JPWO2007126101A1 - Protein chip - Google Patents

Abstract

Provided is a protein chip capable of maintaining an arbitrary protein under conditions close to physiological conditions. A PDZ domain protein is bound on a substrate, and an arbitrary protein having an SLV amino acid sequence can be bound to the C-terminal of the protein that binds to the protein. A protein chip in which a PDZ domain protein is bound on a substrate is characterized by suppressing structural changes or aligning the orientation of the protein.

Description

  The present invention relates to a protein chip using a PDZ domain protein and a protein having a consensus sequence consisting of S / TXV / L / I that specifically binds to the PDZ domain, and a method for producing the same.

  The protein chip is obtained by bonding various proteins on a substrate such as a glass substrate. The function of each protein immobilized on the protein chip is used for various diagnoses, tests, search for molecules that bind to other proteins, and the like.

Methods for immobilizing proteins on substrates include methods of immobilizing proteins on the substrate surface by passive adsorption, methods of coupling proteins to substrates using coupling reagents, and methods of biological capture using protein tags. Etc. Ideally, the immobilization is reproducible, it can be immobilized with the same efficiency even if the protein properties (size, hydrophilicity, hydrophobicity) are different, and it can handle mass processing and automation. Furthermore, it is desired that the activity of the immobilized protein is completely maintained.
As a method of immobilizing protein on the substrate surface by passive adsorption, the substrate is coated with nylon, nitrocellulose, PVDF, polyacrylamide gel, poly-L-lysine, aminopropylsilane, etc., and the protein is adsorbed on the surface. (Non-patent document 1: Hui Ge, 2000, Nucleic Acid Research, vol. 28 (2), e3, Non-patent document 2: Lucy J. Holt et al., 2000, Nucleic Acid Research, vol. 28 ( 15), e72, Non-Patent Document 3: Dmitry Guschin, 1997, Analytical Biochemistry, vol. 250 (2), pp. 203-211). However, although these methods are easy to operate, it is impossible to control the quantitativeness and the direction of protein molecule fixation. Moreover, it is difficult to maintain the activity of the protein to be immobilized, and the reproducibility and efficiency also fluctuate.
Next, as a method for binding a protein to a substrate using a coupling reagent, a method using aldehyde-modified glass and utilizing reactivity with side chains of amino acids constituting the protein (Non-patent Document 4). : Gavin MacBeath, 2000, Science, vol. 289 (5485), pp. 1760-1763) or by modifying the glass surface with an epoxy group and immobilizing the protein on this. These methods can stably immobilize proteins on the substrate, can be applied to various proteins, and have good reproducibility, but the direction of immobilization cannot be completely controlled, and coupling Derivatives of the compounds used in this may change the function of the protein. Therefore, when this method is used, it is necessary to examine a combination of a coupling agent and a support (substrate) surface that does not change or eliminate the function of the protein to be immobilized.
Finally, biological capture methods using proteins as tags include methods using biotin / streptavidin and hexahistidine / nickel interactions. These methods are methods for forming a stable and specific bond between the protein and the surface of the carrier (substrate), and the direction of the bond can be controlled.

The present inventors previously fixed a protein on a substrate by the interaction of a consensus sequence consisting of a PDZ domain and S / TXV / L / I as a protein chip using a unique biological supplementation method. (Patent Document 1: US Pat. No. 6,743,630). That is, by immobilizing a protein having a PDZ domain on a substrate and adding another protein having a consensus sequence consisting of S / TXV / L / I, the other protein can be transferred via the consensus sequence. A chip in which the target protein is immobilized on the substrate by binding to the PDZ domain can be prepared.
The protein chip production method using the interaction of the consensus sequence consisting of PDZ domain and S / TXV / L / I is that FAP-1 (Fas-associated phosphatase-1) Our discovery that it interacts with the C-terminus of the Fas receptor present on the cell surface for transmission into the cell (Non-Patent Document 5: Sato, T., et al. Science 268, 411-415, 1995). ), And subsequent research (Non-patent Document 6: Yanagisawa, J. et al., The Journal of Biolgical Chemistry, 272, pp. 8539-8545, 1997) -It was invented from the finding that a consensus sequence consisting of XV / L / I binds to the PDZ domain of FAP-1.
US Pat. No. 6,743,630 Japanese Patent Laid-Open No. 2002-082116 JP 2003-014747 A JP 2003-153698 A JP 2004-57113 A Japanese Patent Laid-Open No. 2004-230545 JP 2004-347317 A JP 2004-57113 A JP-T-2002-520618 Japanese translation of PCT publication No. 2002-520620 JP-T-2006-511797 International Publication No. 95/04069 Pamphlet International Publication No. 98/05347 Pamphlet Hui Ge, 2000, Nucleic Acid Research, vol.28 (2), e3 Lucy J. Holt et al., 2000, Nucleic Acid Research, vol. 28 (15), e72 Dmitry Guschin, 1997, Analytical Biochemistry, vol.250 (2), pp.203-211 Gavin MacBeath, 2000, Science, vol.289 (5485), pp.1760-1763 Sato, T., et al. Science 268, pp.411-415, 1995 Yanagisawa, J. et al., The Journal of Biolgical Chemistry, 272, pp.8539-8545, 1997)

The protein chip using the interaction between the PDZ domain and the SLV sequence is based on an unprecedented unique immobilization method, and its practical use is expected. However, when a protein consisting of a partial length including the PDZ domain, rather than the full-length protein of FAP-1, is immobilized on the substrate, the binding force between the PDZ domain and the consensus sequence consisting of S / TXV / L / I is sufficient. There was a problem of not. The reason for this is not clear, but one possible reason is that when a protein consisting of a partial length containing a PDZ domain is immobilized on a substrate, the higher-order structure becomes unstable and the function is impaired.
In order to manufacture a practical protein chip, reduction of background noise and cost reduction of the chip are the most important issues to be solved. The background noise is derived from non-specific binding of proteins, but the biggest reason is considered to be due to insufficient purification of the protein immobilized on the substrate. However, when trying to obtain a high-purity protein, it takes a lot of time and materials, and there is a problem that it cannot be compatible with the cost reduction of the chip. As an efficient method for obtaining a high-purity protein, there is a method in which a protein fused with a tag is expressed in cells and the protein is purified by the tag, but depending on the protein, the function is impaired by the tag. Therefore, it is not preferable for producing a high-quality protein chip.
Furthermore, the conventional protein chip requires an ink jet printing apparatus or a dispensing robot equipped with a capillary to finely spot the protein on the substrate. Therefore, it has been desired to develop a protein chip that can array proteins in an arbitrary sequence in a laboratory without requiring a special device.

