JPWO2007000972A1 - New uses as tags - Google Patents

Abstract

An object of the present invention is to provide a novel tag modified to a target protein. In addition, a means for capturing a target protein using such a tag is provided. Specifically, we investigated a novel protein tag based on the knowledge of the molecular mechanism that a DNA-binding protein has a bond with DNA. As a result, DNA-binding proteins, especially DNA-binding transcription factors or DNA-binding nuclear receptor amino acid sequences that have the ability to bind to DNA, can be used as tags for target proteins. We have found that it is possible to purify or detect, as well as label, and have completed the present invention.

Description

The present invention relates to a novel use as a tag, and more particularly to a novel tag for a protein comprising an amino acid sequence having the DNA binding ability of a DNA-binding protein (DNA-binding transcription factor, DNA-binding nuclear receptor). Use of such tags allowed for protein purification, immobilization, capture, detection, labeling, and together provided a novel system for protein chip reagents.
This application claims priority from Japanese Patent Application Nos. 2005-187468 and 2005-286956, which is incorporated herein by reference.

About transcription factors Proteins that compose cells are synthesized through the process of gene (DNA) → mRNA → protein. In particular, the step of synthesizing mRNA from DNA is called transcription reaction or gene expression, and is an important reaction that regulates the amount of protein that is a gene product, and thus controls the amount of protein that is involved in the function and structure of cells. Will be. This transcription reaction is caused by a protein having an affinity for a group of DNAs called transcription factors. For example, RNA polymerase known as a basic transcription factor is a transcription control region called a promoter composed of a TATA box and a transcription initiation site. It binds to DNA having a specific base sequence, forms a transcription complex in which various transcription factors are assembled by incorporating RNA polymerase and initiates mRNA synthesis. A transcription reaction that depends on a promoter has no gene specificity and is called basic transcription.
DNA having a specific base sequence of the transcriptional regulatory region that controls the basic transcription is called a transcriptional regulatory region (sometimes called confused with an enhancer or silencer), and usually exists upstream of the promoter. A transcriptional regulator that binds to this region (or gene-specific transcription factor) interacts with a basic transcription factor either directly or through another transcriptional regulatory cofactor (also called a mediator, cofactor, or mediator) Basic transcription is adjusted.

Nuclear receptors Fat-soluble substances such as fat-soluble vitamins (vitamin A, vitamin D), thyroid hormones, and steroid hormones freely pass through cell membranes and bind to nuclear receptors. The receptor is divided into a hypervariable region, a DNA-binding region, and a hormone-binding region that regulate transcription by the N-terminus, and has a similar structure and forms a superfamily. The DNA-binding region has a zinc finger structure and binds to elements with AGGTCA as the basic motif to promote gene transcription.

On the other hand, proteomic analysis, which analyzes all proteins expressed in vivo, is broadly divided into expression proteomics and functional (interaction) proteomics.
Expression proteomics is a method to comprehensively analyze where and how much a specific protein is expressed in the living body, and functional proteomics is a comprehensive analysis of what molecule a specific protein interacts with. It is a technique to analyze. Conventionally, these analyzes have been performed by two-dimensional electrophoresis, yeast two-hybrid method, surface plasmon method, phage display method, etc., but there are problems such as analysis time, sensitivity, false positives, and the amount of data required for analysis. Development of protein chips that integrate proteins at high density is underway.

  In addition, as a protein chip, serum or the like is brought into contact with an antibody chip, and an antigen-antibody reaction occurring on the chip is detected by a solid-phase enzyme immunoassay (hereinafter sometimes referred to as “ELISA method”). SELDI (Surface Enhanced Laser Desorption / Ionization) protein chip that combines a protein-immobilized chip and a MALDI-TOF MS (matrix-assisted ionization-time-of-flight mass spectrometer: (Non-patent Document 1, Non-patent Document 2)) Systems etc. are being developed.

  In addition, great efforts are currently being made to realize a protein chip. (1) Influence of background noise derived from non-specific binding, (2) Influence of protein solidification on the three-dimensional structure of the protein Because of this, it has not been realized.

  On the other hand, in order to efficiently obtain various proteins required in research or medicine, a cell-free protein synthesis means is attracting attention today. Rabbit reticulocyte extract (Reticulocyte Lysate) was often used for this method. However, recently, based on the elucidation of the destabilization mechanism of wheat germ cell-free system (Wheat Germ Extract), a method of preparing a wheat germ extract having a stable and high translational activity and using the wheat germ extract was used. A high-efficiency cell-free protein synthesis system has been provided and used for various protein synthesis (Non-patent Document 3) (Patent Documents 1 to 3).

  In the wheat germ cell-free protein synthesis system, many hydrophilic proteins involved in protein synthesis are present in the extract. Proteins synthesized in the wheat germ cell-free protein synthesis system undergo the process of synthesizing expressed proteins in an environment mixed with these. Therefore, when purifying an expressed protein, the conventional method uses a column or bead that is synthesized with a known tag (His × 6, GST, etc.) fused and specifically adsorbed to the tag. It was considered effective to carry out the purification. However, some known purification methods using tags have a problem in that some proteins present in the wheat germ extract remain after purification. This is because the protein is derived from germs with the same adsorptivity as each tag, the protein is derived from germs that are adsorbable to the resin and beads of the carrier column, and the buffer is used for purification. It is thought to be caused by germ-derived proteins that wake up. In addition, when proteins are eluted using a high-concentration similar substrate as in the prior art, depending on the reaction conditions, it was necessary to remove those similar substrates from the solution after elution. However, it is not easy to remove them, and a very complicated operation is required.

  In addition, conventional protein labels have used chemical modifications using amino groups that have an irreversible effect on the three-dimensional structure of proteins, radioisotopes, fluorescent proteins, high affinity proteins such as streptavidin, and the like. However, since chemical modification often cannot take its original structure and activity due to irreversible influence on the three-dimensional structure of the protein, it could hardly be used for functional analysis. Radioisotopes require special facilities, and fluorescent proteins have low fluorescence intensity per molecule, and are limited to use only in experimental systems using special equipment. Utilization has caused a decrease in affinity due to modification with other compounds, making free labeling difficult.

  As described above, 1) a tag that can easily capture or label the target protein, and 2) a tag that can be captured in the well of the chip so as not to irreversibly affect the three-dimensional structure of the protein. There was a request for development.

JP 2000-236896 JP Japanese Patent Laid-Open No. 2002-125693 JP 2002-204689 JP Koster, H., et al., Nature Biotechnol., 14, 1123-1128 (1996) Griffin, T. J., et al., Nature Biotechnol., 15, 1368-1372 (1997) Proc. Natl. Acad. Sci. USA, 99: 14652-14657 (2002)

  Many non-specific adsorptive proteins are bound through ionic forces, and therefore can be removed or reduced by washing with a buffer containing an optimum salt. If a tag capable of binding to a specific substance can be developed even at a high salt concentration, the target protein can be preferentially captured and immobilized by washing with a simple salt. Since these methods can be developed to immobilize / capture target proteins with purification processes such as practical protein chip development in proteomics analysis, they can be very important basic technologies. Therefore, an object of the present invention is to provide a novel tag that is modified (expressed together) with a target protein. In addition, a means for purifying, capturing, immobilizing, detecting or labeling the target protein using such a tag is provided.

  The present inventor examined a novel protein tag based on the knowledge of the molecular mechanism that a DNA-binding protein, particularly a transcription factor or a nuclear receptor has a bond with DNA. As a result, the synthesized protein can be easily purified, fixed, captured, detected or labeled by using the DNA binding ability of DNA-binding transcription factor or DNA-binding nuclear receptor as the target protein tag. Furthermore, the inventors have found that a protein chip that can be put into practical use can be produced, and have completed the present invention.

