KR20130084536A - Method for quantitative analysis of real time interactions between hif-1 alpha and p300 proteins - Google Patents

Abstract

PURPOSE: A real time analysis method of the interaction between HIF-1alpha and p300 protein is provided to quantitatively analyze the interaction between HIF-1alpha and p300 protein in real time at a cellula level using a fluorescence resonance energy transfer method and to screen the interaction in a cell in real time. CONSTITUTION: A method for measuring the interaction between HIF-1alpha and p300 protein comprises the steps of: preparing an HIF-1alpha plasmid containing HIF-1alpha of which N terminal, C terminal, or the both terminals is (are) labeled with a first fluorescent material; preparing a p300 protein plasmid containing p300 protein of which N terminal, C terminal, or both terminals is (are) labeled with a second fluorescent material; injecting the prepared HIF-1alpha plasmid and p300 plasmid into eukaryotes; and measuring the fluorescence generated from the transfected cells and calculating the fluorescence transfer efficiency according to equation (FRET index = I_DA- (a_DI_DA)- (a_AI_DA), FRET efficiency = FRET index/255). One of the first and second fluorescent materials is a fluorescent energy donor and the other is a fluorescent energy acceptor. The fluorescence spectrum of the fluorescent energy donor and the fluorescent energy acceptor is partially or entirely overlapped.

Description

METHOD FOR QUANTITATIVE ANALYSIS OF REAL TIME INTERACTIONS BETWEEN HIF-1 ALPHA AND p300 PROTEINS [0002]

The present invention relates to a method for quantitatively analyzing the binding between HIF-1α and p300 proteins in a cell using a fluorescence resonance energy transfer (FRET) method and a method for quantitatively analyzing the binding between HIF-1α and p300 protein Binding inhibitor or promoter in real time in a cell.

Within animal cells, the ability to sense and adapt to the levels of oxygen essential for survival is not only a physiological process such as development, angiogenesis, but also myocardial ischemia, It is known to be involved in various diseases such as cerebralischemia and pulmonary hypertension. HIF-1 (hypoxia inducible factor-1: hypoxia-inducible factor), one of the proteins that plays an important role in the cell response according to oxygen levels, is recognized as a key protein in the above-mentioned diseases .

HIF-1 is composed of alpha (alpha) and beta (beta) subunits in which the beta subunit is always stably expressed, while the HIF-1 alpha subunit is proteosomes in the normoxia state, In the hypoxia state, however, the labeling enzyme recognized by the degrading enzyme does not react to HIF-1α. Therefore, it is stabilized without being degraded, and it is transferred to the nucleus while CBP (cAMP -response element-binding protein) or p300 (E1A binding protein p300) to form a complex. This complex will serve as a vehicle for expressing various genes to adapt to hypoxia.

More specifically, HIF-1 alpha binds to the hypoxia response element (HRE) and activates transcription of target genes such as VEGF, EPO, PDGF-beta (Lando, D., et al., Genes. , 16: 1466-14, 2002). Thus, transcription-activated genes regulated by HIF-1α regulate cell growth, differentiation, and homeostasis. Therefore, when the binding between HIF-1α and CBP or p300 protein is regulated, coronary artery dysfunction, It is possible to obtain very useful information and techniques for the treatment of ischemic diseases such as dysfunction or for the study of cancer treatment through inhibition of angiogenesis.

The part involved in HIF-1α binding to CBP or p300 protein is the Tyr-C (C-Cysteine- / histidine-rich) CH1 domain of p300 and the C-terminal region of HIF- terminal transactivation domain (Kung, AL, et al., Nature Medicine, 6: 1335-1340, 2000). (Freedman, SJ, et al., PNAS, 99: 5367-5372, 2002) of amino acid positions 331 to 418 of p300 protein and amino acid positions 792 to 824 of HIF- (Dames, SA, et al., PNAS, 99: 5271-5276, 2002) of amino acid positions 345 to 439 of mouse CBP protein and amino acid positions 776 to 826 of HIF- , It can be seen that the CH1 region of p300 or CBP serves as a scaffold for folding the C-terminal domain of HIF-1α and is stabilized by various hydrophobic and polar interactions.

 On the other hand, the transcription-assisting proteins CBP and p300 that bind to HIF-1α are known to have very similar structures and functions. However, the gene deletion experiments in mice revealed that CBP-free and p300-free mice have different phenotypes, indicating that they play a somewhat different role (Partamem, A., et al., Int. J Biol., 43: 487-494, 1999). According to a recent report, both proteins are involved in genes that regulate metabolism and development, particularly CBP has been found to be more involved in the regulation of the inhibition (Ramos, YFM, et al., Nucleic Acid Research, 38: 5396- 5408, 2010). Regarding HIF-1α, both proteins are known to bind to the TAD-C portion of HIF-1α, but CBP is also known to bind to the TAD-N portion. However, it is not known exactly how the two proteins bind to transcription-promoting proteins and how their binding to the two proteins results. Most of the in vitro experimental design uses CBP or p300 alone Binding to HIF-1 [alpha].

The expression of HIF-1 dependent target genes is regulated by oxygendependent mechanisms such as proline hydroxylation and asparagine hydroxylation. The hydroxylation of the proline residue in the ODD (oxygendependent domain, oxygen-dependent domain) of HIF-1α induces the degradation of HIF-1α and the asparagine hydroxylation inactivates the C-terminus of HIF- It is known to induce degradation. The protein, FIH (factor-inhibiting HIF; Mahon, PC, et al., Genes Dev., 15: 2675-2686, 2001) involved in the asparagine hydroxylation characterizes the oxygen sensor involved in the regulation of HIF- (Lando, D., et al., Genes Dev., 16: 1466-14, 2002), the asparagine 803 of HIF-1α is hydrolyzed in the normal oxygen state to form the CBP or p300 It is known to interfere with protein binding.

To date, most of the methods for observing the interaction between HIF-1α and p300 or CBP proteins use biochemical or immunological tools. (2) Two-hybrid assay, which is widely used for analyzing protein interactions, (2) Activation of reporter gene by binding of two proteins (Yoshida, E., et al., Genes , And (3) two proteins were labeled with different labeling factors, followed by electrophoresis (SDS-PAGE) and autoradiography (Kung, AL, et al., Nature Medicine, 6: 1335-1340, 2000), (4) coimmunoprecipitation using two antibodies recognizing specific regions of two proteins, And the like.