As a result of intensive investigation and trial and error in order to solve the above problems, the present inventors have found that when an affinity tag is linked to a protein having a PDZ domain, the S / TXV / It has been found that the binding force with the consensus sequence consisting of L / I is not greatly impaired, and the protein chip of the present invention has been invented.
In addition, the affinity tag linked to a protein having a PDZ domain is used for protein purification, and the strong binding force of the consensus sequence consisting of the PDZ domain and S / TXV / L / I is used. Thus, it has been found that high-quality protein chips can be produced at low cost by preventing contamination proteins from binding to the substrate, leading to the invention of the protein chip production method of the present invention. It was.
Furthermore, the present inventors provide a substrate on which an oligonucleotide is fixed, an oligonucleotide having a sequence complementary to the oligonucleotide at an arbitrary position on the substrate, a protein having a PDZ domain, and S / TX- Any protein at any position on the substrate is prepared by preparing a complex of any protein having a consensus sequence consisting of V / L / I, and hybridizing the oligonucleotide at any position and the nucleotide of the complex. Has been found to be arrayed, and the present invention has been completed.

That is, this invention provides the protein chip | tip of following (1)-(14), its manufacturing method, and a kit.
(1) Substrate;
A protein having a PDZ domain linked to an affinity tag; and
Other proteins having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
Including
A protein chip in which the other protein is bound to the PDZ domain via the consensus sequence.
(2) The substrate is a substrate having oligonucleotides immobilized on its surface,
An oligonucleotide having a sequence complementary to the oligonucleotide is bound to the affinity tag,
The protein chip according to (1), wherein the oligonucleotide and an oligonucleotide having a sequence complementary thereto are hybridized.
(3) The protein chip according to (1) or (2), wherein the affinity tag is an affinity tag comprising a fusion protein of GST and avidin.
(4) The protein chip according to any one of (1) to (3), wherein the PDZ domain is a PDZ domain of a human FAP1 protein.
(5) The protein chip according to any one of (1) to (4), wherein the consensus sequence is a sequence consisting of SLV.
(6) A method for producing a protein chip, which comprises the following steps:
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) a step of immobilizing the protein having the PDZ domain purified in the step (C) on a substrate;
(E) Adding the other protein obtained in the step (B) to the substrate on which the protein having the PDZ domain is immobilized in the step (D), and the protein having the PDZ domain and the other protein The process of combining.
(7) A method for producing a protein chip, comprising the following steps.
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) immobilizing oligonucleotides on the substrate;
(E) The protein having the PDZ domain purified in the step (C) is bound to the other protein obtained in the step (B) via the PDZ domain, and further, the protein is passed through the affinity tag. A step of binding an oligonucleotide having a sequence complementary to the oligonucleotide to produce a protein oligonucleotide complex;
(F) A step of adding the protein oligonucleotide complex obtained by the step (E) to the substrate obtained by the step (D) and hybridizing the oligonucleotide on the substrate and the nucleotide of the complex. ;
(8) The method for producing a protein chip according to claim 7,
In the step (B), the other protein is a plurality of other proteins,
In the step (D), the oligonucleotide is a plurality of types of oligonucleotides having different sequences,
In the step (E), the type of nucleotide to be bound via an affinity tag is varied depending on the type of other protein to be bound to the PDZ domain.
(9) A kit comprising a substrate having an oligonucleotide immobilized thereon, and a protein having a PDZ domain to which an oligonucleotide having a sequence complementary to the oligonucleotide is bound.
(10) A PDZ domain protein is bound on a substrate, and any protein having a consensus sequence consisting of S / TXV / L / I can be bound to the C-terminal of the protein that binds to the protein. A protein chip in which a PDZ domain protein is bound on a substrate, which suppresses a change in the three-dimensional structure of the protein due to the placement of the protein on the substrate or aligns the orientation of the protein.
(11) A PDZ domain protein is bound on a substrate, and an arbitrary antibody having a consensus sequence consisting of S / TXV / L / I added to the heavy chain constant region C-terminus is bound to the PDZ domain protein. The binding ability between the antibody and an arbitrary protein is improved by allowing an arbitrary protein to bind to the antibody and suppressing the protein binding site of the antibody from contacting the substrate. A protein in which a consensus sequence consisting of S / TXV / L / I is added to the C-terminus of the heavy chain constant region and bound to the PDZ domain protein, and the complex is bound to the substrate Chip.
(12) A PDZ domain protein linked to an arbitrary oligonucleotide is bound to an arbitrary protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus of the protein, and the oligonucleotide is bound to the oligonucleotide. A protein chip in which complementary nucleotides are arranged and an arbitrary protein-PDZ domain protein-oligonucleotide is arranged.
(13) The PDZ domain protein and the oligonucleotide using the binding of the avidin label and the biotin label by binding the PDZ domain protein with an avidin label or biotin label and binding the biotin label or avidin label to the oligonucleotide; The protein chip according to (12), wherein
(14) The protein chip according to any one of (10) to (13), wherein the consensus sequence composed of S / TXV / L / I is a sequence composed of SLV.

  Here, the meaning of each letter and symbol of S / TXV / L / I is as follows: S is serine, T is threonine, V is valine, L is leucine, and I is isoleucine. , X is any amino acid selected from alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine Indicates. In addition,-(hyphen) indicates a peptide bond, and amino acids shown as options are separated by / (slash). That is, S / T indicates either serine or threonine.

According to the present invention, since the protein is fixed to the substrate through at least the PDZ domain, a change in the three-dimensional structure of the protein, loss of function, or a change caused by the protein directly binding to the substrate, Furthermore, since the functional site of the protein is prevented from being exposed to the substrate surface, a highly sensitive protein chip can be provided.
Further, when a protein containing a PDZ domain is immobilized on a substrate, there is a problem that the binding force between the PDZ domain and a consensus sequence composed of S / TXV / L / I is impaired. Therefore, by linking an affinity tag to a protein containing the PDZ domain, the PDZ domain can be stabilized and the binding force with the consensus sequence consisting of S / TXV / L / I can be maintained firmly. it can. The reason why the binding force between the PDZ domain and the consensus sequence is firmly maintained is not clear, but by binding to the affinity tag, the three-dimensional structure of the PDZ domain is stabilized, or the PDZ domain does not bind directly to the substrate. The reason is that the function is stabilized.
According to the method for producing a protein chip of the present invention, an affinity tag is linked to a protein having a PDZ domain, and the protein having the PDZ domain is purified using the affinity tag as an index. Can be fixed. If another protein having a consensus sequence consisting of S / TX-V / L / I is added, even if the purity of the other protein is low and a lot of contaminating proteins are contained, the PDZ domain And a consensus sequence consisting of S / TXV / L / I, only a protein having a consensus sequence consisting of S / TX-V / L / I is bound to the substrate via the PDZ domain. Since it is fixed, it is possible to prevent the contaminating protein from being fixed to the substrate. That is, a protein chip with low background noise can be produced at a low cost without requiring a complicated purification process. Furthermore, since it is only necessary to add an S / TXV / L / I consensus sequence consisting of only 3 amino acid residues to other proteins that react with the sample, the functions of other proteins that react with the sample. It is possible to manufacture a high-quality protein chip.
Furthermore, according to the present invention, the substrate comprises an oligonucleotide fixed substrate, an oligonucleotide having a sequence complementary to the oligonucleotide, a protein having a PDZ domain, and S / TXV / L / I. By preparing a complex of an arbitrary protein having a consensus sequence, by hybridizing an oligonucleotide on a substrate and an oligonucleotide of a complex complementary thereto, even in a laboratory not equipped with a special device, Arbitrary proteins can be arrayed at positions on any substrate.