In other words, the present invention
“1. Use as a tag for a target protein based on an amino acid sequence expressed together with the target protein (hereinafter referred to as a tag site).
2. 2. The use according to item 1 above, wherein the tag site is a DNA-binding protein comprising an amino acid sequence having DNA binding ability.
3. 3. The use according to item 2 above, wherein the DNA-binding protein is a DNA-binding transcription factor or a DNA-binding nuclear receptor.
4). 4. The use according to item 2 or 3, wherein the DNA-binding protein is selected from any one of the following.
(1) the sequence of SEQ ID NO: 2, 4 or 6;
(2) a sequence having at least about 80% or more amino acid sequence homology with the sequence of SEQ ID NO: 2, 4 or 6;
(3) A sequence having a DNA binding ability, having a mutation such as deletion, substitution, addition or insertion of one to several amino acids with the sequence of (1) above. 2. The use according to item 1, wherein the relationship between the tag site and the tag affinity substance is selected from one or more of the following.
1) Biotin-binding protein / biotin of avidin or streptavidin, 2) Maltose-binding protein / maltose, 3) G protein / guanine nucleotide, 4) DNA-binding protein / DNA, 5) Antigen molecule (epitope) / antibody, 6) 5. Calmodulin binding peptide / calmodulin, 7) ATP binding protein / ATP, 8) Estradiol receptor protein / estradiol The use according to any one of 1 to 5 above, wherein an endopeptidase-recognizing amino acid sequence is added to the tag.
7). A protein capturing method for capturing a fusion protein using the tag according to any one of 1 to 6 above.
8). 8. The method according to item 7 above, wherein the capture is performed by immobilized DNA.
9. 9. The method according to item 7 or 8 above, wherein the capturing is for purification or detection of the fusion protein.
10. 7. A protein labeling method in which a labeling substance is immobilized on a fusion protein using the tag according to any one of items 1 to 6.
11. 11. The method according to any one of 7 to 10 above, wherein the fusion protein is prepared by a cell-free protein synthesis means.
12 12. The method according to item 11 above, wherein the cell-free protein synthesis means uses a wheat germ extract.
13. A protein purification kit comprising the tag according to any one of items 1 to 6 as a fusion protein purification tag.
14 A protein detection kit comprising the tag according to any one of items 1 to 6 as a tag for detecting a fusion protein.
15. A protein labeling kit comprising the tag according to any one of items 1 to 6 as a fusion protein labeling tag.
16. A protein chip reagent using a protein synthesis system, comprising the following elements.
a: the fusion protein is expressed in the well of a container partitioned into one or more regions,
b. A substance having affinity with the tag site of the fusion protein (tag affinity substance) is immobilized on the inner surface of the well and / or the carrier in the well,
c. The fusion protein is captured on the inner surface of the well and / or the carrier in the well through the affinity between the tag site of the fusion protein and the tag affinity substance.
17. The protein chip reagent according to 16 above, wherein two or more types of fusion proteins are expressed in the wells of a container partitioned into one or a plurality of regions.
18. 18. The protein chip reagent according to 16 or 17 above, wherein the relationship between the tag site of the fusion protein and the tag affinity substance is selected from any one or more of the following.
1) Biotin binding protein / biotin of avidin or streptavidin, 2) maltose binding protein / maltose, 3) G protein / guanine nucleotide, 4) polyhistidine peptide / metal ion such as nickel or cobalt, 5) glutathione-S-transferase / Glutathione, 6) DNA binding protein / DNA, 7) antigen molecule (epitope) / antibody, 8) calmodulin binding peptide / calmodulin, 9) ATP binding protein / ATP, 10) estradiol receptor protein / estradiol 19. The protein chip reagent according to any one of items 16 to 18, wherein the fusion protein is expressed in a cell-free protein synthesis system.
20. 20. The protein chip reagent according to item 19, wherein the cell-free protein synthesis system is a cell-free protein synthesis system derived from a wheat germ extract.
21. 21. A protein synthesis kit comprising the protein chip reagent according to any one of 16 to 20 above.
22. 21. A method for examining a substance interacting with a fusion protein comprising the following elements, using the protein chip reagent according to any one of 16 to 20 above.
(1) A detection target substance is added to a well in which the fusion protein is captured, and the presence or absence of an interaction with the fusion protein is confirmed.
(2) Qualitatively or quantitatively determine the interacting substances using markers. "
Consists of.

  The present invention can capture a fusion protein by using a DNA-binding protein, particularly a DNA-binding transcription factor or a DNA-binding nuclear receptor as a novel tag, and can be used for purification, fixation, detection or labeling of a target protein. And enabled new developments as protein tags.

The tag of the present invention is an amino acid sequence (hereinafter sometimes referred to as tag site) that is expressed together with the target protein. The amino acid sequence is a protein shown below or a specific amino sequence (specifically, a repetitive sequence of a certain amino acid). Moreover, the tag part which is this amino acid sequence needs to have affinity with the tag affinity substance shown below.
The fusion protein of the present invention can have a linker, an endopeptidase-recognizing amino acid sequence and the like in addition to a tag and a target protein.

DNA-binding protein used in the tag of the present invention The DNA-binding protein used in the tag of the present invention includes any amino acid sequence that has a DNA binding ability and can bind to DNA non-specifically. Although possible, transcription factors or nuclear receptors are preferably exemplified. A description will be given below with a suitable specific example.

HY5
Nucleic acid sequence (SEQ ID NO: 1)
ATGCAGGAACAAGCGACTAGCTCTTTAGCTGCAAGCTCTTTACCATCAAGCAGCGAGAGGTCATCAAGCTCTGCTCCACATTTGGAGATCAAAGAAGGAATTGAAAGCGATGAGGAGATACGGCGAGTGCCGGAGTTTGGAGGAGAAGCTGTCGGAAAAGAAACTTCCGGTAGAGAATCTGGATCGGCGACCGGTCAGGAGCGGACACAGGCGACTGTCGGAGAAAGTCAAAGGAAGCGAGGGAGGACACCGGCGGAGAAAGAGAACAAGCGGCTGAAGAGGTTGTTGAGGAACAGAGTTTCAGCTCAGCAAGCAAGAGAGAGGAAAAAGGCTTACTTGAGCGAGTTGGAAAACAGAGTGAAAGACTTGGAGAACAAAAACTCTGAACTTGAAGAGCGACTCTCTACTCTTCAGAACGAGAACCAGATGCTTAGACATATTCTGAAGAACACAACAGGAAACAAGAGAGGAGGTGGTGGTGGTTCTAATGCTGATGCAAGCCTTTGA
Amino acid sequence (SEQ ID NO: 2)
MQEQATSSLAASSLPSSSERSSSSAPHLEIKEGIESDEEIRRVPEFGGEAVGKETSGRESGSATGQERTQATVGESQRKRGRTPAEKENKRLKRLLRNRVSAQQARERKKAYLSELENRVKDLENKNSELEERLSTLQNENQMLRHILKNTTGNKRGGGGGSNADASL

FTZ-F1 (SF-1, NR5A1)
Nucleic acid sequence (SEQ ID NO: 3)

Amino acid sequence (SEQ ID NO: 4)
MDYSYDEDLDELCPVCGDKVSGYHYGLLTCESCKGFFKRTVQNNKHYTCTESQSCKIDKTQRKRCPFCRFQKCLTVGMRLEAVRADRMRGGRNKFGPMYKRDRALKQQKKAQIRANGFKLETGPPMGVPPPPPPAPDYVLPPSLHGPEPKGLAAGPPAGPLGDFGAPALPMAVPGAHGPLAGYLYPAFPGRAIKSEYPEPYASPPQPGLPYGYPEPFSGGPNVPELILQLLQLEPDEDQVRARILGCLQEPTKSRPDQPAAFGLLCRMADQTFISIVDWARRCMVFKELEVADQMTLLQNCWSELLVFDHIYRQVQHGKEGSILLVTGQEVELTTVATQAGSLLHSLVLRAQELVLQLLALQLDRQEFVCLKFIILFSLDLKFLNNHILVKDAQEKANAALLDYTLCHYPHCGDKFQQLLLCLVEVRALSMQAKEYLYHKHLGNGMPRNNLLIEMLQAKQT

FTZ-F1β (NR5A2)
Nucleic acid sequence (SEQ ID NO: 5)

Amino acid sequence (SEQ ID NO: 6)
MLPKVETEALGLARSHGEQGQMPENMQVSQFKMVNYSYDEDLEELCPVCGDKVSGYHYGLLTCESCKGFFKRTVQNNKRYTCIENQNCQIDKTQRKRCPYCRFQKCLSVGMKLEAVRADRMRGGRNKFGPMYKRDRALKQQKKALIRANGLKLEAMSQVIQAMPSDLTISSAIQNIHSASKGLPLNHAALPPTDYDRSPFVTSPISMTMPPHGSLQGYQTYGHFPSRAIKSEYPDPYTSSPESIMGYSYMDSYQTSSPAGIPHLILELLKCEPDEPQVQAKIMAYLQQEQANRSKHEKLSTFGLMCKMADQTLFSIVEWARSSIFFRELKVDDQMKLLQNCWSELLILDHIYRQVVHGKEGSIFLVTGQQVDYSIIASQAGATLNNLMSHAQELVAKLRSLQFDQREFVCLKFLVLFSLDVKNLENFQLVEGVQEQVNAALLDYTMCNYPQQTEKFGQLLLRLPEIRAISMQAEEYLYYKHLNGDVPYNNLLIEMLHAKRA

PPAR (peroxisome proliferator-activated receptor)
It is a nuclear receptor belonging to the steroid hormone receptor family, and is involved in the development of lifestyle-related diseases, such as endocrine, sugar and lipid metabolism related to diabetes, as well as various circulatory and carcinogenic mechanisms such as vascular function and inflammation It is known as a transcription factor that regulates the expression of target genes.

p53
Following the RB gene, it is the second tumor suppressor gene identified in 1989. The p53 gene is located on chromosome 17p13.1, and its gene product is a nuclear protein with a molecular weight of 53 kD. Initially, the p53 gene was thought to be an oncogene similar to myc, but then the p53 protein has wild type and mutant type, and the wild type has a function to control cell growth function, and the RB gene It became clear that it is a tumor suppressor gene with a similar function.

