing a protein added with an arbitrary nucleic acid sequence at an arbitrary position by utilizing hybridization of nucleic acids. It also relates to protein complexes prepared using the method.

  In recent years, expression analysis and interaction analysis techniques using protein chips have attracted attention. However, it is difficult to say that the technology for immobilizing proteins has been established. In general, spotting takes time and has problems such as protein drying and loss of activity.

  Moreover, it cannot be said that the preservation | save method of the chip | tip after spotting protein is also established. Proteins often lose activity during storage, and it is said that they are preferably immobilized immediately before use. Therefore, a method for quickly immobilizing a protein at an arbitrary position immediately before use has been desired.

  In recent years, protein localization research has become active, and it is clear that various proteins are three-dimensionally localized in cells and interact with each other while maintaining positional relationships with each other. It is becoming. In addition, many molecules exist as complexes, and many molecules in which they work as a unit have been confirmed. For example, it has been clarified that MAPKKK and MAPKK are present as a complex in a molecule involved in the MAP kinase cascade. Furthermore, in metabolic systems, energy transfer systems, etc., enzymes often exist while maintaining their positions relative to each other.

    In these molecules, positional relationships are very important and need to be reconstructed when studying. However, there are many cases where a complex cannot be formed simply by mixing proteins, and a technique for effectively coordinating protein molecules at an arbitrary distance is required.

    For the reasons described above, it has been required to immobilize proteins at arbitrary positions. That is, an object of the present invention is to provide a technique for immobilizing an arbitrary protein at an arbitrary position. Another object of the present invention is to provide a method for preparing a protein to which a single-stranded nucleic acid is bound.

    In order to achieve the above-mentioned object, the present inventors have repeatedly studied and established a method for binding a single-stranded nucleic acid to a protein, and that the protein can be immobilized by a protein immobilization method using nucleic acid hybridization. As a result, the present invention has been completed.

    That is, the present invention comprises the following items.

  1. A method of immobilizing a protein at an arbitrary position by mixing one or more proteins to which a single-stranded nucleic acid is bound, and utilizing nucleic acid hybridization.

  2. One or more proteins bound to the first single-stranded nucleic acid and an object bound to the second single-stranded nucleic acid are subjected to hybridization between the single-stranded nucleic acids using the protein. The method according to Item 1, wherein the method is immobilized at a specific mutual position.

  3. Item 3. The method according to Item 2, wherein the object is a protein.

  4). Item 3. The method according to Item 2, wherein the object is a carrier.

  5. The carrier is a film, a bead, a gel or a substrate of a material selected from resin, nylon, nitrocellulose, polysaccharide, glass or metal, and the carrier is on a support such as glass, ceramics, metal or plastic as necessary. Item 5. The method according to Item 4, wherein

  6). A plurality of single-stranded nucleic acids having different sequences, which are designed to generate two or more single-stranded nucleic acid portions by hybridization, are hybridized and have a sequence complementary to the single-stranded nucleic acid portion. And immobilizing two or more proteins at specific positions by mixing and hybridizing two or more single-stranded nucleic acids bound with the protein. .

  7. Hybridizing a plurality of single stranded nucleic acids having different sequences designed to produce two or more single stranded nucleic acid moieties upon hybridization and further complementary to one of the single stranded nucleic acid moieties By mixing and hybridizing a carrier bound to a single-stranded nucleic acid having a sequence and at least one single-stranded nucleic acid having a sequence complementary to another single-stranded nucleic acid portion and bound to a protein, Item 5. The method according to Item 1, 2, or 4, wherein the method is immobilized on a carrier, and the carrier and at least one protein are immobilized at specific mutual positions.

  8). Item 8. The method according to any one of Items 1 to 7, wherein the nucleic acid is any one of DNA, RNA, and PNA.

  9. Item 9. The method according to any one of Items 1 to 8, wherein the protein is any one of an enzyme, a receptor, a ligand, an antibody, a lectin, and an affinity protein.

  10. Item 10. The method according to any one of Items 1 to 9, wherein the binding of the nucleic acid to the protein is a covalent bond.

  11. Item 11. The method according to any one of Items 1 to 10, wherein the binding of the nucleic acid to the protein is formed by a method via cell-free protein synthesis.

  12 Item 10. The method according to any one of Items 1 to 9, wherein the binding of the nucleic acid to the protein is affinity binding.