It is explanatory drawing which shows typically that the orientation of the protein for analysis couple | bonded with PDZ domain protein is equal. It is a schematic diagram which shows the protein chip which fixed the protein which has the PDZ domain which connected the affinity tag to the board | substrate. A PDZ domain protein is bound on a substrate, an arbitrary antibody having an SLV amino acid sequence added to the heavy chain constant region C-terminus is bound to the PDZ domain protein, and the protein to be analyzed can be bound to the antibody. It is explanatory drawing which shows typically having improved the binding capability of the said antibody and the protein of analysis object by suppressing that the protein binding site of the said antibody contacts the said board | substrate. It is explanatory drawing which shows typically the protein chip | tip which arrange | positions SLV protein-PDZ domain protein-oligonucleotide. It is explanatory drawing which shows typically that the binding capability of the said antibody and the protein (antigen) to be analyzed fell because the antigen binding site of the antibody contacted the substrate. It is explanatory drawing which shows typically that the three-dimensional structure of the said protein raise | generates by arrange | positioning on a board | substrate, and the sensitivity with the molecule | numerator etc. which should be couple | bonded has fallen. It is a figure which shows the degradability of the protein which has a PDZ domain. It is a figure which shows the capture ability of ribosome protein-SLV in the cell lysate by the PDZ domain fixed to the plate. It is a figure which shows the capture ability of ribosome protein-SLV in the cell lysate by the PDZ domain fixed to the plate. It is a figure which shows the SLV specificity of the capture ability of GFP-SLV by the PDZ domain fixed to the plate. It is a figure which shows the capture of GFP-SLV via a biotinylated oligonucleotide.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Substrate 2 ... Protein to be analyzed 3 ... Antibody that binds to the protein to be analyzed 4 ... Molecules that bind to the protein to be analyzed 5 ... PDZ domain protein 5 '... Affinity tag 6. .. Peptide that is the sequence of SLV 6 '... Antibody having peptide that is the sequence of SLV at the C-terminus of heavy chain constant region 7... Avidin label 8.
10 ... Oligonucleotide 2 complementary to oligonucleotide 1
11 ... Substrate on which oligonucleotides are immobilized

The best mode for carrying out the present invention will be described below, but the present invention is not limited to this mode.
The protein chip of the present invention binds a protein having a PDZ domain as a capture agent for immobilizing an arbitrary protein on a substrate directly or via a nucleotide or the like on the substrate, and S / T on the C-terminus. -Arbitrary protein by which the arbitrary protein which the consensus sequence which consists of XV / L / I couple | bonds is couple | bonded via the said PDZ domain, and the said arbitrary protein arrange | positions directly on a board | substrate. The present invention provides a protein chip capable of suppressing the three-dimensional structure change of the above or aligning the orientation of the arbitrary protein on the substrate.
Furthermore, the protein chip of the present invention has the problem that, when a protein containing a PDZ domain is immobilized on a substrate, the binding force between the PDZ domain and a consensus sequence consisting of S / TXV / L / I is impaired. This is solved by linking an affinity tag to a protein having a domain to stabilize the PDZ domain.

Here, the PDZ domain used in the present invention is not particularly limited as long as it is a PDZ domain capable of binding to a consensus sequence consisting of S / TXV / L / I. For example, human FAP1 (Fas The PDZ domain of associated phosphatase-1) can be used. Human FAP1 protein has 6 PDZ domains, and among these, it is preferable to use the 3rd, 4th and 5th PDZ domains counted from the N-terminus, and more preferably to use the 3rd PDZ domain. Good.
The PDZ domain used in the protein chip of the present invention is a PDZ domain consisting of the amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence having 80% or more, more preferably 90% or more homology with the amino acid sequence. PDZ domains having can be used.
The homology of amino acid sequences in the present specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF).
In the present invention, the protein having a PDZ domain may be a protein in which another protein or peptide is fused to the PDZ domain, or a protein obtained by shortening the human FAP1 protein so as to include the PDZ domain. Also good. A protein containing a plurality of PDZ domains can also be used, but a protein having one PDZ domain is used in order to produce a protein chip that is difficult to degrade and has a certain binding ability for each array. It is preferable.

The consensus sequence consisting of S / TXV / L / I used in the present invention is a sequence consisting of 3 amino acids, where S is serine, T is threonine, V is valine, and L is leucine. And I represents isoleucine, respectively. X is an amino acid selected from alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine. Show. In addition,-(hyphen) indicates a peptide bond, and amino acids shown as options are separated by / (slash). That is, S / T indicates either serine or threonine. V / L / I is the C-terminal amino acid of the protein.
The C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR), but is a carboxyl group (—COOH). It is preferable.

Among the consensus sequences consisting of S / TXV / L / I, the sequence consisting of SLV is particularly preferable because it most strongly binds to the PDZ domain. Here, the amino acid sequence of SLV is serine-leucine-valine (COOH).
From the analysis of the Fas / FAP-1 interaction involved in the apoptosis signal transduction system, the present inventors have found that the 3 amino acids (SLV) at the carboxyl group (C) terminal of Fas bind tightly to the PDZ domain of FAP1. The protein-protein binding consensus motif (S / TXV / L / I-PDZ domain) was found (Non-patent Document 6: Yanagisawa, J. et al., The Journal of Biolgical Chemistry, 272, pp.8539-8545, 1997). At present, it is considered that there are about 500 types of proteins having a PDZ domain in the human genome, and among them, the binding property of the SLV-PDZ domain (FAP1) has been found to be the strongest. The probability that the same sequence as this 3 amino acid sequence is at the C-terminus of any protein is theoretically about 40 or less per about 300,000 total genome proteins (about 300,000 / 20 × 20 × 20). Therefore, by using this binding specificity, it is possible to construct an assay system for target drug screening of protein binding inhibitors as well as the development of next-generation protein chips.

  The affinity tag to be linked to the PDZ domain used in the present invention is not particularly limited as long as it is a substance having affinity with other substances, but a peptide tag having 20 amino acid residues or more is preferable, and GST (glutathione S-transferase) ), Avidin, maltose-binding peptide, biotinylated peptide, cellulose binding protein, Strep tag-containing peptide, thioredoxin and the like. A polypeptide in which two or more of these tags are fused can also be used as an affinity tag. Particularly preferred as the affinity tag used in the present invention is an affinity tag comprising a fusion protein of GST and avidin. Here, as avidin, it is particularly preferable to use streptavidin with little non-specific binding.