In the two-way hybridization assay, the DNA binding domain (DBD) of the yeast GAL4 transcription factor is fused to the CBP or p300 protein and the transcriptional activation domain (TAD) to HIF-1α The use of reporter genes such as GFP (green fluorescent protein), luciferase, and beta -galactosidase to measure transcription activity by binding of proteins can be more easily measured have. However, these methods are disadvantageous in that they require a large amount of sample, are complicated, require long experiment time, use radioisotope, and are difficult to measure in real time in a cell.

On the other hand, a simpler analysis method is required to quantitatively screen the interaction between HIF-1α and p300 or CBP protein. As a part of this study, a 96-well plate-based assay was developed in which the binding between HIF-1α and p300 proteins can be observed in a short time without using a radioactive element (Kung, AL, et al, Cancer Cell, 6: 33-43, 2004). In this method, a biotin is attached to a site corresponding to a minimal p300 binding domain of HIF-1α and fixed on a multiwell plate coated with streptoavidin, The resulting GST-CH1 (p300) protein is treated and europium-conjugated anti-GST antibody is treated to measure the amount of the bound protein with a time-resolved fluorescence spectrometer . Although these assays can be used to screen for compounds that inhibit the interaction of HIF-1α and p300 proteins, they have the disadvantage of using plates coated with expensive avidin, antibodies and spectrometers capable of measuring time-resolved fluorescence. The methods listed above measure HIF-1α and p300 in real time in a cell by measuring binding in a test tube using two purified proteins or by-products through binding of two proteins in a cell. There is no report yet on the direct observation of protein binding.

Fluorescence Resonance Energy Transfer (FRET) is used to measure the binding of two proteins in real time in living cells (Truong, K., et al., Current Opinions in Structural Biology 11: 573-578, 2001). The fluorescence resonance energy transfer efficiency is determined by the distance between two molecules, and the distance that can be measured using fluorescence resonance energy transfer is as small as about 5-6 nm, so that the distance between two molecules, the protein folding phenomenon, do. To measure this, two different phosphors are required to be measured: a fluorescent donor capable of fluorescence by absorbing light, and a fluorescent acceptor capable of absorbing the fluorescence and emitting light again . Fluorescent proteins are mainly used because they are limited in the method of labeling specific proteins in cells (Sapsford, KE, et al. , Angew. Chem. Int. Ed 45: 4562-4599, 2006)

 Recently, the TAD partial domain of HIF-1α and the CH1 or CH3 region of the CBP region were labeled with fluorescent proteins, respectively, and the degree of binding of the two proteins was measured by fluorescence resonance energy transfer (FRE) The measured papers were reported. (Ruas, JL et al., J. Biol. Chem., 285: 2601-2609, 2010), but this paper was used as a tool to identify only specific binding sites, It can not be seen as observing the overall interaction. In particular, it is known that CBP also binds to CH3 of CBP and TAD-N (N-terminal transactivation domain) of HIF-1α, as well as the C-terminal transactivation domain (TAD-C) binding of CH1 and HIF-1α in CBP. Therefore, if the actual total protein binding is measured, it is theoretically possible that two CBPs are bound to one HIF-1α. In order to observe both of them at the same time, the total HIF-1α and CBP . In addition, since the binding of HIF-1α and CBP was measured, the binding between HIF-1α and p300 is different.

HIF-1? And HIF-1? (Wotzlaw, C. et al., PMC Biophysics, 3: 5- 19, 2010) was also reported in the cell using fluorescence resonance energy transfer phenomenon, because HIF-1α actually migrates from the intracellular to the nucleus and binds to p300 or CBP and then expresses a gene related to hypoxia This is not a direct combination of HIF-1α and p300, as it is intended to look at the processes required in all the steps that take place to achieve the goal of hydration and dimerization.

Thus, the inventors of the present invention have found that when an acceptor that receives fluorescence energy by p300 that labels a donor that transmits fluorescence energy at its N-terminus (and / or C-terminus) -Terminal binding of HIF-1α to the complex of HIF-1α and p300 protein by measuring the binding degree in real time in living cells by developing a method for quantitatively analyzing the degree of binding between two proteins from the energy transfer efficiency between HIF- The present invention has been accomplished by confirming that it can be usefully used to directly measure the efficacy when an inhibitor or an accelerator that inhibits the formation of a cell is applied in a cell.

Accordingly, one example of the present invention provides a method for quantitatively analyzing the interaction between HIF-1 alpha and p300 protein in a cell in real time.

Another example provides a method of screening for a binding modulator (promoter or inhibitor) between HIF-1 alpha and p300 protein using the method.

The present invention relates to a method for quantitatively analyzing the binding between HIF-1.alpha. And p300 protein by using a fluorescent resonance energy transfer method and a method for quantitatively analyzing the binding between HIF-1.alpha. And p300 protein binding regulator (inhibitor or promoter) Screening method. More specifically, the assay method of the present invention is a method of analyzing a p300 labeled with an N-terminal (and / or C-terminal) donor (for example, ECFP) (For example, EYFP) at the N-terminus (and / or the C-terminus), and quantitatively analyzes the degree of binding of the two proteins. Since the assay method according to the present invention can measure the degree of binding in real time in living cells, it is possible not only to measure in real time the complexity and degree of HIF-1α and p300 protein complexes, but also to measure HIF-1α and p300 Screening of modulators (inhibitors or promoters) that regulate protein complex formation and to directly measure the efficacy of such modulators in cells.

Hereinafter, the present invention will be described in detail.

The present invention measures the binding of HIF-1α and p300 proteins labeled with a fluorescent substance such as a fluorescent protein or a labeled protein (labeling the substrate corresponding to a tag with a fluorescent dye) through fluorescence energy transfer measurement method Provide a way to do it. According to the method of the present invention, it is possible to quantitatively analyze not only the binding between HIF-1α and p300 protein but also the degree of binding.

Specifically, the binding measurement method comprises the steps of:

1) preparing an HIF-1? Plasmid comprising N-terminal, C-terminal or HIF-1? Labeled with a first fluorescent substance at both terminals;

2) preparing a p300 protein plasmid containing the p300 protein whose N-terminus, C-terminus or both ends are labeled with a second fluorescent substance;

3) injecting the prepared HIF-1? Plasmid and p300 protein plasmid into cells to transfect cells; And

4) measuring the fluorescence emitted from the transfected cells to determine fluorescence transmission efficiency

.