NFκB
Controls gene expression of cytokines (eg, IL-1, IL-2, IL-6, IL-8, GM-CSF, TNF, etc.), chemokines, interferons, MHC molecules, growth factors, cell adhesion molecules, etc. It is known as a disease-related transcriptional regulator that plays an important role in the immune system. For example, it is attracting attention as a target molecule for therapeutic agents such as immune diseases such as rheumatoid arthritis and osteoarthritis, inflammatory diseases, and cancer. Yes.

AP-1
It is a complex consisting of Fos and Jun family of transcriptional regulators and is known to be involved in cell proliferation and differentiation, especially cell canceration and inflammatory diseases. For example, therapeutic agents for inflammatory diseases and cancer It is attracting attention as a target molecule.

HIF-1
It is a gene transcription factor activated by hypoxia and has been shown to be closely involved in the control of angiogenesis through the control of gene expression of vascular endothelial growth factor and the like. NFκB regulates gene expression of cytokines (IL-1, IL-2, IL-6, IL-8, GM-CSF, TNF), chemokines, interferons, MHC molecules, growth factors, cell adhesion molecules, etc. It is known as a transcription factor that plays an important role in the immune system.

CREB (Cyclic AMP Respones Element Binding protein)
It is known as a disease-related transcriptional regulator that plays an important role in cell growth, differentiation, learning and memory processes, nervous system adaptation to drug addiction, and metabolic pathway control such as gluconeogenesis by insulin and glucagon. It is attracting attention as a target molecule for therapeutic drugs for central diseases such as memory impairment, circulatory diseases, and diabetes.

Smad
TGFβ is a transcription factor that plays a central role in intracellular signal transduction of the TGFβ superfamily and is involved in cell growth, differentiation, extracellular matrix formation, and cell death, and is particularly involved in diseases such as cell canceration and tissue fibrosis Attention has been paid. Smads are classified into three types, called R-Smad (Receptor-regulated Smad), Co-Smad (Common-mediator Smad) and I-Smad (Inhibitory Smad).

In addition, examples of DNA-binding proteins that can be used in the tag of the present invention, particularly transcription factors or nuclear receptors are listed below, but the transcription factors or nuclear receptors that can be used in the present invention are limited to these. is not.
selectivity factor 1 (SL1: TAFI110, TAFI63, TAFI48), upstream binding factor (UBF), TATA-binding protein (TBP), TFIID / TAF250, TFIID / TAF150, TFIID / TAF130 (135), TFIID / TAF105, TFIID / TAF100 , TFIID / TAF80 (70), TFIID / TAF68, TFIID / TAF55, TFIID / TAF31 (32), TFIID / TAF30, TFIID / TAF28, TFIID / TAF20 (15), TFIID / TAF18, TFIIAα / β, TFIIAγ, TFIIB, TFIIEα, TFIIEβ, TFIIF / RAP30, TFIIF / RAP74, ERCC3, ERCC2, p62, p52, p44, MO15, MAT1, p34, TFIIIB 90 / BRF, GCN5, Srb, C / EBP, 1-Oct, ACF, Gcn5, PCAF (p300 / CBP-associated factor), SRC-1 (steroid receptor coactivator 1), TAFII250, CBP, p300, MBF1α / EDF-1, MBF1β, Sp1, BTEB1, BTEB2, EKLF, E2F-1, E2F-2, E2F -3, E2F-4, E2F-5, E2F-6, DP-1, DP-2, NFI / CTF-1, E1A (E1AF), CREB-H, ATF-1, ATF-2, ATF-3, ATF-4, ATF-5, ATF-6, ATF-7, C / EBPα, C / EBPβ, C / EBPγ, C / EBPδ, C / EBPε, CHOP, HSF1, HSF2, HSF4, IRF-1, IRF- 2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, ICSBP / IRF-8, p48 / IRF-9, GRα, GRβ, ERα, ERβ, VDR Ah receptor, Arnt, Nrf2, SREBP-1c, SREBP-2, STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, STAT6, GATA-1, GATA-2, GATA-3, GATA-4, GATA-5, Ikaros , MyoD1, HES1, p45 (NF-E2), MAFK, TCF-1, TCF-4, LEF-1, NeuroD, Ad4BP / SF-1, MITF, TFE3, TFEB, TFEC, RARα, RARβ, RARγ, RXRα, RXRβ, Hoxb-1, EGR1, EGR2, EGR3, NGFI-C, Gli, Gli3, gsc, gscl, Pax-6, Brachyury, SRY, c-maf, mafB, NRL, mafK, mafF, mafG, Pit-1 / GHF-1, PROP1, c-myc, N-myc, L-myc, Fos, fra-1, fra-2, c-Jun, JunB, JunD, c-myb, PEBP2α, PEBP2B / AML-1, PEBP2αC, Rb, p53, NF-κB2, NF-κB1, WT1, Smad1, Smad2, Smad3, Smad4, Smad5, Smad6, Smad7, Smad8, Smad9, Nurr1, Nor1.

The amino acid sequence of the transcription factor or nuclear receptor used in the present invention is about 30%, preferably about 50%, more preferably about 70%, most preferably the amino acid sequence shown in SEQ ID NO: 2, 4 or 6. May be an amino acid sequence consisting of about 80%, more preferably about 95% homologous sequences. Furthermore, a sequence having a mutation such as deletion, substitution, addition or insertion of 1 to several amino acids in the amino acid sequence shown in SEQ ID NO: 2, 4 or 6 and DNA binding ability is also suitable.
Deletion, substitution, addition or insertion means are known per se, for example, site-directed mutagenesis, gene homologous recombination, primer extension or polymerase chain amplification (PCR), alone or in combination, for example, Edited by Sambrook et al. [Molecular Cloning, Laboratory Manual Second Edition] Cold Spring Harbor Laboratory, 1989, edited by Masami Muramatsu [Lab Manual Genetic Engineering] Maruzen Co., Ltd., 1988, Ehrlich, HE. Chapter [PCR Technology, Principles and Applications of DNA Amplification] It can be carried out according to the methods described in Stockton Press, 1989, etc., or by modifying those methods. For example, Ulmer's technology (Science, 219, 666, 1983).
In the introduction of mutations as described above, from the viewpoint of carrying DNA binding ability, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids) Etc.) are easily assumed.

The transcription factor or nuclear receptor that can be used in the present invention is not particularly limited as long as it has a DNA binding site. However, the length of the base sequence is advantageously shorter than that in genetic manipulation. . Suitably, the length of the amino acid sequence is 3 to 300, more preferably 3 to 200, and still more preferably 3 to 150.
In addition, if desired, for example, an endopeptidase recognition amino acid sequence can be added between the target protein sequence and the tag sequence in order to separate the target protein and the tag after achieving the purpose.

In addition, the tag of the present invention is not limited to those derived from a transcription factor or a nuclear receptor containing the amino acid sequence having the DNA binding ability described above. In the tag of the present invention, the tag part of the fusion protein only needs to have affinity with a substance having affinity with the tag part. Here, the substance having affinity with the tag site (referred to as tag affinity substance) is selected from any one or more of the following, based on the affinity relationship with the tag.
Avidin or streptavidin biotin-binding protein (tag, same below) / biotin (tag affinity substance, same below), maltose binding protein / maltose, G protein / guanine nucleotide, DNA binding protein / DNA, antigen molecule (epitope) / Antibodies, calmodulin binding peptide / calmodulin, ATP binding protein / ATP, estradiol receptor protein / estradiol.
Further, polyhistidine peptide / metal ions such as nickel or cobalt, glutathione-S-transferase / glutathione are not suitable for purification of the fusion protein, but can be suitably used for detection and labeling of the fusion protein.
In addition, the tag affinity substance is immobilized on the inner surface of the well and / or the carrier in the well. The tag affinity substance only needs to be arranged with the inner surface of the well and / or the carrier in the well. The method for forming is not limited, and examples thereof include coating.

  The tag as described above is expressed together with the target protein, and this tag can be used for capturing or labeling the target protein. By this capture, the fusion protein can be used as a means for purification, detection or immobilization. The synthesis system of the fusion protein is not particularly limited, but a system in which DNA does not exist as a contaminant is preferable. As a suitable example, a protein prepared by a cell-free protein synthesis means is exemplified. Even more preferably, the cell-free protein synthesis means is a system utilizing wheat germ.

Immobilization of DNA on a carrier One aspect of the present invention is when capture using a tag is used for protein purification or immobilization. In this case, as one embodiment, the target protein is captured by a tag using DNA immobilized on a carrier. The DNA sequence of such immobilized DNA may be any sequence that can bind to the transcription factor or nuclear receptor. The DNA immobilization carrier used in the present invention is a commercially available one in which a cellulose resin (manufactured by Amersham Pharmacia Biotech) is used as an example of the carrier, and a calf genomic DNA sequence is immobilized thereon, but is not limited thereto. . In addition, a DNA-immobilized carrier can be prepared by immobilizing a sequence capable of binding to the above transcription factor or nuclear receptor on a conventionally known carrier and washing the unfixed DNA with a buffer as appropriate. Not. Furthermore, a DNA-immobilized column can be easily produced by packing the carrier into the column. Specific examples include (DNA-Cellulose Column (native), manufactured by Amersham Pharmacia Biotech: Calf genomic DNA is immobilized).
The equilibration of the carrier as a pretreatment for capturing the protein with the DNA-immobilized carrier is not particularly limited, and can be prepared according to a known method for washing the DNA-immobilized carrier. For example, a DNA immobilization column resin packed in an Eppendorf tube is washed with MilliQ water, then the supernatant is removed by centrifugation, and further washed with an appropriate buffer, followed by centrifugation. However, the present invention is not limited to this.