Examples of the substrate that can be used in the protein chip of the present invention include glass, metal, resin, fiber, quartz, semipermeable membrane, and a substrate using a biological material such as collagen or chitin.
If necessary, the surface of the substrate can be coated with a substance having affinity for a protein such as a nitrocellulose film, a PVDF film, or a three-dimensional polyacrylamide gel. In addition, the surface of the substrate can be modified with an aldehyde group, an epoxy group, or the like that reacts with a protein.
The shape of the substrate is not particularly limited, and may be a plate shape with a smooth surface, or a multi-well plate or a micro-well plate in which recesses are arranged vertically and horizontally. Also, it may be a special shape such as a sphere.

As a method of immobilizing a protein containing a PDZ domain on a substrate, there are a method of directly binding to a substrate and a method of binding via an oligonucleotide or the like.
First, a method for directly immobilizing a protein having a PDZ domain on a substrate is described below. This method includes immobilization by covalent bond and immobilization by non-covalent bond.
Examples of the immobilization by noncovalent bonding include, for example, nitrocellulose (Non-patent Document 1: Hui Ge, 2000, Nucleic Acid Research, vol. 28 (2), e3) or PVDF membrane (Non-patent Document 2: Lucy J. Holt). et al., 2000, Nucleic Acid Research, vol. 28 (15), e72) includes a physical adsorption method in which a protein containing a PDZ domain is spotted in the manner of a dot blot. In addition, there is a method in which a polyacrylamide pad having a thickness of 10 to 100 μm is bonded onto a slide glass and proteins are spotted on the pad (Non-patent Document 3: Dmitry Guschin, 1997, Analytical Biochemistry, vol. 250 (2 ), pp.203-211). Immobilization can also be carried out in the pores of HydroGel ^™ (Perkin Elmer) based on a three-dimensional polyacrylamide gel. Further, biotin / streptavidin or hexahistidine / nickel interaction using a modified protein can also be used as a biological binding method.

  Examples of the covalent immobilization include, for example, a method of utilizing reactivity between side chains of amino acids constituting a protein using aldehyde-modified glass (Non-patent Document 4: Gavin MacBeath, 2000, Science, vol. .289 (5485), pp.1760-1763) and the method of immobilizing proteins on the glass surface with an epoxy group. Also, on the surface of the slide glass, carbides such as hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide, tungsten carbide, zirconium carbide, or a mixture of these carbides with metals, ceramics, etc. The surface treatment layer comprising a laminate is formed by sputtering or the like, and the surface treatment layer is activated by immobilizing a group having an activated ester group bonded to the end of the hydrocarbon group via an amide bond. It is also possible to covalently bond the amino group at the protein end to this activated ester group (Patent Document 2: Japanese Patent Laid-Open No. 2002-082116, Patent Document 3: Japanese Patent Laid-Open No. 2003-014747).

 Other proteins having a consensus sequence consisting of S / TXV / L / I at the C-terminus used in the present invention are not particularly limited, and proteins such as transcription factors, signal transduction factors, receptors, and enzymes can be used. The protein chip can be produced by fixing to a substrate via the PDZ domain. As a method of using the protein chip, for example, the protein to be assayed is labeled with a radioisotope or a fluorescent dye, and the labeled protein is spotted on the protein chip, whereby the protein to be assayed and the protein on the protein chip are separated. Binding can be measured by radiation or fluorescence.

FIG. 1 is an explanatory view schematically showing a protein chip in which an arbitrary protein is bound to a PDZ domain protein immobilized on a substrate by the above method.
As shown in FIG. 1, on this protein chip, unlike the conventional protein chip shown in FIG. 6, since the three-dimensional structure of the bound protein is maintained and the orientation is aligned, a highly sensitive binding analysis is performed. Is possible.
FIG. 2 is an explanatory diagram schematically showing a protein chip in which an affinity tag is further linked to a protein having a PDZ domain in order to stabilize the PDZ domain. The reason why the binding force between the PDZ domain and the consensus sequence is firmly maintained when the affinity tag is linked is not clear, but the three-dimensional structure of the PDZ domain is stabilized by binding to the affinity tag, or the PDZ domain The reason is that the function is stabilized by not being directly coupled to the substrate.

In the protein chip of the present invention, an arbitrary antibody in which a PDZ domain protein is bound on a substrate and a consensus sequence consisting of S / TX-V / L / I is added to the heavy chain constant region C-terminus, By binding to the PDZ domain protein, a protein chip on which antibodies are arrayed can be produced.
If a protein chip to which this antibody is immobilized is used, for example, a substance contained in a sample can be detected and quantified by another type of antibody on the chip.

FIG. 3 shows that an arbitrary antibody in which a PDZ domain protein is bound on a substrate and a consensus sequence consisting of S / TXV / L / I is added to the heavy chain constant region C-terminus is added to the PDZ domain protein. It shows a protein chip that can be bound and can bind an arbitrary protein to the antibody. In this protein chip, the binding ability between the antibody and an arbitrary protein is improved by suppressing the protein binding site of the antibody from contacting the substrate.
As shown in FIG. 3, on this protein chip, unlike the conventional protein chip shown in FIG. 5, the binding ability between the antibody and an arbitrary protein does not decrease, so a highly sensitive binding analysis should be performed. Is possible.

Here, as a method of adding a consensus sequence consisting of S / TXV / L / I to the C-terminal of the heavy chain constant region of the antibody, the heavy chain constant region C-terminal is obtained by genetic engineering techniques in the antibody-producing cell. And a method using a protein production / modification technique using transgenic mice (including knock-in mice), and the like.
Here, instead of so-called immunoglobulin, an antibody comprising a polypeptide corresponding to a part of the antigen recognition site of the antibody may be used. This polypeptide is a polypeptide that can bind to a target substance, and can be obtained, for example, by screening using a phage display method.

As a method of immobilizing a protein containing a PDZ domain on a substrate, there is a method of binding via an oligonucleotide in addition to a method of immobilizing directly on a substrate.
That is, the present invention relates to a substrate on which an oligonucleotide is fixed, a consensus sequence comprising an oligonucleotide having a sequence complementary to the oligonucleotide, a protein having a PDZ domain, and S / TXV / L / I. A protein having a PDZ domain is immobilized on the substrate via the oligonucleotide, by preparing a complex of the other protein having a protein, and hybridizing the oligonucleotide on the substrate and the oligonucleotide of the complex. Provided is a protein chip in which another protein is immobilized through binding of a PDZ domain and a consensus sequence consisting of S / TXV / L / I.