In another example, a HIF-l alpha plasmid comprising an N-terminus, a C-terminus, or HIF-l [alpha] at both ends labeled with a first fluorescent material; A p300 protein plasmid comprising a p300 protein whose N-terminus, C-terminus or both ends are labeled with a second fluorescent substance; And a kit for measuring the binding between HIF-1 alpha and p300 protein, comprising fluorescence measurement means. The kit may further comprise cells for transfection and expression of HIF-l [alpha] plasmids and p300 protein plasmids.

The method and kit for measuring the binding between HIF-1.alpha. And p300 protein according to the present invention have an advantage that they can be measured even in living cells.

Since the first fluorescent material and the second fluorescent material are for measuring fluorescence energy transfer phenomena, they may be the same or different. In a preferred example, one of them is a donor that transmits fluorescence energy and the other is a fluorescent energy And the like.

As the fluorescent material, a bioluminescent protein, a tag protein, a fluorescent dye, a quantum dot, or the like including a fluorescent protein can be used.

In the present invention, the donor and acceptor used as the first fluorescent protein and the second fluorescent protein can basically be used as long as there is an overlap of the fluorescence spectrum of the fluorescent energy donor and the absorption spectrum of the fluorescent energy acceptor. The overlapping of the spectrum means that the fluorescence (emission) spectrum range of the donor is partially or entirely included in the absorption spectral range of the acceptor.

Accordingly, in the present invention, the donor is selected from the group consisting of bioluminescent proteins including fluorescent proteins, fluorescent dyes, quantum dots and the like, and the acceptor is a fluorescent protein, a tag, Proteins, fluorescent dyes, bioluminescent proteins, quantum dots, and the like, and may have an absorption wavelength range in a range overlapping the fluorescence wavelength range of the donor.

For reference, Fig. 1 shows the absorption and fluorescence spectra of ECFP and EYFP. For example, in the fluorescence resonance energy phenomenon using ECFP-EYFP, the fluorescence spectrum of ECFP as a fluorescent energy donor is from about 440 nm to 700 nm, and the absorption spectrum of EYFP as a fluorescence energy acceptance spectrum exists from about 440 nm to 550 nm. In particular, when we look closely at the spectra from about 450 nm to 525 nm, we can see that the fluorescence of ECFP overlaps the absorption of EYFP. The portion painted in green in Fig. 1 is the part where the spectrum overlaps. Even if the same fluorescent protein exists, if there is a certain degree of overlapping between the absorption spectrum and the fluorescence spectrum, one can be used as a relative donor and the other as a relative supplement using the same fluorescent protein. At this time, it is difficult to determine which one acts as a donor or donor because it is the same fluorescent protein.

As mentioned above, fluorescence resonance energy transfer phenomenon can be measured by using two fluorescent proteins if spectrum overlapping exists basically. There are currently more than 40 known fluorescent proteins, and since the development of fluorescent proteins with better mineralizability, that is, high fluorescence efficiency, light stability, high absorbance efficiency, etc., is ongoing, both donor and acceptor pairs are listed It is difficult to do. For reference, the wavelengths having the maximum value of absorption of fluorescence protein, which is presently known in Table 1 below, are referred to as Blue Fluorescent Protein (BFP), Cyan Fluorescent Protein (CFP), Green Fluorescent Protein (GFP) Fluorescent Protein) and RFP (Red fluorescent protein) - series.

Fluorescent system Fluorescent protein Maximum absorption wavelength (nm) Maximum fluorescence wavelength (nm) Absorption wavelength range
(nm) Fluorescence wavelength range
(nm) Blue
Fluorescent
Proteins
(BFP) Sapphire 399 511 300-450 400-600 T-sapphire 399 511 300-450 400-600 Enhanced Blue Fluorescent Protein (EBFP) 383 447 300-450 400-600 EBFP2 383 448 300-450 400-600 Azurine 384 450 300-450 400-600 Cyan
Fluorescent
Proteins
(CFP) Enhanced Cyan Fluorescent Protein (ECFP) 439 476 300-480 440-700 mECFP (monomeric Cyan Fluorescent Protein) 433 475 300-480 440-700 Cerulean 433 475 300-480 440-700 Cypet 435 477 300-480 440-700 AmCyan1 458 489 300-480 460-700 mTFP1 462 492 300-500 470-700 Green
Fluorescent
Proteins
(GFP) GFP 475 509 400-550 480-650 Enhanced Green Fluorescent Protein (EGFP) 484 507 400-550 480-650 AcGFP (Aequorea coerulescens GFP) 480 505 400-550 480-650 TurboGFP 482 502 400-550 480-650 Emerald 487 509 400-550 480-650 Max Green 492 505 400-530 450-650 ZsGreen 493 505 400-530 450-650 Yellow
Fluorescent
Proteins
(YFP)


Enhanced Yellow Fluorescent Protein (EYFP) 514 527 400-550 500-650 Topaz 514 527 400-550 500-650 Venus 515 527 400-550 500-650 mCitrine 516 529 400-550 500-650 YPet 517 530 400-550 500-650 PhiEYFP 525 537 400-600 540-730 ZsYellow1 529 539 400-600 540-730 mBanana 540 553 400-600 540-730 mOrange 548 562 400-600 540-730 mKO 548 559 400-600 540-730 Orange / Red
Fluorescent
Proteins
(RFP) dTomato 554 581 400-600 540-730 DsRed 558 583 450-630 550-800 mTangerine 568 585 450-630 550-800 TagRFP 555 584 450-630 550-800 mStrawberry 574 596 450-630 550-800 mRFP 584 607 450-630 550-800 mCherry 587 610 450-650 560-800 mRasberry 598 625 450-630 550-800 mKate 588 635 450-630 550-800 mplum 590 649 450-650 570-800 AQ143 595 655 450-650 570-800