  13. Item 13. The method according to Item 12, wherein the affinity binding is any one of avidin-biotin reaction, GST-glutathione, MBP (maltose binding protein) -amylose, chelate, or antibody-antigen reaction.

  14 Two or more proteins are immobilized by hybridization of a single-stranded nucleic acid to two or more single-stranded portions of a nucleic acid complex having three or more single-stranded portions, and are immobilized on a carrier via one single-stranded portion. A protein complex.

  15. Item 15. The complex according to Item 14, wherein at least one protein is an enzyme.

  16. Item 16. A method comprising adding a substrate to the protein complex of Item 15, and performing an enzyme reaction.

  17. Item 15. A method for measuring an interaction between proteins of a protein complex according to Item 14.

  According to the present invention, any protein can be bound to a single-stranded nucleic acid that can hybridize with a single-stranded nucleic acid (or a single-stranded nucleic acid moiety) immobilized on an object (protein or carrier), By changing the type of protein bound to a single-stranded nucleic acid that can hybridize, the protein can be immobilized at an arbitrary position in an arbitrary combination.

The present invention is a method of immobilizing a protein at an arbitrary position using one or more proteins to which a single-stranded nucleic acid is bound, utilizing nucleic acid hybridization. One or more proteins mixed with a single-stranded nucleic acid having a plurality of different sequences designed to generate a nucleic acid sequence and further bound with a single-stranded nucleic acid having a complementary sequence are mixed with the nucleic acid sequence. The protein is immobilized at an arbitrary position by hybridization.
In this specification, “immobilization” refers not only to the case where a protein and a solid phase (carrier) are immobilized in a specific positional relationship as shown in FIGS. 2 and 4, but also two or more proteins as shown in FIGS. The case where they are maintained (fixed) in a specific mutual positional relationship is also included.

  Specifically, as shown in FIGS. 1 and 2, utilizing the property of hybridization between single-stranded nucleic acid A and A ′ having a complementary sequence thereto, proteins are immobilized at fixed positions, or In this method, an arbitrary protein is immobilized at an arbitrary position on a solid phase (carrier) on which A ′ is immobilized.

  Specifically, as shown in FIGS. 3 and 4, single-stranded nucleic acids having a plurality of different sequences designed to generate one or more single-stranded nucleic acid sequences at any position were hybridized. In this method, a plurality of proteins are immobilized in a fixed mutual positional relationship using a nucleic acid complex, or a plurality of proteins are simultaneously immobilized at arbitrary positions on a solid phase.

  The nucleic acid used for this purpose is preferably DNA, RNA, or PNA. The length of the nucleic acid is not particularly limited, but the length necessary for hybridization is required, and preferably 15 bases, more preferably 20 bases or more, for each hybridization part. Moreover, when experimenting by changing the distance between proteins, it can set arbitrarily.

  The solid phase or carrier for immobilizing the protein is a film, bead, gel or substrate of a material selected from resin, nylon, nitrocellulose, polysaccharide, glass or metal, and the carrier is glass if necessary. It is on a support such as ceramics, metal or plastic.

  The protein used in the method of the present invention is not particularly limited, but enzymes, receptors, ligands, antibodies, lectins, affinity proteins and the like are preferably used. Also, a fusion protein or the like is preferably used, and a fusion protein with a plurality of fluorescent proteins can also be used. For example, fluorescence resonance energy transfer (FRET) can be used.

  The nucleic acid can be bound to the protein by a chemical binding method via a functional group on the protein surface. Preferably, an amino group, a carboxyl group, a thiol group, or the like is used and reacted with a functional group introduced at the end of the nucleic acid to be bonded. However, when this method is used, there is a high possibility that the activity of the protein will be impaired, so it is necessary to conduct experiments in advance.

  Therefore, it can be said that it is more preferable to immobilize the nucleic acid through cell-free protein synthesis.

  The non-natural amino acid introduction method, which synthesizes non-natural amino acids having various functional side chains and introduces them in a position-specific manner in the same way as natural amino acids, can introduce various functional groups with almost no loss of protein function. It can be said that it is excellent in that point. Compared with the chemical modification method, the position at which the functional group is introduced can be arbitrarily determined, so that the risk of impairing the original function of the protein can be greatly reduced.