FIG. 4 is a diagram schematically illustrating a method for producing a protein chip in which the above-described arbitrary SLV protein-PDZ domain protein-oligonucleotide is arranged.
Here, in order to bind an arbitrary oligonucleotide on the substrate, the same technique as the above-described protein immobilization method by covalent bond or non-covalent bond can be used. Further, the length of the oligonucleotide may be sufficient to bind with a complementary oligonucleotide, and there may be an error of several bases as long as it can be sufficiently bonded. Unlike the case where protein is placed directly on the substrate, there is already a technology to accumulate oligonucleotides on the substrate at a high density in the protein chip on which any SLV protein-PDZ domain protein-oligonucleotide is placed. Thus, there is an advantage that an arbitrary protein is accumulated on the substrate at a high density.

Hereinafter, the method for producing a protein chip of the present invention will be described in detail.
The method for producing a protein chip of the present invention includes the following steps.
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) a step of immobilizing the protein having the PDZ domain purified in the step (C) on a substrate;
(E) Adding the other protein obtained in the step (B) to the substrate on which the protein having the PDZ domain is immobilized in the step (D), and the protein having the PDZ domain and the other protein The process of combining.

In the steps (A) and (B), examples of a method for expressing a protein in a cell or synthesizing it enzymatically include the following methods. That is, as a method of obtaining a protein by expressing it in cells, the target protein is produced by transforming a host with an expression vector containing DNA encoding the target protein and culturing the resulting transformant. The method of doing is mentioned. As an enzymatic synthesis method, in vitro synthesis using a cell-free protein translation system comprising rabbit reticulocyte lysate, wheat germ lysate, Escherichia coli lysate, etc., using RNA corresponding to the DNA encoding the target protein as a template. The method of doing is mentioned.
In the above method, if DNA encoding a consensus sequence consisting of S / TXV / L / I is used for DNA encoding a protein of interest, S / TXV Other proteins having a consensus sequence consisting of / L / I can be easily obtained.

In the step (C), as a method for purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index, for example, beads having an affinity substance bound to the affinity tag Or the method of refine | purifying using a column is mentioned.
In the step (D), the method for immobilizing the protein having the PDZ domain on the substrate is as described in detail above.
In the step (E), in order to bind a protein having a PDZ domain and another protein, a consensus sequence comprising S / TXV / L / I is attached to a substrate on which the PDZ domain is fixed. A solution containing other protein having C at the terminal may be added, and impurities other than other protein may be mixed in the solution. As described above, in the production method of the present invention, it is not always necessary to purify other proteins, so that the cost can be reduced and only 3 amino acid sequences need be added to other proteins. It is possible to provide a high quality protein chip without impairing the function of the protein.

Next, a method for producing the protein chip of the present invention in which the PDZ domain is linked to the substrate via an oligonucleotide will be described. The manufacturing method includes the following steps.
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) immobilizing oligonucleotides on the substrate;
(E) The protein having the PDZ domain purified in the step (C) is bound to the other protein obtained in the step (B) via the PDZ domain, and further, the protein is passed through the affinity tag. A step of binding an oligonucleotide having a sequence complementary to the oligonucleotide to produce a protein oligonucleotide complex;
(F) A step of adding the protein oligonucleotide complex obtained by the step (E) to the substrate obtained by the step (D) and hybridizing the oligonucleotide on the substrate and the nucleotide of the complex. .

About the process of said (A)-(C), it can implement by the method mentioned above.
In the step (D), as a method for immobilizing the oligonucleotide on the substrate, a known method for producing a DNA array can be used. As a known method for producing a DNA array, there are a method in which DNA is immobilized while synthesizing probe DNA one base at a time on a substrate by photolithography technology or ink jet technology, and a method in which probe DNA synthesized separately in advance is mechanically used. Examples thereof include a method of immobilizing on a substrate by a micro spotting technique or an ink jet technique.
In the step (E), as a method for binding an oligonucleotide to a protein having a PDZ domain, for example, a method in which avidin is used as an affinity tag and a biotin-labeled oligonucleotide is linked via the avidin. Can be mentioned.
Examples of a method for binding biotin to an oligonucleotide include a method using an enzyme (Patent Document 12: International Publication No. 95/04069, Patent Document 4: Japanese Patent Application Laid-Open No. 2003-153698, Patent Document 6: Special). No. 2004-230545).

In one embodiment of the present invention, in the step (D), a plurality of oligonucleotides having different sequences are arranged on a substrate, and a complex is produced in the step (E). By using an oligonucleotide complementary to the oligonucleotide at this location, any protein can be immobilized at any location on the substrate.
The present invention also provides a kit comprising a substrate on which an oligonucleotide is immobilized and a protein having a PDZ domain to which an oligonucleotide having a sequence complementary to the oligonucleotide is bound. This kit makes it possible to produce a protein chip in which an arbitrary protein is arrayed at a position on an arbitrary substrate even in a laboratory without a special device.

  As mentioned above, although the specific embodiment was shown and demonstrated about the protein chip | tip of this invention, this invention is not limited to these. Those skilled in the art can make various modifications and improvements to the above-described embodiments of the protein chip without departing from the scope of the present invention.

Construction of PDZ domain protein and PDZ domain-streptavidin protein fusion production system
CDNA derived from FAP-1 (Non-patent Document 2: Sato, T., et al. Science 268, pp.411-415, 1995), HFAPD (FAP-1, 3,898-5,712 (start codon is at position +64)) Using the pGEX plasmid containing (SEQ ID NO: 2) as a template, a DNA fragment containing the PDZ domain was prepared by PCR. The streptavidin region (Stv) was prepared by PCR using Streptomyces avidinii genomic DNA (ATCCF # 27149) as a template. PDZ and Stv are once subcloned into pBluescript II SK (Stratagene) pSK [KpnI / XhoI / HindIII] -Stv-PDZ, and the prepared plasmid as a template pGEX-6P-1 [BamHI / SmaI] (Amersham Bio A PCR fragment for insertion into (Science) was prepared and tethered. The primers used for PCR are listed below.

pSK [KpnI / XhoI / HindIII] -Stv-PDZ production
Stv-PDZ Stv part (Forward): 5'-GGGGTACCACCATGGGCATCACCGGCACCTGG-3 '(SEQ ID NO: 3)
Stv-PDZ Stv part (Reverse): 5'-CCGCTCGAGAGCACCTTGGTGAAGGTGTC -3 (SEQ ID NO: 4)
PDZ part of Stv-PDZ (Forward): 5'-CCGCTCGAGAAGATGAATTTTAAAACT-3 '(SEQ ID NO: 5)
PDZ part of Stv-PDZ (Reverse): 5'-CCCAAGCTTATCATCACTGTGGGGTTACCGGGAC-3 '(SEQ ID NO: 6)
pGEX-6P-1 [BamHI / SmaI] -Stv-PDZ
(Forward): 5'-CGGGATCCGGCGAATTGGGTACCACCATG-3 '(SEQ ID NO: 7)
(Reverse: M13 reverse): 5′-GGAAACAGCTATGACCATG-3 ′ (SEQ ID NO: 8)