Immobilization of DNA in a high molecular weight polymer In the present invention, 1) the target protein does not come into direct contact with the atmosphere, 2) it gives spatial clearance around the target protein, and 3) simple. Since a technique of reducing and removing unnecessary substances by washing is necessary, a high molecular polymer that can satisfy these conditions was used for immobilization. By mixing a general polymer and DNA, it is possible to form a space in which DNA is stretched in a mesh shape in which direct contact with the air is blocked. After capturing a target protein fused with a tag capable of binding to DNA in this space, the target protein can be specifically immobilized by simple washing. In the present invention, agarose (Takara Bio, SeaKem Gold Agarose) is used as a high molecular polymer, salmon sperm DNA is added to the agarose, fluidized by heat, mixed, and agarose containing salmon sperm DNA is placed on a glass plate. By spreading it out, a space in which DNA was immobilized was formed on the flat plate. However, it is not limited to this method. In addition to the polymer, it can be fixed with ceramics, carbon, activated carbon or the like. When capturing proteins on a flat carrier, DNA is mixed with a polymer that has fluidity temporarily, followed by agarose, polyacrylamide, various hydrogels, water-absorbing polymers, and hydrophobic polymers. It is preferable to immobilize them.

Preparation of Tag-Modified Protein In the present invention, the tag-modified protein means a protein in which a tag and a target protein are fused (sometimes referred to as a fusion protein).
Examples of fusion proteins using DNA-binding protein tags include DNA-binding transcription factor fusion proteins and DNA-binding nuclear receptor fusion proteins. The transcription-translation in this preparation is exemplified by a method using a cell-free synthesis means as described below as a suitable method. A transcription template refers to DNA that can be used as a template molecule for in vitro transcription reaction, and has at least a base sequence encoding a fusion protein (tag + target protein) downstream of an appropriate promoter sequence. An appropriate promoter sequence refers to a promoter sequence that can be recognized by RNA polymerase used in a transcription reaction, and examples thereof include SP6 promoter, T7 promoter and the like. The DNA encoding the fusion protein means DNA including the DNA encoding the tag protein and the DNA encoding the target protein, and may optionally include DNA encoding the endopeptidase recognition sequence. Furthermore, a linker sequence can be introduced between the tag sequence and the target protein sequence in order to maintain an appropriate distance between the two. In addition, either the N-terminus or C-terminus of the tag sequence may be linked to the target protein. A DNA encoding a target protein means a DNA encoding a protein to be expressed in a cell-free protein synthesis system.
The transcription template preferably has a base sequence having an activity of controlling translation efficiency between the promoter sequence and the base sequence encoding the fusion protein. For example, the transcription template is derived from RNA virus-derived 5 such as Ω sequence derived from tobacco mosaic virus. 'Untranslated regions and / or Kozak sequences can be used. Furthermore, the transcription template preferably includes a 3 ′ untranslated region including a transcription termination region and the like downstream of the base sequence encoding the fusion protein. As the 3 ′ untranslated region, about 1.0 to about 3.0 kilobases downstream from the stop codon is preferably used. These 3 ′ untranslated regions are not necessarily native to the gene encoding the fusion protein. In a preferred embodiment of the method of the present invention, the reaction product obtained by amplifying and synthesizing the DNA encoding the fusion protein by the PCR method can be used as it is as a transcription template without purification. In order to prevent the production of short-chain DNA (resulting in yield loss of target product and low-molecular translation product noise) generated by non-specific amplification, use a promoter-splitting primer described in International Publication WO02 / 18586. You can also.
The transcription template DNA thus obtained may be subjected to a transcription reaction after being purified by chloroform extraction or alcohol precipitation, but the reaction solution after the PCR reaction can be used as it is as a transcription template solution.
A transcription reaction is performed in vitro from transcription template DNA encoding a fusion protein prepared using a method known per se or transcription template DNA encoding a fusion protein prepared without purifying DNA amplified and synthesized by PCR. A step of generating mRNA as a translation template by a transcription reaction. This step includes a solution containing a transcription template provided in a reaction system (eg, a commercially available container such as a 96-well titer plate), preferably a PCR reaction solution, and an RNA polymerase (eg, SP6) compatible with the promoter in the transcription template. After mixing with a solution (also referred to as a “transcription reaction solution”) containing components necessary for the transcription reaction such as RNA polymerase and RNA synthesis substrate (four kinds of ribonucleoside triphosphates), about 20 ° C. It is carried out by incubating the mixture at about 60 ° C., preferably about 30 ° C. to about 42 ° C. for about 30 minutes to about 16 hours, preferably about 2 hours to about 5 hours.
In addition, a gene DNA (Chiu, W. – L., et al., Curr. Biol. 6, 325-330 (1996)) encoding a fusion protein was inserted into mRNA as a preferred translation template. Based on the pEU-vector (Sawasaki, T. et al., PNAS, 99 (23), 14652-7 (2002)), the Ω sequence portion was replaced with the base sequence of SEQ ID NO: 136 described in WO 03/056009 Transcription can also be performed using SP6 RNApolymerase (Promega) with the plasmid DNA as a template.
Next, the transcription solution is added directly to the cell extract for protein synthesis. The cell extract for protein synthesis used herein may be any cell extract that can translate a translation template to produce a protein encoded by the template. Specifically, Escherichia coli, Plant seed germs, cell extracts such as rabbit reticulocytes, and the like are used. These may be commercially available, or a method known per se, specifically, for Escherichia coli extract, Pratt, JM et al., Transcription and Translation, Hames, 179-209, BD & Higgins, SJ, eds), IRL Press, Oxford (1984). Examples of commercially available cell extracts for protein synthesis include those attached to E. coli S30 extract system (Promega) and RTS 500 Rapid Translation System (Roche) from E. coli. Examples include those attached to Rabbit Reticulocyte Lysate System (manufactured by Promega), and those derived from wheat germ that are attached to PROTEIOS ^™ (manufactured by TOYOBO). Among them, it is preferable to use a plant seed germ extract system, and plant seeds are preferably gramineous plants such as wheat, barley, rice and corn, and seeds such as spinach, and in particular, wheat seed germ extract. Those using are suitable. Furthermore, a wheat seed germ extract from which the endosperm components and low molecular weight protein synthesis inhibitors mixed in the extract preparation step are substantially removed is more preferable. In the translation reaction, the transcription solution obtained by the preparation is directly added to the cell extract for protein synthesis, and the amino acid, energy source, various ions, buffer solution, ATP regeneration system, nuclease inhibitor, Add a solution containing components necessary or suitable for translation reaction such as tRNA, reducing agent, polyethylene glycol, 3 ', 5'-cAMP, folate, antibacterial agent, etc. This can be done by incubating for a period of time.

Capture and recovery of tag-modified protein on DNA-immobilized carrier Tag-modified protein, a fusion protein prepared by translation reaction, is contacted with DNA-immobilized carrier in the state of translation reaction solution, and tag-modified protein is immobilized on DNA. Captured and recovered by the support. Specifically, after the protein synthesis reaction is completed, a purification column such as an Eppendorf tube is filled and equilibrated with a DNA immobilization carrier, and a translation reaction solution (cell extract) containing a fusion protein is developed in this, for example. Further, a capture buffer is introduced to perform a contact reaction. By contact, the DNA of the DNA-immobilized carrier and the tag-modified protein are bound via the DNA binding site of the tag. Thereafter, after washing with a washing buffer, the affinity between the DNA and the tag is decreased with the elution buffer, and the tag-modified protein is released from the DNA-immobilized carrier and eluted. Elution can be achieved, for example, by changing the salt concentration in the eluate. As another method, by containing DNase in the eluate, the DNA of the DNA-immobilized carrier can be decomposed and the tag-modified protein can be eluted.
The following method is also useful. If a capture carrier to which an affinity substance is bound is prepared, the target protein can be captured and detected using this system. A preferred example is a system using biotinylated DNA as a reagent and immobilized streptavidin as a capture carrier. A biotinylated DNA reagent is allowed to act on immobilized streptavidin as a capture carrier, and the tag-modified target protein is contacted and reacted to form streptavidin-biotinylated DNA-tag-modified target protein. The target protein can be captured. By this capture, the target protein is selectively captured, and its presence can be confirmed and detected by a binding reaction between streptavidin and biotin. Such a system can be applied as a protein immobilization technique such as a protein chip.