More detailed properties of fluorescent proteins are found in Wang et al. In one embodiment of the present invention, the donor comprises BFP, ECFP, GFP, EYFP, Orange-red, and the like (Wang, Y., et al., Ann. Rev. Biomed. Eng 10: 1-38, 2008) At least one selected from the group consisting of ECFP, GFP, EYFP, RFP and Far-red RFP may be used as the acceptor, but the present invention is not limited thereto. In a specific example of the present invention, a pair of ECFP (donor) / EYFP (recipient) was used.

tag (O⁶-alkylguanine-DNA alkyltransferase 변형체), CLIP^TM tag (SNAP

tag 의 변형), Halo tag

(Haloakane dehalogenase 변형체) 같은 단백질을 발현시킨 후, 그 tag에 해당하는 기질에 염료를 달아 넣어주면, 기질에 달린 염료의 종류에 따라 HIF-1α이나 p300을 표지 할 수 있게 된다. 이러한 경우 염료의 종류에 따라 다양한 공여체/수여체 쌍을 이용할 수 있다. 그 예로 상용되는 SNAP tag 형광기질은 SNAP-cell

360 (BFP 대체), SNAP-cell

430 (ECFP 대체), SNAP-cell

505 (GFP 대체), SNAP-cell

Fluorescein (GFP 대체), SNAP-cell

Oregon Green (GFP 대체), SNAP-cell

TMR-star (RFP 대체) 등이 있다. 또한 Halo tag의 경우, Halo Tag

TMR (RFP대체), Halo Tag

Oregon Green (GFP대체), Halo Tag

diAcFAM (GFP대체), Halo Tag

coumarin (BFP대체), Halo Tag

Alexa Fluoro 488 (GFP대체), Halo Tag

R110 (RFP대체) 등이 상용되고 있다. 마찬가지로, 두 염료의 스펙트럼 겹침 현상이 존재한다면, 즉 공여체의 형광 스펙트럼 범위가 수여체의 흡광 스펙트럼 범위에 일부 혹은 전체가 포함되는 경우, 형광 공명 에너지 현상을 관측 할 수 있다. 다만 두 단백질의 결합 정도를 나타내는 에너지 전달 효율 값 자체는 사용되는 염료, 혹은 형광 단백질에 따라 달라질 수 있는데, 그 정도는 스펙트럼 겹침 현상을 비롯한 다양한 인자들에 따라 달라지게 된다. (아래 효율 계산 식 참고)On the other hand, fluorescent labeling of the intracellular proteins includes not only fluorescent proteins but also labeling methods using direct dyes or labeled proteins. For example, SNAP

tag (O ⁶ -alkylguanine-DNA alkyltransferase variant), CLIP ^TM tag (SNAP

tag), Halo tag

(Haloakane dehalogenase variant), and then adding a dye to the substrate corresponding to the tag, it is possible to label HIF-1α or p300 depending on the type of dye attached to the substrate. In this case, a variety of donor / acceptor pairs may be used depending on the type of dye. For example, commonly used SNAP tag fluorescent substrates are SNAP-cell

360 (BFP replacement), SNAP-cell

430 (ECFP replacement), SNAP-cell

505 (GFP replacement), SNAP-cell

Fluorescein (GFP replacement), SNAP-cell

Oregon Green (GFP replacement), SNAP-cell

And TMR-star (RFP replacement). In the case of Halo tags, Halo Tag

TMR (Alternative to RFP), Halo Tag

Oregon Green (GFP replacement), Halo Tag

diAcFAM (GFP replacement), Halo Tag

coumarin (BFP replacement), Halo Tag

Alexa Fluoro 488 (GFP replacement), Halo Tag

R110 (substitute RFP), and the like are commercially available. Similarly, fluorescence resonance energy phenomena can be observed if there is a spectral overlapping phenomenon between the two dyes, that is, when the fluorescent spectrum range of the donor includes some or all of the absorption spectrum range of the acceptor. However, the value of the energy transfer efficiency, which indicates the degree of binding of the two proteins, may vary depending on the dye or fluorescent protein used. The degree of the energy transfer efficiency depends on various factors including the spectrum overlapping phenomenon. (Refer to efficiency calculation formula below)

The fluorescence energy donor or fluorescence energy donor may be labeled on either HIF-1α or p300 protein. However, the p300 protein is always present in the nucleus in the cell, and HIF-1α is eliminated. When hypoxia is induced, It is preferable to label the p300 protein with a fluorescent energy donor and HIF-1? With a fluorescent energy donor in order to increase the fluorescence intensity of the acceptor by increasing the energy.

The peptide sequences of the HIF-1.alpha. And p300 proteins in the present invention are not particularly limited. In a specific example of the present invention, human HIF-1α (Genebank accession number AAC50152; gene (mRNA) sequence: U22431, SEQ ID NO: 3) of SEQ ID NO: 1 and Human p300 protein (Genebank accession number AAA18639 (2414aa) In yet another example of the present invention, the p300 protein has a full-length amino acid sequence of the p300 protein (Genebank, Accession No. AAA18639, SEQ ID NO: 2), and the like. ; 1013 amino acids from the 323rd to 423rd positions in which the CH3 domain binding to at least HIF-1 alpha is present in the amino acid sequence described in the gene (mRNA) sequence: U01877, SEQ ID NO: 4) It may be a polypeptide comprising 101 to 2414 consecutive amino acids including 101 amino acids from the 323 th to 423 th amino acids in the amino acid sequence of SEQ ID NO: 2. In addition, H IF-1.alpha. Contains 41 amino acids from the 786th to 826th of the HIF-1α C-terminal side binding to at least the p300 protein among the HIF-1α full-length amino acid sequence (GenBank entry number AAC50152, SEQ ID NO: 1) And may be, for example, a polypeptide comprising 41 to 826 consecutive amino acids including 41 amino acids 786 to 826 in the amino acid sequence of SEQ ID NO: 1. In an embodiment of the present invention, HIF- A p300 protein comprising the HIF-1 alpha full length amino acid sequence of SEQ ID NO: 1 and the amino acid sequence of 1-528 of the full length of SEQ ID NO: 2 as the p300 protein was used.

The vector used for the production of the plasmid is not limited as long as it can be expressed in eukaryotic cells. It is also possible to produce a stable cell line that always expresses HIF-1α or p300 protein inserted in eukaryotic cells including a gene having antibiotic resistance It is better if it is.