  E. coli BL21 transformed with the resulting plasmid was cultured (37 ° C, 3.5 hours, 0.1 mM IPTG added and then at 25 ° C for 4.5 hours), suspended in PBS, treated with lysozyme (final concentration 1 mg / mL), ultrasonic After crushing, Triton X-100 (final concentration 1%) was added and allowed to stand at 4 ° C. for 30 minutes, followed by centrifugation to prepare a supernatant as a cell lysate. Glutathione Sepharose 4B (Amersham Biosciences) was added to this and mixed overnight at 4 ° C., then the beads were precipitated by centrifugation, washed with PBS (5 times), 50 mM HEPES pH 7.4, 150 mM NaCl, 5 mM. Suspended in DTT, 10% Glycerol (50% slurry), this was used as glutathione beads to which each GST fusion protein was bound. A portion was suspended and heated in a sample buffer, fractionated by 10% SDS-PAGE, and the production of the fusion protein was confirmed by CBB staining (Quick-CBB, Wako Pure Chemical Industries, Ltd.). As shown in FIG. 7, GST-HFAPD was partially decomposed, but GST-Stv-PDZ had a relatively high molecular weight.

・ Construction of production system for human ribosomal protein with SLV sequence at C-terminus
An expression plasmid for cultured cells of human ribosomal protein was prepared as an arbitrary protein having an SLV sequence at the C-terminus. Human ribosomal protein-SLV is reverse-transcribed from Human Reference Total RNA (Stratagene) using a primer set that contains a restriction enzyme recognition site on the 5 'side and a codon for SLV, a termination codon, and a restriction enzyme recognition site on the 3' side. A PCR fragment was prepared using the enzymatically prepared cDNA as a template, and inserted into EcoRI-XhoI or BamHI-XhoI of the expression vector pCMV-Tag-2B (Stratagene). The primers used for PCR are listed below.

Sa
Forward: 5'-GGGAATTCACCATGTCCGGAGCCCTTGATG-3 '(SEQ ID NO: 9)
Reverse: 5'-CCGCTCGAGTCATTACACCAGAGACCAGTCAGTGGTTGCTC-3 '(SEQ ID NO: 10)
S4X
Forward: 5'-GGGAATTCACCATGGCTCGTGGTCCCAAG-3 '(SEQ ID NO: 11)
Reverse: 5'-CCGCTCGAGTCATCACACCAGAGACCCACTGCTCTGTTTGGCCG-3 '(SEQ ID NO: 12)
S12
Forward: 5'-CGGGATCCACCATGGCCGAGGAAGGCATTG-3 '(SEQ ID NO: 13)
Reverse: 5'-CCGCTCGAGTCATCACACCAGAGATTTCTTGCATTTGAAATACT-3 '(SEQ ID NO: 14)
S14
Forward: 5'-GGGAATTCACCATGGCACCTCGAAAGGGG-3 '(SEQ ID NO: 15)
Reverse: 5'-CCGCTCGAGTCATCACACCAGAGACAGACGGCGACCACGGCGAC-3 '(SEQ ID NO: 16)
S15
Forward: 5'-GGGAATTCACCATGGCAGAAGTAGAGCAG-3 '(SEQ ID NO: 17)
Reverse: 5'-CCGCTCGAGTCATTACACCAGAGACTTGAGAGGGATGAAGCGGG-3 '(SEQ ID NO: 18)
L5
Forward: 5'-GGGAATTCACCATGGGGTTGTTAAAGTTG-3 '(SEQ ID NO: 19)
Reverse: 5'-CCG CTCGAG TCATTACACCAGGCTCTCAGCAGCCCGCTCCT-3 '(SEQ ID NO : 20)
L9
Forward: 5'-GGGAATTCACCATGAAGACTATTCTCAGC-3 '(SEQ ID NO: 21)
Reverse: 5'-CCG CTCGAG TCATTACACCAGAGATTCATCAGCCTGCTGAACAG-3 '(SEQ ID NO : 22)
L13a
Forward: 5'-GG GAATTC ACCATGGCGGAGGTGCAGGTCC-3 '(SEQ ID NO: 23)
Reverse: 5'-CCG CTCGAG TCATCACACCAGAGAGACCAGGAGTCCGTGGGTCT-3 '(SEQ ID NO : 24)
L17
Forward: 5'-GG GAATTC ACCATGGTTCGCTATTCACTTG-3 '(SEQ ID NO: 25)
Reverse: 5'-CCGCTCGAGTCATAACACCAGAGACTCCCGTGCCATAAGTTTTT-3 '(SEQ ID NO: 26)
L18
Forward: 5'-GGGAATTCACCATGGGAGTGGACATCCGCC-3 '(SEQ ID NO: 27)
Reverse: 5'-CCGCTCGAGTCATTACACCAGAGAGTTTTTGTAGCCTCGGCTGG-3 '(SEQ ID NO: 28)
L28
Forward: 5'-GGGAATTCACCATGTCTGCGCATCTGCAATG-3 '(SEQ ID NO: 29)
Reverse: 5'-CCGCTCGAGTCATCACACCAGGGAGCTCTTGGTGGGGCGGG-3 '(SEQ ID NO: 30)
L32
Forward: 5'-GGGAATTCACCATGGCCGCCCTCAGAC-3 '(SEQ ID NO: 31)
Reverse: 5'-CCGCTCGAGTCACTACACCAGAGACTCATTTTCTTCACTGCGCA-3 '(SEQ ID NO: 32)

  The resulting human ribosomal protein-SLV expression plasmid was transfected into human kidney-derived 293T cells using FuGENE HD (Roche), and the recovered cells were 150m NaCl, 20 mM Tris- containing protease inhibitor cocktail (Roche). A cell lysate was prepared by suspending and centrifuging in HCl pH7.4, 1 mM EDTA, 1% Triton X-100. Each human ribosomal protein-SLV was fractionated by 15% SDS-PAGE, Western blotted, anti-FLAG antibody (Sigma, M2) recognizing a vector-derived FLAG peptide, and anti-mouse IgG-HRP as a secondary antibody. After immersion in (Bio-Rad), detection was performed with ECL. Expression was detected although there was a difference in expression depending on the type of ribosomal protein.

(2) Construction of GFP protein production system with SLV at C-terminus
The expression of GFP (Green Fluorescent Protein) was performed by using a plasmid pGLO (Bio-Rad) expressed in E. coli as a template and a PCR fragment encoding an amino acid sequence with SLV added to the C-terminus of GFP amplified with the following primers: , Treated with EcoRI and replaced with the XhoI-EcoRI fragment of pGLO.