In the present invention, since the tag-modified protein is recovered as described above, a sequence that can be cleaved by an enzyme such as an endopeptidase between the tag and the target protein is used for the separation of the target protein. If introduced beforehand, the target protein can be easily separated. Such sequences are known per se and are widely applicable. The introduction can be performed, for example, by linking a base sequence encoding an amino acid sequence (endopeptidase recognition sequence) cleaved with endopeptidase into a base sequence of DNA encoding a DNA-binding protein. This makes it possible to separate and sort the target protein and the DNA-binding protein as a tag by causing various known endopeptidases to act. The endopeptidase may be any peptidase that can cleave the target protein and the DNA-binding protein. For example, precision protease {endopeptidase that selectively cleaves the sequence of precision site (Leu-Glu-Val-Leu-Phe-Gln ↓ Gly-Pro) at the position of arrow: manufactured by Amersham Biosciences}, Thrombin, Factor Xa etc. can be used.

  Another embodiment of the present invention can be used for detecting a reaction of a multi-analyte protein using the above tag. For example, a target protein is captured in a polymer polymer containing DNA (sometimes called a polymer polymer in which DNA is encapsulated) and used to detect the reaction of a chemical substance containing the target protein or other proteins. it can. A suitable example is to create a protein reaction space that is blocked from the outside air by DNA-encapsulated agarose on a flat plate such as a glass slide, capture the fluorescently labeled protein with a modified tag, and examine the fluorescence intensity. The target protein can be quantified. In addition, it is possible to capture the tag-modified phosphorylated protein, remove excess protein (non-captured protein) and the like with a washing buffer, and detect the activity of the phosphorylated protein. Furthermore, as another preferred example using the same DNA-encapsulated agarose, a vaccine is prepared by fusing a tag with a protein of a pathogenic organism, arranging it on the flat plate, and reacting with serum collected from a diseased patient. Candidate or marker proteins can be screened. Such a system can be applied as a protein immobilization technique such as a protein chip.

Method for Immobilizing Labeling Substance to Fusion Protein Using Tag Another embodiment of the present invention uses the above tag to label a target protein with a labeling substance such as a low-molecular affinity substance or a fluorescent dye via DNA. be able to. Examples of the labeling substance include biotin, FITC which is a fluorescent dye known per se, rhodamine, Texas red, and a low molecular weight substance. For example, as a suitable example of labeling with a low molecular affinity compound, biotinylated DNA is used as a reagent and the target protein is biotinylated and labeled. A solution containing the target protein for tag modification can be brought into contact with biotinylated DNA and reacted to obtain a target protein for tag modification with biotinylated DNA binding. Furthermore, this conjugate can be captured on a plate with a slide glass immobilized with streptavidin. By this capture, the target protein is selectively captured, and its presence can be confirmed by a binding reaction between streptavidin and biotin. Such a system can be applied as a protein immobilization technique such as a protein chip or a protein labeling technique.

Chip for cell-free protein synthesis with integrated transcription / translation At least another aspect of the present invention is 1) a vector in which a PCR product of the base sequence encoding the tag and the target protein is introduced, or 2) RNA Components necessary for transcription, such as polymerase, SP6, RNA synthesis substrate (4 types of ribonucleotide tetraphosphate), 3) amino acids, energy sources, various ions, ATP regeneration system, nuclease inhibitor, tRNA, reducing agent , Polyethylene glycol, cAMP, folate, antibacterial and other necessary components for translation, 4) protein synthesis cell extract, preferably wheat germ extract, added to a container known per se A chip for cell-free protein synthesis. The chip can be suitably dried by a known drying method, particularly a freeze drying method, to obtain a transcription / translation integrated dry cell-free protein synthesis dried chip. As a suitable method, a chip manufacturing method described in International Publication No. WO 2005/015212 A1 can be used. In addition, by dividing the container, which is a chip, into several regions, and by adding a PCR product of a base sequence encoding a different target protein or a vector into which the encoded base sequence is introduced into the partitioned region at once, Many types of tag fusion proteins can be expressed on the chip.
The protein synthesis method of the dried and produced chip for transcription-translation integrated cell-free protein synthesis of the present invention includes a transcription / translation non-conjugated type and a transcription / translation conjugated type, and any method can be used. For example, in the transcription / translation non-conjugated type, first, a magnesium concentration solution suitable for the transcription reaction is added to the chip, and the transcription reaction is first performed. Subsequently, in order to obtain a temperature and magnesium concentration suitable for the translation reaction, a solution for adjusting the temperature of the chip and diluting the magnesium concentration with a heater or the like is added to the chip. As a result, the conditions inside the chip are suitable for the translation reaction, and only the translation reaction substantially proceeds. In the transcription / translation conjugate type, conditions suitable for both transcription and translation are set, and transcription and translation are performed simultaneously.
In addition, if DNA is immobilized on the bottom of the chip in advance, the translated tag fusion protein binds to the DNA. Thus, if the inside of the chip is washed with an appropriate washing solution, components other than the tag fusion protein bound to the DNA can be removed. After that, 1) the tag fusion protein can be purified by changing the salt concentration in the chip, and 2) the tag fusion protein bound to the DNA immobilized on the bottom of the chip will remain after the completion of the translation reaction. Therefore, since the fusion protein is in an active state, the fusion protein can be suitably used for screening a substance showing reactivity with the fusion protein in such a state.

Purification Kit In the present invention, at least one of the reagents used in the protein purification method as described above can be provided as a reagent kit for protein synthesis means. The kit provides a novel method capable of purifying and separating a target protein using a DNA-binding protein as a tag.

Detection Kit In the present invention, at least one of the reagents used for protein detection can be provided as a protein testing reagent kit. The kit provides a novel method capable of examining a target protein using a DNA-binding protein as a tag.

Labeling Kit In the present invention, at least one reagent used for protein labeling can be provided as a protein labeling reagent kit. The kit provides a novel method capable of labeling a target protein using a DNA-binding protein as a tag.

  The protein chip of the present invention means a chip in which one or two or more fusion proteins are captured or immobilized together or separately on a suitable substrate (well surface) or a chip present in a well. To do. Then, the fusion protein is captured on the inner surface of the well and / or the carrier in the well via the affinity between the tag site of the fusion protein and the tag affinity substance. The protein chip is used to analyze the binding and interaction of proteins with proteins, nucleic acids, low molecular compounds, sugar chains, etc. (hereinafter, this may be referred to as “proteome analysis”). . The expression method of the fusion protein is preferably a cell-free protein synthesis system, particularly a wheat germ extract-derived, E. coli-derived, or rabbit reticulocyte extract-derived synthesis system. Insect cell-derived synthesis systems, chemical synthesis systems (solid phase synthesis), and the like can also be used.

Method for producing protein chip reagent using cell-free protein synthesis system The reagent contains at least the following elements.
the fusion protein is expressed in the well of a container partitioned into one or more regions,
b. A substance having affinity with the tag site of the fusion protein (tag affinity substance) is immobilized on the inner surface of the well and / or the carrier in the well,
c. The fusion protein is captured on the inner surface of the well and / or the carrier in the well through the affinity between the tag site of the fusion protein and the tag affinity substance.
The fusion protein is preferably expressed using a cell-free protein synthesis system, particularly derived from a wheat germ extract.

Preparation of protein library The reagent and / or kit of the present invention is a protein obtained by expressing, capturing, and immobilizing one or more different fusion proteins in a well of a container partitioned into one or a plurality of regions. It can be prepared as a library preparation kit.

Method for Inspecting Interactive Substance The method for inspecting an interactive substance can be implemented by including at least the following steps.
(1) The detection target substance is added to the well in which the fusion protein is captured, and the presence or absence of interaction with the fusion protein is confirmed.
(2) Qualitatively or quantitatively determine the interacting substances using markers.

  As the well used, those described above can be used, but can be appropriately selected according to the detection method used for proteome analysis. Specifically, those made of a material such as plastic, polycarbonate, complex carbohydrate, acrylic resin, nitrocellulose, glass, or silicon can be used. In addition, when using the surface plasmon method (Cullen, DC, at al., Biosensors, 3 (4), 211-225 (1987-88)) for proteome analysis, gold, silver on a transparent substrate such as glass In addition, a metal thin film made of platinum or the like is used.

  The translation template contained in the translation reaction solution is appropriately selected depending on the purpose of proteome analysis using the protein chip prepared by the protein chip synthesis kit of the present invention. Specifically, for example, when used for functional analysis of a protein, mRNA having a sequence encoding a functional protein is selected. For example, ion channel, transporter, growth factor, hydrolase, synthetic enzyme, oxidase / reductase, protein inhibitor, bioactive peptide, peptide toxin, receptor such as G protein coupled receptor, ligand, antibody Etc. The analyte is not particularly limited as long as it is a substance that interacts with a specific protein and / or a translation template of the specific protein, and examples thereof include proteins, nucleic acids, low molecular compounds, and sugar chains.

  There is no particular limitation as to which fusion protein is expressed in the well, but it is preferable that the position (coordinates) of the well and the type of the fusion protein correspond to each other.