  A natural protein is composed of 20 kinds of amino acids as described above, and the amino acid sequence is defined by the gene encoding the protein. A gene designates an amino acid by a genetic code (codon) consisting of a combination of three consecutive bases on DNA. There are four types of bases, adenine (A), thymine (T), cytosine (C), and guanine (G). The total number of 3-base codons consisting of these combinations is 4 × 4 × 4 = 64, but most of them are A certain 61 kinds of codons are assigned to 20 kinds of natural amino acids. The remaining three codons, TAG, TAA, and TGA, are called stop codons, do not correspond to any of the 20 amino acids, and define the end of the amino acid sequence of the protein.

  In order to introduce non-natural amino acids into proteins in a position-specific manner, dedicated codons that specify them are required. However, as described above, information for specifying all of the 64 types of codons that can be composed of three bases is defined.

  Schultz et al. (Noren, C. J. et al. (1989) Science, 244, 182-188) and Chambarlin et al. (Bain, J. D. et al. (1989) J. Am. Chem, Soc., 111, 8013-8014) report assigning TAG, one of the stop codons, to an unnatural amino acid. An arbitrary codon on the protein is replaced with TAG, a tRNA having an anticodon CUA corresponding to the stop codon TAG, which does not exist in nature, is synthesized, and the unnatural amino acid is supported and the unnatural amino acid is introduced in a position-specific manner.

  Another method for determining the position of an unnatural amino acid is to use a 4-base codon that is an extension of a normal 3-base codon (Hohsaka, T., et al. (1996) ibid., 118, 9778). -9779). In this method, for example, when a 4-base codon composed of CGGG is translated by an artificially synthesized tRNA having a 4-base anticodon, a full-length protein into which an unnatural amino acid has been introduced is synthesized. On the other hand, when translated by a natural tRNA having a 3-base anticodon for only 3 bases of CGG, the reading frame of the subsequent codon shifts, and the synthesis of the protein is terminated halfway by encountering a stop codon. Become. Thus, the method using a 4-base codon is characterized in that it can define an unnatural amino acid virtually independently of the usual three-base codons present in 64 types, and is more in comparison with the method using a stop codon. It is also possible to introduce various types of unnatural amino acids into the same protein molecule in a position-specific manner and to allow functional expression. In fact, as an example, there is a report of synthesizing a mutant protein in which a non-natural amino acid nitrophenylalanine having an electron accepting group at position 54 of streptavidin and a non-natural amino acid anthranilamidoalanine having a fluorescent group at position 84 are introduced (Hohsaka T., et al. (1999) ibid., 121, 12194-12195). The synthesized unnatural double mutant protein showed fluorescence quenching as expected. More recently, it has become clear that unnatural amino acids can also be specified for 5-base codons (Hohsaka, T., et al., Submitted.).

  The unnatural amino acid introduction method is usually performed using a cell-free protein synthesis system because it is difficult to express tRNA carrying any unnatural amino acid in the cell. The cell-free protein synthesis system is a technology that has recently attracted attention as a protein synthesis method having various advantages. The advantages of this cell-free protein synthesis method are compared with the methods using recombinant microorganisms or cultured cells as the means of protein synthesis that have been used so far. Is relatively easy to synthesize 2) The conditions can be determined easily (when using microorganisms, it usually takes at least one month), 3) Non-natural The type amino acid can be used. For this reason, cell-free protein synthesis is expected to be used in a wide range of applications, such as when it is necessary to synthesize many types of proteins in a short time, and for the synthesis of proteins that are difficult to produce by recombinant organisms. ing. At present, the cell-free protein synthesis system mainly uses extracts from living cells, and among them, those derived from Escherichia coli, wheat germ, and rabbit reticulocytes are particularly widely used. In particular, a system derived from Escherichia coli may be relatively easy to supply raw materials, and is more commonly used than the other two systems. However, Escherichia coli is a prokaryotic cell, and when synthesizing a protein derived from a eukaryotic cell, particularly a protein derived from human, there are many examples in which the synthesis system is not well adapted and the synthesis fails. Among them, the signal transduction proteins that are particularly well studied as causative factors such as receptors, such as receptors, protein kinases, protein phosphatases, and transcription factors, are the proteins that are best at cell-free protein synthesis systems derived from E. coli. However, on the contrary, it is also a protein with a high demand for introducing the most unnatural amino acid for application to the functional analysis. Therefore, it can be said that the synthesis of these proteins is preferably a wheat germ system or a system using rabbit reticulocytes.