The cells may be all eukaryotic cells, living cells, and cells derived from, for example, human, monkey, rodent (mouse, rat, etc.). In addition, the cell may be a cell existing in vivo, or may be a cell isolated from a living body when it is a human-derived cell. In the examples of the present invention, HeLa cells and PC3 cells were used, but the present invention is not limited thereto.

There is no particular limitation on the method for injecting the plasmid into the eukaryotic cell, and methods such as electroporation, gene-gun, chemical methods (cationic liposome or cationic polymer) can be used Depending on the type of eukaryotic cell used, a particular method well known in the art may be selected.

The present invention relates to a p300 protein plasmid expressing a fusion protein of the p300 protein with a fluorescent energy donor (for example, ECFP, etc.), HIF-1α expressing a fusion protein of HIF-1α and a fluorescent energy acceptor (eg, EYFP, etc.) The plasmid is injected into a cell (for example, HeLa cell), and then the binding between the two proteins is calculated using fluorescence energy transfer efficiency. In this case, HIF-1α can be treated with DFO (Desferoxamine), CoCl _2, etc. to induce HIF-1α binding to p300 without degrading only under hypoxic conditions.

In the present invention, a fluorescent substance (a fluorescent energy donor and a fluorescent energy acceptor) can independently bind to the N-terminal, C-terminal, or both terminals of the p300 protein and HIF-1α. HIF-1? N-ECFP, p300 N-ECFP, p300 N-EYFP, p300 C-ECFP, p300 Y-ECFP, HIF- ECFP and the like can be manufactured and used.

The fluorescence energy transfer efficiency was different depending on whether the fluorescent substance was present at the N-terminal and / or C-terminal and the combination of the two proteins. When the energy donor and the donor were both at the same terminal, that is, (eg, p300 C-ECFP + HIF-1α C-EYFP), or both the donor and the donor are present at the N terminus of the p300 protein and HIF-1α , p300 N-ECFP + HIF-1α N-EYFP). The reason for this is that the fluorescence energy transfer efficiency is affected by various factors but is sensitive to the distance of the two proteins. Therefore, the above results suggest that the two fluorescent materials are structurally located closer to each other when they are at the same terminal . Therefore, in a preferred specific example of the present invention, the fluorescent energy donor and the fluorescent energy acceptor are respectively bound to the same terminal of the p300 protein and HIF-1?, For example, the C terminal.

The fluorescent signal may be generated by expressing a fluorescent substance such as a fluorescent protein or a labeled protein in the transfected cells. This step can be carried out by culturing the cells under normal conditions. The culture medium and culture conditions may be appropriately selected depending on the type of the cell, and this is easily understood in the technical field to which the present invention belongs. For example, HeLa cells can be cultured for 18-24 hours at 37 < 0 > C using DMEM medium to express the protein, but are not limited thereto.

The fluorescence measurement method (means) is not particularly limited, but a fluorescence microscope capable of measuring the shape and position of a cell and the intensity of each fluorescence signal can be used. Here, a confocal microscope, an epi-microscope, a time-resolved fluorescence imaging microscope, etc., which can reduce the noise or background effect by a fluorescence microscope, can be used. It is also more effective to use a laser as a light source for exciting a fluorescent substance (e.g., fluorescent protein, dye), but a lamp and an excitation bandpass filter for selecting a specific wavelength may also be used. However, when using a lamp, it is important to select a filter in a narrow region to selectively excite only specific fluorescent proteins. In the embodiment of the present invention, the intensity of fluorescence was measured using a confocal microscope or an epi-microscope, but the fluorescence decay time of the fluorescent protein was measured through fluorescence imaging using a time-resolved fluorescence imaging microscope to obtain fluorescence energy transfer efficiency May be more preferable.

The fluorescence energy transfer efficiency can be specifically obtained from the following equation. When the obtained fluorescence energy transfer efficiency (FRET efficiency) is 0.1-0.2, it can be judged that p300 protein and HIF-1? Here, FRET efficiency has a value between 0 and 1, and the larger the value, the closer the distance between the two proteins is. A more detailed formula is shown in Example 2. The fluorescence energy transfer efficiency depends on the distance between the two proteins, assuming that the various physical properties shown in the equation are constant. Even if the binding between the two proteins is stronger, the fluorescence energy transfer efficiency It does not change.

[expression]

FRET index = I _DA - (a _{DA D} I) - (I a _{A DA)}

FRET efficiency = FRET index / 255

(255: maximum value for _IDA in 8 bit images)

Where a _D and a _A denote the leak rate of the fluorescence of the donor and the fluorescence rate due to the water absorption, respectively. Fluorescence leakage of the donor and calculation of fluorescence ratio by absorbance of the receiving body are described in more detail in FIG. 2 and in the examples. I _DA is booty and to donor mean results of analysis at the same time, the donor after here within the expressing cells can booty fluorescent segieul actual fluorescence energy transfer efficiency is (1) a direct where leakage of the donor itself, the fluorescence intensity of the donor (a _D I _DA ), and (2) the fluorescence intensity (a _A I _DA ) after the donor was excited directly at the donor excitation wavelength. Specific efficiency calculations are made in the following examples. When the binding of p300 C-ECFP + HIF-1? C-EYFP was examined by treating DFO in HeLa cells, the average fluorescence resonance energy transfer efficiency was about 15% on average (see Examples). not have the three-dimensional structure of p300 with HIF-1α is known because actually p300 C-ECFP + HIF-1α C-EYFP in predictable distance of ECFP and EYFP, but on the basis of the above experiments R ₀ value of ECFP and EYFP (energy efficiency 50%) is about 5 nm, it is expected that the distance between the two molecules is about 6.7 nm from this transfer efficiency value. These measurements are average values of many molecules in the cell. Therefore, when the fluorescence energy transfer efficiency is higher than the measured value of 15%, (1) the more molecules are bonded on average, (2) You may think that the distance gets closer. However, since the distance between two molecules does not depend on the strength of the bond, it can be considered that more molecules are bonded on average. Therefore, the increase and decrease of fluorescence energy transfer efficiency can be used as a measure of how many molecules of p300 and HIF-1α bound to each other are bound to each other.

For real-time imaging in live cells, equipment other than fluorescence microscopy is needed, first a temperature controller to maintain the cells at 37 ° C, and a chamber containing carbon dioxide to adjust the cell culture pH. These devices were purchased from Live Cell Instrument (Korea). In an embodiment of the present invention, DFO (desferrioxamine) is treated to produce conditions similar to hypoxia. In order to actually induce hypoxia, experiments are carried out by flowing a gas with controlled concentrations of carbon dioxide and oxygen.