  The method for qualitatively or quantitatively determining the substance that interacts with the fusion protein is not particularly limited as long as it is a method that can detect the interaction between the fusion protein and the specimen, and specifically, a solid-phase enzyme immunoassay method. In the case of analysis by (Enzyme Linked Immunosorbent Assay (ELISA): Crowthjer, JR, Methods in Molecular Biology, 42, (1995)), a plastic microtiter plate usually used by the ELISA method is preferable. When the surface plasmon resonance method (Cullen, DC et al., Biosciences, 3 (4), 211-225 (1987-88)) is used, gold, silver, platinum, etc. on a transparent substrate such as glass That in which the metal thin film of this was comprised is preferable. In the case of using evanescent field molecular imaging method (Funtsu, T., et al., Nature, 374, 555-559 (1995)), a transparent body such as glass is preferable, more preferably made of quartz glass. Is used. When the fluorescence imaging analysis method is used, a nitrocellulose membrane, nylon membrane, plastic microtiter plate, etc., which are usually used for capturing and immobilizing proteins and the like can also be used.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples should be regarded as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is limited by the following examples. Is not to be done.

Preparation of transcription templates for DNA-binding protein fusion proteins (HY5, NR5A1, NR5A2, HY5-GFP, HY5-eIF4B, HY5-CaMk2δ, HY5-GS, HY5-NEK2) The mRNA used as the translation template is the HY5 gene DNA (sequence) PEU-HY5 vector with only number 1), pEU-NR5A1 vector with only NR5A1 gene DNA (SEQ ID NO: 3), pEU-NR5A2 vector with only NR5A2 gene DNA (SEQ ID NO: 5), HY5 PEU-HY5-GFP vector into which gene DNA (SEQ ID NO: 1) and GFP gene DNA (Chiu, W. – L., et al., Curr. Biol. 6, 325-330 (1996)) have been inserted PEU-HY5-elF4B vector, HY5 gene DNA (SEQ ID NO: 1) and CaMK2δ gene DNA (GenBank Accession No. AF071569) inserted with HY5 gene DNA (SEQ ID NO: 1) and elF4B gene DNA (GenBank Accession No. AB076839) PEU-HY5-CaMk2δ vector into which is inserted, HEU gene DNA (SEQ ID NO: 1) and pEU-HY5 into which GS gene DNA (GenBank Accession No. S70004) is inserted A transcription reaction solution [final concentration, 80 mM HEPES-KOH pH7. Using a pEU-HY5-NEK2 vector into which a GS vector, HY5 gene DNA (SEQ ID NO: 1) and NEK2 gene DNA (GenBank Accession No. NM_002497) are inserted as a template. 8, 16 mM magnesium acetate, 10 mM dithiothreitol, 2 mM spermidine, 2.5 mM 4NTPs (4 types of nucleotide triphosphates), 0.8 U / μl RNase inhibitor, 1.6 U / μl SP6 RNA polymerase] at 37 ° C. The reaction was allowed for 3 hours. The obtained RNA was purified by ethanol precipitation and used.
In addition, transcription templates for DHFR (dihydrofolate reductase), GST-GS (GST: glutathione-S-transferase), GST-NEK2, and GST-CaMK2δ were prepared in the same manner as described above.

Translation of DNA-binding protein fusion proteins (HY5, NR5A1, NR5A2, HY5-GFP, HY5-eIF4B, HY5-CaMk2δ, HY5-GS, HY5-NEK2) For protein synthesis containing 5.8 μl of a prepared wheat germ extract-containing solution Reaction solution (each at a final concentration of 29 mM HEPES-KOH (pH 7.8), 95 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nacalai Tectonics), each 0.23 mM L-type amino acid, 2.9 mM dithiothrei Toll, 1.2 mM ATP (Wako Pure Chemical Industries, Ltd.), 0.25 mM GTP (Wako Pure Chemical Industries, Ltd.), 15 mM creatine phosphate (Wako Pure Chemical Industries, Ltd.), 0.9 U / μl RNase inhibiter (TAKARA), 50 ng / 25 μl of μl tRNA (Moniter, R., et al., Biochim. Biophys. Acta., 43, 1- (1960)), 0.46 μg / l creatine kinase (Roche) was prepared. To this reaction solution, translation template mRNAs prepared in Example 1 (HY5, NR5A1, NR5A2, HY5-GFP, HY5-eIF4B, HY5-CaMk2δ, HY5-GS, HY5-NEK2, DHFR (for control), GST-GS ( (For control), GST-NEK2 (for control), and GST-CaMK2δ (for control)) were added at 8 μg / μl and incubated at 26 ° C. for 48 hours.

Equilibration of column resin
The column resin, which is a DNA-Cellulose Column (native) (Amersham Pharmacia Biotech: Calf genomic DNA is immobilized), was washed with 5 times the amount of MilliQ water, and centrifuged at 10,000 rpm for 1 minute. Subsequently, the column resin was washed with a 5-fold amount of a wash buffer (Table 1) and centrifuged at 10,000 rpm for 1 minute. The above operation was repeated once more.

Assay of DNA-binding protein 20 μl each of the column resin equilibrated in Example 3 and each DNA-binding protein prepared in Example 2 (HY5, 11 types of human nuclear receptors) in an Eppendorf tube (total 240 μl) and twice the amount of binding buffer (Table 1) were introduced, capped, and then stirred at 10 ° C. for 2 hours at 4 ° C. Thereafter, centrifugation was performed at 10,000 rpm for 1 minute, and 720 μl of the supernatant obtained by centrifugation was used as a sample (lane 1 in FIG. 1). Subsequently, the column resin was washed three times with 1 ml of wash buffer (Table 1), eluted with 50 μl of elution buffer (1) (Table 1), and 20 μl of the eluate obtained by elution was used as a sample (see FIG. 1). Lane 2). Subsequently, elution was performed with 50 μl of elution buffer (2), and 20 μl of the eluate obtained by elution was used as a sample (lane 3 in FIG. 1). Each sample was subjected to SDS-PAGE under reducing conditions using a 12.5% polyacrylamide gel and stained with CBB (FIG. 1).

Results of DNA-binding protein assay The HY5 band was confirmed from the results of lane 2 in FIG. Furthermore, HY5 was confirmed as an almost single band by elution with a salt concentration of 500 mM in lane 3 of FIG. Therefore, HY5 transcription factor was used in the following examples.

Purification of HY5 transcription factor In Example 4, HY5 that appears to bind to DNA even when the sequence specificity is particularly low was selected as a DNA-binding protein. Into an Eppendorf tube, the column resin equilibrated in Example 3 and 80 μl of HY5 prepared in Example 2 and twice its binding buffer (Table 1) were introduced. After capping, 2 hours at 4 ° C. Stir (10 rpm). Thereafter, centrifugation was performed at 10,000 rpm for 1 minute, and 240 μl of the supernatant obtained by centrifugation was used as a sample (total in the lane in FIG. 2). Subsequently, the column resin was washed 3 times with 1 ml of wash buffer (Table 1), eluted with 50 μl of elution buffer (1) (Table 1), and 20 μl of the eluate obtained by elution was used as a sample (see FIG. 2). Lane 1). Subsequently, elution buffer (2) (Table 1) was eluted with 50 μl, and 20 μl of the eluate obtained by elution was used as a sample (lane 2 in FIG. 2). Each sample was subjected to SDS-PAGE under reducing conditions using 12.5% polyacrylamide gel and stained with CBB (FIG. 2).
The same procedure was performed for DHFR as a control (FIG. 2).

Results of Purification of HY5 Transcription Factor Similar to the results of Example 4, HY5 could be purified with an elution buffer containing salt. In particular, a dense band was detected in the elution buffer (2) having a high salt concentration (lane 2). Further, in DHFR that is not a DNA-binding protein (DNA-binding transcription factor), there was no band representing the molecular weight of DHFR. Therefore, HY5, which is a DNA-binding transcription factor, was bound to the DNA-immobilized column in a non-sequence specific manner.

Purification of HY5 transcription factor and HY5 transcription factor fusion protein using DNase-containing elution buffer Column resin equilibrated in Example 3 in an Eppendorf tube, HY5, HY5-GFP and HY5 prepared in Example 2 -elF4B 100 μl and a double amount of binding buffer (Table 1) were introduced, and after capping, the mixture was stirred at 4 ° C. for 2 hours (10 rpm). Thereafter, the mixture was centrifuged at 10,000 rpm for 1 minute, and then the column resin was washed three times with 1 ml of wash buffer (Table 1). Four column resins were prepared, each eluted with the following elution buffer, and 20 μl of the eluate obtained by elution was used as a sample (FIG. 3).
1. elution buffer (1) (Table 1) 50μl (lane 1 in Fig. 3)
2. wash buffer (Table 1) 49ml + DNase 1μl (lane 2 in Fig. 3)
3. wash buffer (Table 1) 40 ml + DNase 10 μl (lane 3 in FIG. 3)
4). wash buffer (Table 1) 46.5 ml + DNase 1 μl + 25 mM EDTA (lane 4 in Fig. 3)
Each sample was subjected to SDS-PAGE under reducing conditions using a 12.5% polyacrylamide gel and stained with CBB (FIG. 3).
The same procedure was performed for DHFR as a control (FIG. 3).