In the method of the present invention, a system for introducing a nucleic acid into a protein using a cell-free protein synthesis method is not particularly limited. However, a tRNA carrying an amino acid bound to a single-stranded nucleic acid is directly converted into a cell-free protein synthesis method. Therefore, it can be said that it is preferable to use the method shown in FIG. That is, as shown in FIG. 5, for example, an aminoacyl-tRNA synthase is allowed to act on an amber suppressor tRNA ^lys (hereinafter referred to as tRNA ^{Sup lys} ) to introduce lysine, and then a functional group (hereinafter referred to as an amino group on the side chain) is introduced. SAC (Specific Acceptor for Coupling) is introduced (SAC-Lys-tRNA ^{Sup lys} ). As a template for cell-free protein synthesis, a gene in which the above-mentioned TAG sequence (stop codon) is introduced at an arbitrary position of a gene (cDNA clone) encoding a target protein (Nonsense-mutated mRNA) is prepared, and SAC- Lys-tRNA ^{Sup lys} is mixed at the time of synthesis and introduced into the protein at the TAG sequence portion (SAC Protein).

  An amber mutation is one of nonsense mutations, and a codon of a certain amino acid is mutated to become an amber codon (UAG) as a termination codon. The protein is terminated by the amount of UAG, and the protein remains immature. An amber suppressor is one of the nonsense suppressors. By reading an amber codon (UAG) as a codon corresponding to a certain amino acid, it is possible to recover protein synthesis that should be interrupted or terminated. For example, when the anticodon sequence (AAG) of lysine is mutated to UAG, the suppressor tRNA (Lys) is recognized and the amber stop codon is read as Lys. In one embodiment of the present invention, the suppressor tRNA is charged with an amino acid and further modified.

On the other hand, it is necessary to prepare a nucleic acid. As an example, 5′-terminal aminated synthetic oligo DNA (“NH ₂ -5′-oligo” in FIG. 5) has a functional group (hereinafter referred to as SDC (Specific Donor for Coupling)) that can be coupled to SAC. For example, SDC-NHS is reacted to synthesize SDC-oligo DNA (SDC-oligo).

Next, the SAC-introduced protein thus obtained is coupled with SDC oligo DNA to synthesize a protein having a DNA side chain ([SAC-SDC-oligo] -Protein). No particular limitation is imposed on the SAC, and the like SHA (Salicylhydroxamic acid) and ^{Versalinx TM SA (OMe) -Y-} NHS ( manufactured PROLINX Co.). Most preferably, reagents optimized to react with compounds having an amino terminus are sold by PROLINX (www.prolinx.com) and are preferably used. Details are given in the examples.

  On the other hand, the SDC is not particularly limited, but PBA (phenyl boronic acid) or the like is preferably used. In particular, PFP- (PBA) n sold by PROLINX is preferably used.

The SAC binding protein and SDC binding nucleic acid are then coupled under appropriate conditions. For example, a protein to which SHA (Salicyhydroxamic acid) or Versalinx ^™ SA (OMe) -Y-NHS is bound and a nucleic acid to which PBA (Phenyl boronic acid) is bound are mixed and allowed to stand for several tens of minutes. Complex can be formed. Moreover, this reaction can be dissociated again by using appropriate conditions, which is very convenient.

  The protein-nucleic acid complex synthesized as described above can be used as it is, but it is more preferable to substitute it with an appropriate buffer.

  The present invention is also a protein immobilization method characterized in that the nucleic acid binding to the protein is affinity binding. Specifically, it is preferable to use a combination of streptavidin-biotin, maltose-binding protein-amylose, glutathione transferase-glutathione, antibody-antigen and the like. In particular, the streptavidin-biotin combination is preferable because biotinylated nucleic acids and streptavidin fusion proteins are relatively easily available in general laboratories. The principle is shown in FIG.

Biotinylated oligonucleic acid is synthesized using a synthesizer. In general, biotin is added to the 5 ′ end, but it may also be the 3 ′ end. On the other hand, the protein is expressed as a fusion protein with streptavidin. Streptavidin is preferably added to the end without impairing its activity according to the properties of the target protein. In some cases, an appropriate linker sequence can be inserted.
In this method, the nucleic acid and the target protein need only be mixed. However, it is preferable to remove unreacted nucleic acid by an appropriate method.