Forward: 5'-ACAAACTCGAGTACAACTATA-3 '(SEQ ID NO: 33)
Reverse: 5'-CGAATTCATTACACCAGAGATTTGTAGAGCTCATCCAT-3 '(SEQ ID NO: 34)

  The GFP protein can be easily expressed by pGLO (expressing wild-type GFP) and the constructed plasmid (GFP-SLV) in Escherichia coli BL21 in an arabinose-containing medium and hydrophobic chromatography (Bio-Rad Macro-Prep HIC carrier). Purified. The obtained crude protein fluoresced both GFP and GFP-SLV.

Capture of Fas molecules in PDZ domain by pull-down assay
Suspend Jurkat cells expressing Fas molecules with SLV at the C-terminus in cell lysis buffer (150 mM NaCl, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% Triton X-100, protease inhibitor cocktail tablet [Roche]) After turbidity, the supernatant was prepared by centrifugation, the glutathione bead suspension prepared in Example 1 was added to the tube containing the cell lysate, and the tube was rotated and mixed at 4 ° C. overnight. The beads collected by centrifugation are suspended in the cell lysis buffer and centrifuged to repeat washing. Then, the tube containing the remaining buffer and beads is heated, and the solution is separated by 10% SDS-PAGE. After painting, it was transferred to a nitrocellulose membrane (Hybond C super, Amersham Biosciences). After blocking the filter (soaked in 5% skim milk or BSA), soaked in the primary antibody (anti-Fas antibody, sc-715, Santa Cruz) and secondary antibody labeled with HRP (anti-rabbit IgG, BD Farmingen) ) And then detected by ECL. Fas molecules were also confirmed in GST-HFTVD and GST-Stv-PDZ, and it was confirmed that Fas molecules were captured and pulled down in the PDZ domain.

Capture of ribosomal protein-SLV of PDZ domain by pull-down assay To add SLV to any protein and check whether protein-SLV is captured by PDZ, ribosomal protein is used as an example by PCR to ribosomal protein cDNA. SLV was added by amplifying with a primer having a sequence encoding SLV at the C-terminal, the PCR product was incorporated into an expression vector, expressed in cultured cells by transformation with the constructed plasmid, prepared as a crude protein, We investigated whether each ribosomal protein-SLV in the protein was captured by PDZ. The expression of each ribosomal protein to which SLV was added in cultured cells was confirmed in advance by Western blotting as shown in Example 2 (1). GST-Stv-PDZ beads or GST-PDZ (HFAPD) beads were added to the lysate of each cell whose expression was confirmed, pulled down, and Western blotted, and ribosomal protein-SLV on the filter was detected with an anti-FLAG antibody. As a result, binding between various ribosomal proteins-SLV and PDZ domain was confirmed.

Capability test of various protein-SLV using PDZ domain immobilized on microplate Glutathione beads bound with GST fusion protein prepared in Example 1 were eluted with 50mM Tris-HCl pH8.0, 10m reduced glutathione. The collected solution was dialyzed against PBS-T to prepare each GST fusion protein, and then 1 ug / 100 uL per well was added to a 96-well microplate and incubated overnight at 4 ° C. to solidify the GST fusion protein. After removing the solution, washing with PBS-T and blocking, an arbitrary protein having SLV at the C-terminal was added and fixed (at room temperature for 2 hours). For detection, after binding and washing the primary antibody that recognizes the captured antigen, binding and washing the HRP-labeled secondary antibody, coloring with ABTS Peroxidase Substrate (KPL), and stopping the reaction with 0.1% SDS Then, absorption at 405 nm was measured with a plate reader (infinite M200, Tecan Co.). For detection of GFP, anti-GFP antibody-HRP (Miltenyi Biotec) was used, and no secondary antibody was used.

(1) Capability of capturing ribosomal protein-SLV in cell lysate by PDZ domain immobilized on plate
Cell lysate prepared from E. coli BL21 expressing GST-Stv-PDZ was bound to glutathione beads, and eluted and dialyzed GST-Stv-PDZ was fixed to a 96-well microplate, and serially diluted ribosomal protein-SLV was added. The expressing 293T lysate was added. The captured ribosomal protein was detected with anti-FLAG antibody. As shown in FIG. 8, it was confirmed that the expression level was high and the ribosomal protein Sa-SLV in which a large amount of pull-down was observed was captured on the plate. In addition, it was confirmed that S14, S15, and L9-SLV were captured on the PDZ-immobilized plate even in the plate prepared by immobilizing GST-HFAPΔ prepared in the same manner (FIG. 9).

・ SLV specificity of capture ability of GFP-SLV by PDZ domain immobilized on plate
Cell lysate prepared from E. coli BL21 expressing GST-Stv-PDZ was bound to glutathione beads, the eluted GST-Stv-PDZ was fixed to a 96-well microplate, and purified GFP and GFP-SLV were serially diluted. In addition, the captured GFP was detected with an anti-GFP antibody. As shown in FIG. 10, although non-specific binding was observed with the addition of 100 ug of protein in the GST-immobilized plate, almost no GFP bound to the GST-Stv-PDZ plate, but specific in GFP-SLV. Binding was confirmed.

GST-Stv-PDZ biotin oligonucleotide-mediated immobilized polystyrene microplate with each well containing double-stranded DNA solution (50 ng of DNA encoding ribosomal proteins S8, S12, S30, 450 ng of ssDNA, 95 ° C 50 uL and 50 uL of immobilization buffer (1.5 N NaCl, 0.3 M Tris-HCl pH 7.5, 0.3 M MgCl ₂ ) were added and incubated at 37 ° C. overnight. After removal of the solution, air-dried for 30 minutes at 37 ° C., UV fixing (UV Stratalinker, Stratagene Inc.) after, 0.1 M NaCl to each well, 0.1M Tris-HCl pH9.3, 2mM MgCl 2, at 0.1% Tween20 Washed 3 times.
Add 100uL of prehybridization solution (6x SSPE, 0.05% Tween, 20mM EDTA, 5x Denhaltz, 100ng / uL ssDNA, 0.05% SDS) to each well, hold at 45 ° C for 30 minutes, and then soak in 5% BSA for 1 hour at room temperature did. GST-Stv-PDZ Oligonucleotide labeled with biotin at the 5 ′ end having S12 sequence in 50 ug (10 nmole):
5'-TTTTTTTACGTGGAATTCGCGAAGCTGCCAAAGCCTTAGACA-3 '(SEQ ID NO: 35)
2 nmole (20 uL of 100 uM solution) was mixed and allowed to stand at room temperature for 1 hour. Subsequently, 50 ug of GFP-SLV was added and allowed to stand at room temperature for 1 hour. , Deionized formamide, 100 ng / uL ssDNA, 0.05% SDS) to 300 uL, and 100 uL was dispensed into each well. After incubation at 45 ° C. for 3 hours, the plate was washed with 6 × SSPE, 0.05% Tween at the same temperature, and then washed with PBST, and anti-GFP antibody-HRP was bound and detected after the color reaction. As shown in FIG. 11, the high binding property in the well of S12 suggested that GFP-SLV was captured via a biotinylated oligo having the sequence of S12.