Results of purification method of HY5 transcription factor and HY5 transcription factor fusion protein using DNase-containing elution buffer
Purification was possible in all HY5 transcription factors and HY5 transcription factor fusion proteins. Furthermore, purification was also possible by elution with an eluate containing DNase. This was because DNA immobilized on the column was degraded by DNase. In addition, the eluate containing EDTA could not be purified because DNase activity was inhibited.

Washing under high salt concentration conditions Into an Eppendorf tube, 50 μl of the column resin equilibrated in Example 3, 150 μl of HY5-GFP prepared in Example 2 and 300 μl of the binding buffer 300 μl (double amount) were introduced. After capping, the mixture was stirred (10 rpm) at 4 ° C. for 2 hours. Thereafter, the mixture was centrifuged at 10,000 rpm for 1 minute, and 3 μl of the supernatant obtained by centrifugation was used as a sample (lane 1 in FIG. 4). Subsequently, the column resin was washed twice with 1 ml of wash buffer (Table 2), and 10 μl of the washing solution was used as a sample (lane 2 in FIG. 4). Subsequently, elution was performed with 50 μl of elution buffer (Table 2), and 10 μl of the eluate obtained by elution was used as a sample (lane 3 in FIG. 4). Subsequently, elution was performed with 50 μl of elution buffer (Table 2), and 10 μl of the eluate obtained by elution was used as a sample (lane 4 in FIG. 4). Each sample was subjected to SDS-PAGE under reducing conditions using a 12.5% polyacrylamide gel and stained with CBB (FIG. 4).

Results of washing under high salt concentration conditions
When HY5-GFP was washed under high salt conditions, no other bands were detected. That is, the protein could be purified with high purity by washing under high salt concentration conditions.

Purification with undiluted translation solution and versatility of HY5 transcription factor purification tag The versatility of the HY5 transcription factor purification tag was confirmed by the following binding conditions 1 and 2. Details are as follows.
Join condition 1
Into an Eppendorf tube, 25 μl of the column resin equilibrated in Example 3 and 240 μl of each HY5-GFP, HY5-CaMK2δ, and HY5-GS prepared in Example 2 were introduced and capped (about 100 mM KOAc). In addition, it did not dilute with binding buffer.
Join condition 2
In an Eppendorf tube, 25 μl of column resin equilibrated in Example 3, 120 μl of each HY5-GFP, HY5-CaMK2δ, and HY5-GS prepared in Example 2 and 240 μl of the double amount of binding buffer (Table 3) Introduced and covered (about 33 mM KOAc).
Binding conditions 1 and 2 were stirred (10 rpm) at 4 ° C. for 2 hours. Thereafter, the mixture was centrifuged at 10,000 rpm for 1 minute, and 4.5 μl of the supernatant obtained by centrifugation was used as a sample (FIG. 5: Lane 1 of binding conditions 1 and 2). Subsequently, the resin for the column was washed twice with 500 μl of wash buffer (Table 3), and 12 μl of the washing solution was used as a sample (FIG. 5: Lane 2 of binding conditions 1 and 2). Subsequently, elution buffer 1 (Table 3) was eluted with 25 μl, and 12 μl of eluate obtained by elution was used as a sample (FIG. 5: lane 3 of binding conditions 1 and 2). Subsequently, elution buffer 2 (Table 3) was eluted with 25 μl, and 12 μl of eluate obtained by elution was used as a sample (FIG. 5: lane 4 of binding conditions 1 and 2). Each sample was subjected to SDS-PAGE under reducing conditions using a 12.5% polyacrylamide gel and stained with CBB (FIG. 5).

Purification with undiluted translation solution and versatility of HY5 transcription factor purification tag Single band of each HY5 transcription factor fusion protein in both condition 1 where the translation solution was not diluted and condition 2 where the translation solution was diluted Could be detected. That is, HY5 was found to be a highly versatile purification tag.
Thereby, the DNA-binding transcription factor fusion protein-containing translation solution can be purified as it is without dilution.

Preparation of Biotinylated DNA The base sequence of SEQ ID NO: 7 was synthesized and amplified by the PCR means shown in Table 4. The synthesized base sequence is biotinylated at the 5 'end and carries a base sequence (A, C, G-Box) that can bind to HY5, a DNA transcription factor. 1 μl of exonuclease I was added to the PCR product and incubated for 1 hour at 37 ° C. The mixture was further incubated at 80 ° C. for 15 minutes, prepared with a G-2 microspin column using Buffer 1 (Table 5), and used as biotinylated DNA.

Preparation of HY5-specific binding DNA carrier using streptavidin-biotin bond and capture of HY5-modified protein Prepare 25 μl of streptavidin-fixed magnetic beads (MagneSphere: Promega) as a carrier for immobilizing biotinylated DNA, and 50 μl of Buffer 1 (Table 5) was used to wash with 3 times the amount of beads. 20 μl of biotinylated DNA prepared in Example 9 was added thereto. Subsequently, it was incubated at 4 ° C. for 30 minutes (mixed every 5-10 minutes). Subsequently, the plate was washed with 25 μl of Buffer 1, 45 μl of the post-translation solution (HY5-GS, GST-GS) prepared in Example 2 was added, and incubated at 4 ° C. for 1 hour. In addition, 1 μl of the solution before addition was used as a sample (FIG. 6: each lane 1). Next, using Magneto Magnetic Separation Stand (Promega), 4.5 μl of the obtained supernatant was used as a sample (FIG. 6: each lane 2). The protein was then eluted by washing 5 times with 50 μl Buffer 2 (Table 5), then adding 20 μl Buffer 3 (Table 5) and incubating for 10 minutes at rt. In addition, 17 μl of this eluate was used as a sample (FIG. 6: each lane 3). Subsequently, the SDS-added PAGE sample preparation solution was added to the column and boiled. In addition, 17 μl of this boiled solution was used as a sample (FIG. 6: each lane 4).
The same procedure was performed with non-biotinylated DNA (HY5-GS) as a control (dsDNA in FIG. 6).

Results of capture with HY5 specific binding DNA carrier
The DNA immobilization carrier was able to capture HY5-GS. On the other hand, GST-GS, a protein fused with GST that is not a DNA-binding protein (DNA-binding transcription factor), could not be captured. In addition, HY5-GS could not be detected with a DNA-immobilized carrier (dsDNA) that was not biotinylated. This is because non-biotinylated DNA could not bind to streptavidin.
As described above, the target protein can be captured and detected by using a DNA-binding transcription factor and biotinylated DNA.

Preparation of DNA-encapsulated agarose gel slide
After adding 0.2% SeaKem Gold Agarose (Takara Bio Inc.) to 20 mM Hepes-KOH (pH 7.6), 2 mM Magnesium Chloride Buffer and dissolving, final concentration of Salmon testes DNA (native) (Sigma Aldrich) 0.1 μg / ml was added and thinned into a non-fluorescent slide glass to prepare an agarose gel slide enclosing DNA.

Capture of HY5-modified protein on DNA-encapsulated agarose gel slide 0.5 μl of the translated solution (HY5-GFP) prepared according to Example 2 was spotted on the DNA-encapsulated agarose gel slide of Example 11, and 50 ml of Buffer 1 ( Table 5). And the fluorescence of GFP of each time was detected with the fluorescence image analyzer (Tyhoon) (FIG. 7: left in a slide).
As a control, GST-GFP was used in the same manner as above (FIG. 7: right in the slide).

Results of capture of HY5-modified protein on DNA-encapsulated agarose gel slide According to the results of FIG. 7, only DNA-binding transcription factor-fused GFP was immobilized on the gel slide.
As described above, when a DNA-binding transcription factor and a DNA-encapsulated agarose gel slide are used, the target protein can be captured, detected, and quantitatively detected.

Detection of autophosphorylation activity of captured HY5-modified protein on DNA-encapsulated agarose gel slide 5 μl of the post-translational solution (HY5-NEK2, HY5-CaMK2δ, HY5-GS) prepared according to Example 2 was placed on the DNA-encapsulated agarose gel slide. 0.25 μl was spotted and left in 50 ml Buffer 1 (Table 5) for 2 hours. After thoroughly wiping off the moisture on the slide glass, phosphorylation solution containing [γ- ³² P] ATP {50 mM Tris-HCl pH 7.8, 100 mM sodium chloride, 10 mM magnesium chloride, 0.1 mM dithiothreitol} 25 μl Was mounted in an agarose gel form and reacted at 37 ° C. for 30 minutes. Next, the mixture was allowed to stand in a phosphorylation reaction buffer not containing [γ- ³² P] ATP for 2 hours to wash away unreacted [γ- ³² P] ATP. Then, an autoradiogram was detected using an image analyzer (Tyhoon) (FIG. 8: slide left).
In addition, GST-NEK2, GST-CaMK2δ, and GST-GS were used in the same manner as above (FIG. 8: slide right).