  As the industrial application of the present invention, protein chip, protein array, bioreactor, protein complex formation and the like can be considered.

  By using the method of the present invention, a protein chip is first prepared as a stable DNA-immobilized plate, and proteins can be chipped (arrayed) without spotting when necessary. As is well known, proteins are unstable and are preferably prepared for use, and the use of this method can lead to more stable results.

  In addition, it is considered that an enzyme system in the same metabolic system can be operated more efficiently by binding at an arbitrary distance of the protein by this method. This can be used for industrial applications such as bioreactors, and will also provide academic knowledge.

  As one embodiment of the present invention, there can be mentioned a cell-free protein synthesis system for introducing SAC, an oligo DNA modifying reagent, and a protein-DNA complex preparation reagent (kit) including a reaction buffer.

  As still another embodiment of the present invention, a binding solid phase (nylon membrane, glass plate, plastic plate, plate) in which DNA designed to complement DNA bound to protein is immobilized is considered. be able to.

The details of the present invention will be described in Examples. The present invention is not limited to these examples.
Example 1 Preparation of Gene for Cell-Free Protein Expression To demonstrate the present invention, two types of genes (6XHIS tag sequence or Myc tag sequence added to the C-terminus of E. coli DHFR (Dihydrofolate Reductase)) are wheat-free. Cloning to the EcoRV-BamHI site of the dedicated vector pEU3-NII of the protein synthesis kit (PROTEIOS Wheat germ cell-free protein synthesis kit (manufactured by Toyobo)). Cloning is performed using DNA fragments obtained by PCR amplification of DNA derived from Escherichia coli using the primers described in Sequences 1, 2, or Sequences 3, 4. Specifically, 100 ng of Escherichia coli genomic DNA is amplified for 30 cycles using KOD-Plus- (manufactured by Toyobo) with the composition according to the protocol. Subsequently, the DNA fragment is purified using the MagExtractor-PCR & Gel Clean up-kit (manufactured by Toyobo). Subsequently, the purified fragment is ligated to pEU3-NII vector digested with EcoRV and BamHI using Ligation high (manufactured by Toyobo), and E. coli is transformed. Next, a plasmid is extracted from the clone in which the introduction of the insert has been confirmed using a general method, the sequence is confirmed, and the target clone (pEU-DHFR / His-tag, pEU-DHFR / Myc-tag) is obtained. .
Example 2 Introduction of Mutation into Gene Next, a TAG sequence is introduced at the position of DHFR protein 76Lys in the pEU-DHFR / 6XHIS and pEU-DHFR / Myc gene sequences obtained in Example 1. Specifically, using the DNAs of SEQ ID NOs: 5 and 6, mutagenesis is carried out using Quickchange site directed mutationation kit (manufactured by Stratagene). Specifically, it is performed according to the instruction manual of the kit. That is, 100ng of the purified plasmid was used and 25 thermal cycles were performed, followed by treatment with the restriction enzyme DpnI and transformation of E. coli using the treatment solution. The obtained clone is used after purifying the plasmid, confirming the sequence, and confirming the presence or absence of mutagenesis. The DHFR gene obtained by this mutation is obtained by replacing the AAG sequence of 226-228 with TAG. Hereinafter, pEU-DHFR76TAG / His-tag and pEU-DHFR76TAG / Myc-tag are used.
Example 3 Aminoacylation of Lysyl tRNA ^{Sup · lys} First, E. coli amber suppressor tRNA ^lys (tRNA ^{Sup · lys} ) (manufactured by Sigma) is aminoacylated using lysine as a substrate. Specifically, about 1500 pmole of tRNA was dissolved in 100 μl of a reaction solution (20 mM imidazole-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM Lysine, 2 mM ATP, 150 mM NaCl), and an excess amount of aminoacyl tRNA-synthase was dissolved. (Sigma) is added and reacted at 37 ° C. for 45 minutes. After the reaction, a phenol / chloroform treatment is performed, 0.1 volume of 3M sodium acetate and 2.5 volumes of ethanol are added, and the mixture is centrifuged at 15,000 for 10 minutes to collect the precipitate. The precipitate is washed with 70% ethanol and dissolved with an appropriate amount of sterile water.