In another example, the present invention provides a method for screening a HIF-1α peptide and a p300 protein binding regulator (inhibitor or promoter) using the fluorescence polarization measurement method.

1) preparing a cell in which the aforementioned N-terminus, C-terminus, or HIF-1α labeled with a first fluorescent substance at the end, and p300 protein whose N-terminus, C-terminus or both ends are labeled with a second fluorescent substance Wherein one of the first fluorescent material and the second fluorescent material is a donor for transmitting fluorescence energy and the other is an acceptor for receiving fluorescence energy; And

2) contacting the candidate compound with the cell and measuring fluorescence energy transfer efficiency after incubation; And

3) comparing the fluorescence energy transfer efficiency measured in the candidate compound-contacted cells with the fluorescence energy transfer efficiency measured in cells not contacted with the candidate compound

. ≪ / RTI >

In this case, the fluorescence energy transfer efficiency may be measured by measuring the fluorescence intensity as described above, or by measuring the fluorescence decay time of fluorescent molecules, and the fluorescence energy transfer efficiency measured in the candidate compound- When the candidate compound is judged to be a binding promoter between the HIF-1 alpha and the p300 protein when the fluorescence energy transfer efficiency measured in the untouched cell is higher than the fluorescent energy transfer efficiency measured in the untouched cell, the fluorescence energy transfer efficiency measured in the candidate compound- When the fluorescence energy transfer efficiency is lower than the fluorescence energy transfer efficiency measured in the untreated cells, the candidate compound is judged to be a binding inhibitor between HIF-1α and p300 protein.

The binding promoter between HIF-1α and p300 proteins screened by the above method is useful as a therapeutic agent for diseases such as cardiac damage caused by anemia among the symptoms caused by lack of oxygen. In addition, HIF-1α screened by the above- p300 protein interbody binding inhibitor is useful as a therapeutic agent for diseases such as ischemic diseases such as coronary artery insufficiency, brain injury, angiopathy, and cancer.

The candidate compound may be any substance used in the development of a new drug, and there is no particular limitation. For example, the candidate compound may be one selected from the group consisting of antibodies (proteins), peptides, oligonucleotides, polynucleotides, synthetic compounds, etc. that can act to compete with HIF-1α and promote or inhibit binding to p300 protein Or more.

In a preferred embodiment of the present invention, a HIF-1α protein genetically modified at three sites (L795P, C800R, and L818S mutants) of the 786-826th amino acid region, which has an important influence on the binding between HIF-1α and p300 in the HIF- Fluorescence energy transfer efficiency between HIF-1α and p300 was calculated. It is known that the above-mentioned modified protein is inhibited in binding with p300 (Gu, J. et al., J. Biol. Chem., 276: 3550-3554, 2001). When compared to HIF-1α without genetic modification, it showed low fluorescence energy transfer efficiency and it was confirmed that the binding between two proteins was inhibited.

From the preferred embodiments of the present invention, inhibitors that inhibit the binding of HIF-1α and p300, which have not been disclosed previously, were screened and the intracellular role of clioquinol (CQ), which is known to affect both binding, was also confirmed.

As described above, the present invention can quantitatively analyze the binding between HIF-1.alpha. And p300 protein in a cell in real time using a fluorescence resonance energy transfer method. Therefore, HIF- 1 < / RTI > and p300 protein binding inhibitors or in vivo in real time.

FIG. 1 is a graph showing the absorption and fluorescence spectra of the fluorescent proteins ECFP and EYFP, respectively. In FIG. 1, a portion where the fluorescence spectrum of the fluorescent energy donor ECFP and the absorption spectrum of the fluorescent energy-receiving EYFP overlap each other This is where the overlap occurs, which affects energy transfer efficiency.
FIG. 2 shows fluorescence images of ECFP in HeLa cells expressing p300 labeled with fluorescent protein ECFP at the C-terminus and fluorescence images of EYFP in cells expressing HIF-1α labeled with the fluorescent protein EYFP at the C-terminus (A _D ) of the donor itself by direct excitation of the donor and the leakage (a _A ) to the donor wavelength of the acceptor fluorescence by the direct excitation of the acceptor.
The top row of FIG. 3 shows the HIF-1α triple mutant (L795P, C800R, L818S) C-EYFP labeled with EYFP and p300 (SEQ ID NO: 1) using HIF-1α, a genetically modified p300 and binding site of HIF- (I _DD ), donor-induced fluorescence intensities (I _DA ), and acceptor fluorescence intensities (I _AA ), respectively, in HeLa cells expressing C- ), And then calculated the fluorescence resonance energy transfer efficiency using a _D , a _A calculated from FIG. 2, and the results (top row) of the fluorescence resonance energy transfer result and the p300 C-ECFP and HIF-1α C-EYFP The donor-induced fluorescence intensities (I _DD ) and donor-induced fluorescence intensities (I _DD Or FRET), and fluorescence intensity (I _AA ) image of the receiving body fluorescence intensity by the receiving body. Then, fluorescence resonance energy transfer efficiency is calculated using a _D and a _A calculated from FIGS. 1 and 2, It shows the result (below column).
Figure 4 shows the donor fluorescence intensities (I _DD ) and donor fluorescence intensities (I _DA (I)) in PC3 cells expressing p300 C-ECFP and HIF-1α C- Or FRET), and fluorescence intensity (I _AA ) image of the receiving body fluorescence intensity by the receiving body. Then, fluorescence resonance energy transfer efficiency is calculated using a _D and a _A calculated from FIGS. 1 and 2, The lower row shows that the addition of CQ (Clioquinole) and zinc ion does not affect the binding of the two binders, so there is no difference in fluorescence resonance energy transfer efficiency.
FIG. 5 shows fluorescence resonance energy transfer efficiency after addition of a compound that affects the binding of p300 C-ECFP and HIF-1α C-EYFP. As the compounds were all dissolved in DMSO, DMSO alone was used as a control The relative value of 1 is fixed, and the degree to which the energy transfer efficiency is changed by other compounds is indicated.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