Results of detection of autophosphorylation activity of captured HY5-modified protein on DNA-encapsulated agarose gel slide Based on the results in FIG. 8, only the DNA-binding transcription factor fusion protein is immobilized on the gel slide and has autophosphorylation activity. Only the known NEK2 and CaMK2δ spots were detected, indicating that autophosphorylation activity can be detected.
As described above, if a DNA-encapsulated agarose gel slide is used, a protein-immobilized chip having a function can be produced.

Capture of biotin-labeled HY5 modified protein on streptavidin-coated slide 1 μl of biotinylated DNA prepared in Example 9 and 5 μl of translated solution (HY5-GFP) prepared in Example 2 were incubated at room temperature for 10 minutes. Biotin-labeled HY5 fused GFP was prepared by binding biotinylated DNA. On the other hand, a slide coated with streptavidin (Gleiner) was prepared as a labeled protein capture chip. Next, 1 μl of a mixture of biotinylated DNA and post-translation solution (HY5-GFP) was spotted on the slide and washed 3 times using 50 μl of Buffer 1 (Table 5). Then, fluorescence of GFP was detected with a fluorescence image analyzer (Tyhoon) (left slide in FIG. 9).
As a control, the same procedure was performed with a mixture of non-biotinylated dsDNA and HY5-GFP (center slide in FIG. 9) and HY5-GFP alone (right slide in FIG. 9) (FIG. 9).

Results of capture of biotin-labeled HY5-modified protein on streptavidin-coated slide According to the results of FIG. 9, only the DNA-binding transcription factor fusion protein is immobilized on the chip.
As described above, a protein-immobilized slide can be prepared by using a DNA-binding transcription factor, biotinylated DNA, and a streptavidin-coated chip.

Purification of human nuclear receptors NR5A1 and NR5A2 In the previous examples, HY5 transcription factor was used, but not only HY5 but also nuclear receptors NR5A1 and NR5A2 have low sequence specificity and bind to DNA. In order to investigate, NR5A1 and NR5A2 were examined. Specifically, the column resin equilibrated in Example 3 and 145 μl of NR5A1, NR5A2 prepared in Example 2 (lane 1 in FIG. 10) were introduced into an Eppendorf tube, and after capping, 2 hours at 4 ° C. Stir (10 rpm). Thereafter, the mixture was centrifuged at 10,000 rpm for 1 minute, and 145 μl of the supernatant obtained by centrifugation was used as a sample (lane 2 in FIG. 10). Subsequently, the column resin was washed 3 times with 1 ml of wash buffer (Table 1), eluted with 15 μl of elution buffer 2 (Table 3), and 15 μl of the eluate obtained by elution was used as a sample (lane 3 in FIG. 10). ). Each sample was subjected to SDS-PAGE under reducing conditions using a 12.5% polyacrylamide gel and stained with CBB (FIG. 10).

Results of purification of nuclear receptors NR5A1 and NR5A2 Similar to the results of Example 4, NR5A1 and NR5A2 could be purified using an elution solution containing a salt. Therefore, NR5A1 and NR5A2 which are the DNA-binding nuclear receptors of the present invention also bind to the genomic DNA immobilization column in a non-sequence-specific manner and can be used as the tag of the present invention.

  The industrial applicability of the present invention is to use a DNA-binding protein as a novel protein tag to capture a novel target protein and provide a purification or detection of the target protein and a labeling method.

The result of the assay of a DNA binding type protein is shown. M represents a molecular weight marker, total represents an unpurified fraction, and an asterisk (*) represents a HY5 band. (Example 4) The purification result of HY5 transcription factor is shown. “marker” indicates a molecular weight marker, “total” indicates an unpurified fraction, and an asterisk (*) indicates a HY5 band. (Example 5) The result of HY5 transcription factor fusion protein is shown. marker represents a molecular weight marker, total represents an unpurified fraction, and an asterisk (*) represents a band of HY5 transcription factor fusion protein. (Example 6) The result of HY5-GFP protein by washing | cleaning on high salt concentration conditions is shown. M represents a molecular weight marker, and total represents an unpurified fraction. (Example 7) The result of the HY5 transcription factor fusion protein by elution with an undiluted translation solution is shown. M represents a molecular weight marker, and an asterisk (*) represents a band of HY5 transcription factor fusion protein. (Example 8) The result of the capture of the tag-modified protein on the DNA carrier is shown. M represents a molecular weight marker. (Example 10) The result of the capture of the tag-modified protein on a DNA-encapsulated agarose gel slide is shown. The left slide shows HY5-GFP and the right slide shows GST-GFP. (Example 11) The results of capturing the tag-modified protein on a DNA-encapsulated agarose gel slide and detecting autophosphorylation activity are shown. The left side of the slide is the HY5 fusion protein, and the right side of the slide is the GST fusion protein. From the top of the slide, spots of NEK2, CaMK2δ, and GS are shown. Example 12 The results of capturing the tag-modified protein with a biotin label and streptavidin slide are shown. The left slide is biotinylated DNA and HY5-GFP, the middle slide is non-biotinylated DNA and HY5-GFP, and the right slide is only HY5-GFP. (Example 13) The purification results of NR5A1 and NR5A2 are shown. M represents a molecular weight marker, and an asterisk (*) represents a band of NR5A1 or NR5A2. (Example 14)

Claims (22)

Use as a tag of a target protein by an amino acid sequence (hereinafter referred to as a tag site) expressed with the target protein. The use according to claim 1, wherein the tag site is a DNA-binding protein comprising an amino acid sequence having DNA binding ability. The use according to claim 2, wherein the DNA-binding protein is a DNA-binding transcription factor or a DNA-binding nuclear receptor. The use according to claim 2 or 3, wherein the DNA-binding protein is selected from any one of the following.
(1) the sequence of SEQ ID NO: 2, 4 or 6;
(2) a sequence having at least about 80% or more amino acid sequence homology with the sequence of SEQ ID NO: 2, 4 or 6;
(3) A sequence having a DNA binding ability, having a mutation such as deletion, substitution, addition or insertion of one or several amino acids from the sequence (1) above. The use according to claim 1, wherein the relationship between the tag site and the tag affinity substance is selected from one or more of the following.
1) Biotin binding protein / biotin of avidin or streptavidin, 2) Maltose binding protein / maltose, 3) G protein / guanine nucleotide, 4) DNA binding protein / DNA, 5) Antigen molecule (epitope) / antibody, 6) Calmodulin Binding peptide / calmodulin, 7) ATP binding protein / ATP, 8) Estradiol receptor protein / estradiol The use according to any one of claims 1 to 5, wherein an endopeptidase recognition amino acid sequence is added to the tag. A protein capturing method for capturing a fusion protein using the tag according to claim 1. The method according to claim 7, wherein the capturing is performed by immobilized DNA. 9. A method according to claim 7 or 8, wherein the capture is for purification or detection of the fusion protein. A protein labeling method in which a labeling substance is immobilized on a fusion protein using the tag according to any one of claims 1 to 6. The method according to any one of claims 7 to 10, wherein the fusion protein is prepared by a cell-free protein synthesis means. The method according to claim 11, wherein the cell-free protein synthesis means uses a wheat germ extract. A protein purification kit comprising the tag according to any one of claims 1 to 6 as a fusion protein purification tag. A protein detection kit comprising the tag according to any one of claims 1 to 6 as a fusion protein detection tag. A protein labeling kit comprising the tag according to any one of claims 1 to 6 as a fusion protein labeling tag. A protein chip reagent using a protein synthesis system, comprising the following elements.
a: the fusion protein is expressed in the well of a container partitioned into one or more regions,
b. A substance having affinity with the tag site of the fusion protein (tag affinity substance) is immobilized on the inner surface of the well and / or the carrier in the well,
c. The fusion protein is captured on the inner surface of the well and / or the carrier in the well through the affinity between the tag site of the fusion protein and the tag affinity substance. The protein chip reagent according to claim 16, wherein two or more kinds of fusion proteins are expressed in wells of a container partitioned into one or a plurality of regions. The protein chip reagent according to claim 16 or 17, wherein the relationship between the tag site of the fusion protein and the tag affinity substance is selected from any one or more of the following.
1) Biotin binding protein / biotin of avidin or streptavidin, 2) maltose binding protein / maltose, 3) G protein / guanine nucleotide, 4) polyhistidine peptide / metal ion such as nickel or cobalt, 5) glutathione-S-transferase / Glutathione, 6) DNA binding protein / DNA, 7) antigen molecule (epitope) / antibody, 8) calmodulin binding peptide / calmodulin, 9) ATP binding protein / ATP, 10) estradiol receptor protein / estradiol The protein chip reagent according to any one of claims 16 to 18, wherein the fusion protein is expressed in a cell-free protein synthesis system. The protein chip reagent according to claim 19, wherein the cell-free protein synthesis system is a cell-free protein synthesis system derived from a wheat germ extract. A protein synthesis kit comprising the protein chip reagent according to any one of claims 16 to 20. 21. A method for examining a substance interacting with a fusion protein comprising the following elements, using the protein chip reagent according to claim 16.
(1) A detection target substance is added to a well in which the fusion protein is captured, and the presence or absence of an interaction with the fusion protein is confirmed.
(2) Qualitatively or quantitatively determine the interacting substances using markers.