Example 4 Lysyl tRNA ^{Sup lys} SA (OMe) -Y conversion 1 nmol of aminoacylated tRNA ^Sup is dissolved in sodium carbonate buffer (pH 8.0) to a concentration of 10 nmol / ml. On the other hand, SA (OMe) -Y-NHS (product of PROLINX) is dissolved in DMF so as to be 50 mM. 90 μl of oligo DNA solution and 10 μl of SA (OMe) -Y-NHS solution are mixed and allowed to react on ice for about 1 hour. Then, it refine | purifies in a gel filtration column (NAP25 column; product made by Pharmacia). Purify and dry under reduced pressure. Dissolve in an appropriate amount of sterile distilled water before use.

Example 5 SA (OMe) -PBA conversion of oligo DNA 5′-aminated oligo DNA (SEQ ID NOs: 7 and 8) is dissolved in 90 μl of 0.1M NaHCO ₃ (pH 8.0) to a concentration of 10 nmol / ml. The oligo DNA of SEQ ID NO: 9 is hereinafter referred to as A, and the oligo DNA of SEQ ID NO: 10 is referred to as B. Thereafter, 1 mg of PFP- (PBA) n (manufactured by PROLINX) is dissolved in 130 μl of DMF, 20 μl is added to aminoacylated tRNA ^sup and allowed to react at room temperature for about 3.5 hours. DMF is evaporated under reduced pressure, dissolved in 1 ml of sterilized distilled water, purified with a gel filtration column (NAP25 column; manufactured by Pharmacia), and dried under reduced pressure. Dissolve in an appropriate amount of sterile distilled water before use.

Example 6 Cell-free protein synthesis Cell-free protein synthesis is performed using a PROTEIOS cell-free protein synthesis kit (manufactured by Toyobo Co., Ltd.). In this method, a vector which has been transcribed with T7 RNA polymerase and purified is used, and the method is also performed according to the instruction manual. The synthesized and purified mRNAs are referred to as mRNA-DHFR / His-tag and mRNA-DHFR / Myc-tag, respectively. The cell-free protein synthesis reaction is also basically performed according to the batch method protocol described in the instruction manual, and each mRNA and Lysyl tRNA ^{Sup lys} synthesized in Example 6 are converted to SA (OMe) -Y into 20 μl reaction. Add to about 1 μg and carry out the reaction at 26 ° C. for about 1 hour.

After the reaction, 20 μl of 2M NH ₂ OH, 2 mM EDTA, 2 mM NaHCO ₃ (pH 10) prepared for use is added and left at 4 ° C. for 4 hours. This treatment exposes active hydroxamic acid groups.

Example 7 Coupling reaction (nucleic acid-protein coupling reaction)
Equal amounts of the samples prepared in Examples 5 and 6 are mixed. After forming the complex at room temperature for about 1 hour, the solution is gel filtered through a NAP-10 column, dried under reduced pressure, dissolved in an appropriate amount of sterile distilled water, and stored at −80 ° C.

The reaction was carried out with a combination of His-tag protein and oligonucleotide A, Myc-tag protein and oligonucleotide B. Hereinafter, they are referred to as protein DNA complex A and protein DNA complex B.

Example 8 Hybridization and Detection 100 μl of 5 ′ biotinylated oligo DNA (10 pmol / μl: sterilized distilled water) was added to a 96-well plate coated with streptavidin in the following combination (SEQ ID NO: 11 is complementary to SEQ ID NO: 9: hereinafter A ′, and SEQ ID NO: 12 is complementary to SEQ ID NO: 10: hereinafter referred to as B ′). That is, A 'is added to wells 1, 2, and 3, B' is added to wells 4, 5, and 6, and A '+ B' is added to wells 7, 8, and 9 and reacted at room temperature for 1 hour. Wash each well 3 times with Tween20 / PBS (-). At this time, two identical plates are prepared (plates I and II). After the reaction, for each of the two plates, protein DNA complex A in wells 1, 4, and 7, protein DNA complex B in wells 2, 5, and 8, and protein DNA complex A in wells 3, 6, and 9, Add 20 μl each of an equal mixture of B. After 1 hour, each well was washed 3 times, and anti-Myc antibody diluted about 1/1000 times in plate I was reacted with anti-Myc antibody diluted about 1/1000 times in plate 2 for 1 hour. Wash wells 3 times. Thereafter, 50 μl of peroxidase-labeled anti-IgG antibody diluted 1/500 times was added to each well, reacted at 37 ° C. for 1 hour, each well was washed 3 times, and 50 μl of TMBZ (manufactured by Dojin) was added. The color is developed at 37 ° C., and the reaction is stopped by adding an equal amount of 1N sulfuric acid (all antibodies used were manufactured by santacruz). Thereafter, the absorbance is measured at 450 nm. The result is shown in FIG. As shown in the drawing, a detection pattern specific to His-tag can be obtained from the A sequence, and a detection pattern specific to Myc-tag can be obtained from the sequence B.