That is, the present invention provides the following protein chips (1) to ( 8 ), a production method thereof and a kit.
(1) Substrate;
A protein having a PDZ domain linked with an affinity tag comprising a fusion protein of GST and avidin ; and
Other proteins having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
Including
A protein having the PDZ domain binds to the substrate via the affinity tag;
A protein chip in which the other protein is bound to the PDZ domain via the consensus sequence.
(2) The substrate is a substrate having oligonucleotides immobilized on its surface,
An oligonucleotide having a sequence complementary to the oligonucleotide is bound to the affinity tag,
The protein chip according to (1), wherein the oligonucleotide and an oligonucleotide having a sequence complementary thereto are hybridized.
(3) The protein chip according to (1) or (2), wherein the PDZ domain is a PDZ domain of a human FAP1 protein.
(4) The protein chip according to any one of (1) to ( 3 ), wherein the consensus sequence is a sequence consisting of SLV.
(5) A method for producing a protein chip, which comprises the following steps:
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag composed of a fusion protein of GST and avidin is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) a step of immobilizing the protein having the PDZ domain purified in the step (C) on a substrate;
(E) Adding the other protein obtained in the step (B) to the substrate on which the protein having the PDZ domain is immobilized in the step (D), and the protein having the PDZ domain and the other protein The process of combining.
(6) A method for producing a protein chip, comprising the following steps.
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag composed of a fusion protein of GST and avidin is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) immobilizing oligonucleotides on the substrate;
(E) The protein having the PDZ domain purified in the step (C) is bound to the other protein obtained in the step (B) via the PDZ domain, and further, the protein is passed through the affinity tag. A step of binding an oligonucleotide having a sequence complementary to the oligonucleotide to produce a protein oligonucleotide complex;
(F) A step of adding the protein oligonucleotide complex obtained by the step (E) to the substrate obtained by the step (D) and hybridizing the oligonucleotide on the substrate and the nucleotide of the complex. .
(7) The method for producing a protein chip according to (6) ,
In the step (B), the other protein is a plurality of other proteins,
In the step (D), the oligonucleotide is a plurality of types of oligonucleotides having different sequences,
In the step (E), the type of nucleotide to be bound via an affinity tag is varied depending on the type of other protein to be bound to the PDZ domain.
(8) A kit comprising a substrate having an oligonucleotide immobilized thereon and a protein having a PDZ domain in which an oligonucleotide having a sequence complementary to the oligonucleotide is bound via an affinity tag comprising a fusion protein of GST and avidin .

Claims (14)

substrate;
A protein having a PDZ domain linked to an affinity tag; and
Other proteins having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
Including
A protein chip in which the other protein is bound to the PDZ domain via the consensus sequence. The substrate is a substrate having oligonucleotides immobilized on its surface;
An oligonucleotide having a sequence complementary to the oligonucleotide is bound to the affinity tag,
The protein chip according to claim 1, wherein the oligonucleotide and an oligonucleotide having a sequence complementary thereto are hybridized.   The protein chip according to claim 1 or 2, wherein the affinity tag is an affinity tag comprising a fusion protein of GST and avidin.   The protein chip according to any one of claims 1 to 3, wherein the PDZ domain is a PDZ domain of a human FAP1 protein.   The protein chip according to any one of claims 1 to 4, wherein the consensus sequence is a sequence consisting of SLV. A method for producing a protein chip comprising the following steps:
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) a step of immobilizing the protein having the PDZ domain purified in the step (C) on a substrate;
(E) Adding the other protein obtained in the step (B) to the substrate on which the protein having the PDZ domain is immobilized in the step (D), and the protein having the PDZ domain and the other protein The process of combining. A method for producing a protein chip, comprising the following steps.
(A) a step of expressing or enzymatically synthesizing a protein having a PDZ domain to which an affinity tag is linked;
(B) a step of expressing or enzymatically synthesizing another protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus;
(C) a step of purifying the protein having the PDZ domain obtained in the step (A) using an affinity tag as an index;
(D) immobilizing oligonucleotides on the substrate;
(E) The protein having the PDZ domain purified in the step (C) is bound to the other protein obtained in the step (B) via the PDZ domain, and further, the protein is passed through the affinity tag. A step of binding an oligonucleotide having a sequence complementary to the oligonucleotide to produce a protein oligonucleotide complex;
(F) A step of adding the protein oligonucleotide complex obtained by the step (E) to the substrate obtained by the step (D) and hybridizing the oligonucleotide on the substrate and the nucleotide of the complex. ; A method for producing a protein chip according to claim 7,
In the step (B), the other protein is a plurality of other proteins,
In the step (D), the oligonucleotide is a plurality of types of oligonucleotides having different sequences,
The method for producing a protein chip, wherein in the step (E), the type of nucleotide to be bound through the affinity tag is varied depending on the type of other protein to be bound to the PDZ domain.   A kit comprising a substrate on which an oligonucleotide is immobilized, and a protein having a PDZ domain to which an oligonucleotide having a sequence complementary to the oligonucleotide is bound.   A PDZ domain protein is bound on a substrate, and an arbitrary protein having a consensus sequence consisting of S / TXV / L / I can be bound to the C-terminal of the protein that binds to the protein. A protein chip in which a PDZ domain protein is bound on a substrate, which suppresses a change in the three-dimensional structure of the protein due to the arrangement on the substrate or aligns the orientation of the protein.   A PDZ domain protein is bound on a substrate, and an arbitrary antibody having a consensus sequence consisting of S / TX-V / L / I added to the heavy chain constant region C-terminus is bound to the PDZ domain protein. An antibody capable of binding an arbitrary protein and binding ability of the antibody to an arbitrary protein is improved by inhibiting the protein binding site of the antibody from contacting the substrate. A protein chip in which an arbitrary antibody to which a consensus sequence consisting of S / TXV / L / I is added to the C-terminus of the chain constant region is bound to a PDZ domain protein, and the complex is bound to a substrate.   A PDZ domain protein linked to any oligonucleotide is bound to any protein having a consensus sequence consisting of S / TXV / L / I at the C-terminus of the protein, and complementary to the oligonucleotide. A protein array on which nucleotides are arranged and an arbitrary protein-PDZ domain protein-oligonucleotide is arranged.   By binding the PDZ domain protein with an avidin label or biotin label, and binding the biotin label or avidin label to the oligonucleotide, the PDZ domain protein utilizing the binding of the avidin label and biotin label is bound to the oligonucleotide. The protein chip according to claim 12.   The protein chip according to any one of claims 10 to 13, wherein the consensus sequence composed of S / TXV / L / I is a sequence composed of SLV.