< Example  1> fluorescent protein Labeled HIF -1α and p300  Preparation of plasmids expressing proteins and HeLa Intracellularly  Injection ( tansfection )

In order to prepare a plasmid expressing the HIF-1.alpha. And the p300 protein labeled with the fluorescent protein ECFP or EYFP, a nucleotide sequence (SEQ ID NO: 3) coding for Human HIF-1α of SEQ ID NO: 1 and a nucleotide sequence (SEQ ID NO: 4) encoding the amino acid sequences 1-528 of human p300 proteins were cloned into pECFP-C1, pECFP-N1, pEYFP-C1 and pEYFP-N1 vectors (Clontech, USA) (Plasmid maxi kit, Qiagen, Germany) were inoculated into DH5a bacteria (Qiagen, Germany), and the plasmids were mass-produced (restriction site XhoI and BamHI for p300, KpnI and BamHI for HIF- Germany). As a result, plasmids having the following four different structures were prepared for each of Human HIF-1α of SEQ ID NO: 1 and Human p300 of SEQ ID NO: 2.

HIF-1α N-EYFP (pEYFP-C1 vector), HIF-1α N-ECFP (HIF-1α coding gene is cloned into pECFP-C1 vector and thus ECFP is present at the N terminus of HIF- HIF-1α coding gene clone, thus EYFP is present at the N-terminus of HIF-1α), HIF-1α C-ECFP (HIF-1α coding gene cloning into pECFP-N1 vector, And HIF-1α C-EYFP (HIF-1α coding gene cloning into the pEYFP-N1 vector, thus EYFP is present at the C-terminus of HIF-1α)

p300: p300 N-ECFP (p300 protein coding gene cloning into pECFP-C1 vector, thus ECFP is present at the N terminus of p300), p300 N-EYFP (cloning p300 protein coding gene into pEYFP-C1 vector, p300 C-ECFP (p300 protein coding gene cloning in pECFP-N1 vector, thus ECFP is present at the C terminus of p300), and p300 C-EYFP (located in the N terminus of p300) cloning of the p300 protein coding gene, thus EYFP is present at the C terminus of p300)

HeLa cells were cultured in DMEM medium (Invitrogen, USA) containing 10% FBS (Invitrogen, USA) and 1% Penicillin / Streptomycin (Invitrogen, USA) at 37 ° C. Microscopic observations were made one day before plasmid injection About 3 × ^{10 4} cells were attached to the container (1.5 cm ² , SPL, Korea) of the cover glass bottom for easy handling. 0.5 mg of the plasmid prepared above was injected into the above cells using cation liposome (Lipofectamine 2000, Invitrogen, USA) or electroporator (Neon transfection system, Invitrogen, USA). Detailed methods of injecting followed the method provided by Invitrogen.

In this example, p300 was labeled with a fluorescent energy donor (ECFP), and HIF-1α was labeled with a fluorescent energy acceptor (EYFP). For the calculation of the actual FRET efficiency, only donor (p300-ECFP) transfected cells, recipient only (HIF 1α-EYFP) transfected cells, and donor (p300-ECFP) and recipient (HIF 1α-EYFP) The transfected cells were prepared. As a control, pure ECFP and EYFP not labeled with a specific protein and ECFP and EYFP transfected cells were prepared.

< Example  2> Using fluorescence energy transfer efficiency HeLa Intracellularly HIF  Measurement of binding between 1a and p300

The confocal fluorescence microscope used in the experiment was Fluoview2000 from Olympus and laser wavelengths of 405 nm and 514 nm were used for excitation of ECFP and EYFP, respectively. ECFP fluorescence image and EYFP fluorescence image were obtained using 480-495 nm bandpass filter (BP) and 535-565 nm BP, respectively. Using the transfected cells mentioned in Example 1, I _DD (detection of donor-stimulated donor fluorescence intensity), I _DA (detection of donor-excited fluorescence intensity, that is, intensity at the fluorescence energy transfer wavelength Detection), and I _AA (fluorescence intensities detection of water bodies). The results thus obtained are shown in Fig. 1 and Fig.

Image processing for FRET calculation was performed using NIH's free program ImageJ, and actual calculation was performed using ImageJ's plugin program 'FRET and colocalization analyzer'. To calculate the actual FRET efficiency, the following equation was used.

FRET index image _DA = I - (a _{DA D} I) - (I a _{A DA)}

FRET efficiency = FRET index / 255

(255: maximum value for _IDA in 8 bit images)

Here, a _D and a _A respectively refer to the leakage ratio of the fluorescence of the donor and the fluorescence ratio by the absorber absorption. I _DA is means a booty and a result of the donor can then this donor in the same time expressing cells booty fluorescent segieul detected, the actual fluorescence energy transfer efficiency is (1) leakage of the donor itself fluorescence caused by direct excitation of the donor (a _{D IDA} ), and (2) fluorescence intensities (a _A I _DA ) were subtracted after the donor was directly excited at the donor excitation wavelength.

Elements a _D and a _A, which take into account the leakage of the donor and fluorescence by direct excitation of the recipient, were obtained using cells labeled only with donor and cells labeled donor only. The detailed process is as follows.

1. Leakage Fluorescence Leakage Calculations

2. Direct fluorescence calculation by water absorption

3. Actual FRET efficiency calculation considering above 1,2

1. Leakage Fluorescence Leakage Calculations

After obtaining the images of I _DD and I _DA using cells expressing only the donor, a graph showing the I _DD values and the I _DA values on the x axis and the y axis was obtained for each pixel. The slope of the straight line obtained here is a _d Lt; / RTI &gt; If the two values are similar, then the slope is close to 1. The above image was repeated 10 times to obtain the average of the slopes (see the upper part of FIG. 2).

2. Water body On absorption  Fluorescence

The images of I _DA and I _AA were obtained by using cells expressing only water bodies, and a graph showing the I _AA value and the I _DA value of each pixel was obtained. The slope of the obtained straight line is a a _A value immediately. The above image was repeated 10 times to obtain the average of the tilt (refer to the bottom of FIG. 2).