1 schematically illustrates one embodiment of immobilizing a protein by hybridization of a single stranded nucleic acid. 1 schematically shows an embodiment in which a protein is immobilized on a carrier (solid phase) by hybridization of a single-stranded nucleic acid. A method of hybridizing a plurality of polynucleotides having a non-complementary portion (corresponding to a single-stranded nucleic acid portion) to form a plurality of single-stranded nucleic acid portions, and then immobilizing a protein to the portion by hybridization Is shown schematically. 1 schematically shows an embodiment in which a plurality of proteins are immobilized on a carrier (solid phase). A system for introducing a nucleic acid into a protein using a cell-free protein synthesis method according to an amber suppressor mutation is illustrated. A method for linking proteins and nucleic acids using a streptavidin-biotin system is schematically shown. His-tag and Myc-tag bound to single-stranded nucleic acid are bound to oligo DNA (A ′, B ′, A ′ + B ′) bound on the substrate and can be recognized by the corresponding antibody. Showing).

Claims (17)

A method of immobilizing a protein at an arbitrary position by mixing one or more proteins to which single-stranded nucleic acids are bound, and utilizing nucleic acid hybridization. One or more proteins bound to the first single-stranded nucleic acid and an object bound to the second single-stranded nucleic acid are subjected to hybridization between the single-stranded nucleic acids using the protein. The method according to claim 1, wherein the method is fixed at a specific mutual position. The method according to claim 2, wherein the object is a protein. The method according to claim 2, wherein the object is a carrier. The carrier is a film, a bead, a gel or a substrate of a material selected from resin, nylon, nitrocellulose, polysaccharide, glass or metal, and the carrier is on a support such as glass, ceramics, metal or plastic as necessary. 5. The method of claim 4, wherein: A plurality of single-stranded nucleic acids having different sequences, which are designed to generate two or more single-stranded nucleic acid portions by hybridization, are hybridized and have a sequence complementary to the single-stranded nucleic acid portion. And the two or more single-stranded nucleic acids bound with the protein are mixed and hybridized to immobilize the two or more proteins at specific mutual positions. Method. Hybridizing a plurality of single stranded nucleic acids having different sequences designed to produce two or more single stranded nucleic acid moieties upon hybridization and further complementary to one of the single stranded nucleic acid moieties By mixing and hybridizing a carrier bound to a single-stranded nucleic acid having a sequence and at least one single-stranded nucleic acid having a sequence complementary to another single-stranded nucleic acid portion and bound to a protein, The method according to claim 1, 2, or 4, wherein the method is immobilized on a carrier, and the carrier and at least one protein are immobilized at specific mutual positions. The method according to any one of claims 1 to 7, wherein the nucleic acid is any one of DNA, RNA, and PNA. The method according to any one of claims 1 to 8, wherein the protein is any one of an enzyme, a receptor, a ligand, an antibody, a lectin, and an affinity protein. The method according to any one of claims 1 to 9, wherein the binding of the nucleic acid to the protein is a covalent bond. 11. The method according to any one of claims 1 to 10, wherein the nucleic acid binding to the protein is formed by a method via cell-free protein synthesis. The method according to any one of claims 1 to 9, wherein the nucleic acid binding to the protein is affinity binding. The method according to claim 12, wherein the affinity binding is any one of avidin-biotin reaction, GST-glutathione, MBP (maltose binding protein) -amylose, chelate, or antibody-antigen reaction. Two or more proteins are immobilized by hybridization of a single-stranded nucleic acid to two or more single-stranded portions of a nucleic acid complex having three or more single-stranded portions, and are immobilized on a carrier via one single-stranded portion. A protein complex. 15. The complex according to claim 14, wherein at least one protein is an enzyme. A method comprising adding a substrate to the protein complex according to claim 15 and performing an enzyme reaction. The method of measuring the interaction between the proteins of the protein complex of Claim 14.

Images