FIG. 2 shows fluorescence images of ECFP in HeLa cells expressing p300 labeled with fluorescent protein ECFP at the C-terminus and fluorescence images of EYFP in cells expressing HIF-1α labeled with the fluorescent protein EYFP at the C-terminus (A _D ) of the donor itself due to direct excitation of the donor and the leakage (a _A ) to the donor wavelength of the acceptor fluorescence due to the direct excitation of the acceptor. First, the donor's own fluorescence leakage (a _D ) due to the direct excitation of the donor was about 1 from the slope. This means that even if there is no fluorescence and FRET of the donor itself, the leakage of fluorescence by the donor at the acceptor fluorescence wavelength is almost the same value. In the case of water bodies, the fluorescence value of the receiving body by direct absorption of water bodies is not so large, so the slope value is close to zero. Therefore, it can be concluded that fluorescence leakage of the donor can have a significant effect on the efficiency when the actual fluorescence resonance energy efficiency is calculated.

3. Considering 1 and 2 above Practical FRET  Efficiency calculation

Expression FRET index image _DA = I - (a _{DA D} I) - calculating the FRET efficiency by using a (I a _{A DA)} was determined for FRET efficiency image directly. At this time, the colocalization FRET based on the fluorescence coexisting in the minimum value, the maximum value, and the donor and accceptor of the FRET is calculated and shown in FIG.

The top row of FIG. 3 shows the HIF-1α triple mutant (L795P, C800R, L818S) C-EYFP labeled with EYFP and p300 (SEQ ID NO: 1) using HIF-1α, a genetically modified p300 and binding site of HIF- (I _DD ), donor-induced fluorescence intensities (I _DA ), and acceptor fluorescence intensities (I _AA ), respectively, in HeLa cells expressing C- ) Image, and then calculates a fluorescence resonance energy transfer efficiency using a _D , a _A calculated from FIG. 2, and shows the result of the image representation. In the case of a triple mutant, the energy transfer efficiency is close to zero. The bottom row of FIG. 3 shows the donor-induced fluorescence intensities (I _DD ), donor-induced fluorescence intensities (I _DA ), and donor-induced fluorescence intensities, respectively, in HeLa cells expressing p300 C-ECFP and HIF- , And the fluorescence intensity (I _AA ) image of the receiving body was measured, and then the fluorescence resonance energy transfer efficiency was calculated using a _D and a _A calculated from FIGS. 1 and 2, will be.

These fluorescence energy transfer efficiencies are theoretically a function of the distance between the donor and acceptor, and the distance between the two proteins can be deduced from the energy transfer efficiency.

Where R is the distance between the two proteins, E is the fluorescence energy transfer efficiency, and R ₀ is the Forster constant, which is the distance when the energy transfer efficiency reaches 50%.

Where n is the refractive index of the solution, k is the angle between the two fluorescent molecule dipoles, φ _D is the fluorescence yield of the donor, and J is the degree of overlap between the absorbance spectrum of the donor and the fluorescence spectrum of the acceptor. As can be seen from the above equation, the exact distance can be calculated only by considering various factors such as the fluorescence yield of the donor, the overlap between the fluorescence of the donor and the absorption spectrum of the acceptor, and the topographical elements of the two fluorescent proteins and the refractive index of the solution.

In the case of FIG. 3, when HIF-1α is stabilized by treatment with DFO (desferoxamine, 150 mM, 6 hours) when p300 C-ECFP and HIF-1α C-EYFP are expressed, And the color of the colocalized FRET of the last picture shows that the efficiency is about 15%. (As shown in the color change diagram on the right, blue is blue when energy efficiency is less than 5%, violet-red when it is about 5-10%, and red-orange when it is about 10-15% In this case, HIF-1α is not degraded only in hypoxic conditions in HeLa cells, but is bound to p300. When the energy donor and donor were both at the C-terminus (p300 C-ECFP + HIF-1α), the fluorescence energy transfer efficiency was different depending on whether the fluorescent protein was present at the N- or C- C-EYFP) have high energy transfer efficiency.

The binding site with p300 in HIF-1 alpha is known to contain amino acid sequence 786-826. HIF-1 alpha triple mutant (HIF-1 alpha L795P, C800R, L818S mutant, Gu, J. et al., J. Biol. Chem., 276: 3550-3554, 2001) genetically modified in three amino acid sequences (HIF-1α C-EYFP triple mutant) was inserted into the pEYFP-N1 (clontech, USA) plasmid. The HIF-1.alpha. Was stabilized by injecting HeLa cells simultaneously with p300 (p300C-ECFP) and DFO (Desferoxamine, 150 mM, 6 hours) by using EYFP-labeled plasmid in the above-mentioned modified gene, And the fluorescence energy transfer efficiency of the binding of the two proteins was calculated. The obtained results are shown in the upper row of Fig. When DFO is treated to inhibit the degradation of HIF-1α, fluorescence energy efficiency is 0 or more, and most of the fluorescence energy transfer efficiency is 0 when the binding of two proteins is inhibited.

As shown in FIG. 3, when HIF-1α is stabilized by treating DFO with p300 C-ECFP and HIF-1α C-EYFP, fluorescent energy transfer due to binding of two proteins occurs in the nucleus, And about 15%, respectively. However, fluorescence energy transfer was not observed in the case of genetically modified HIF-1α. This is an example showing that the binding of two proteins can be confirmed by using fluorescence energy transfer phenomenon.

As shown in the above examples, the degree of influence of the binding of two proteins can be measured from the fluorescence energy transfer efficiency. If the fluorescent protein is applied to the stable cell line that always expresses two fluorescent proteins, The compounds of the present invention can be used for screening compounds which are useful for screening compounds.

< Example  3> Addition of metal ions and compounds HIF -1α and p300  Bond change

CH1 domain portion of p300 binding to HIF-1α is known to have the proper conditions to combine the three Zn ² to stabilize the structure by binding to ^{+ HIF-1α (Freedman, SJ} , et al, PNAS, 99.: 5367-5372, 2002). Therefore, adding a compound that chelates Zn ^{2 +} is thought to alter the structure of p300 and inhibit binding of the two proteins. Clioquinol (CQ), while the Zn ^{2 +} chelation at the same time are known to carry the Zn ^{2 +} into the cells is also known to inhibit the activity of PHD2 which is involved in the hydroxylation of HIF-1α as well. In the examples of the present invention, the influence of CQ and Zn ^{2 +} on the binding of HIF-1α and p300 in cells was examined using fluorescence resonance energy phenomenon.

FIG. 4 shows fluorescence energy resonance efficiency before and after treatment of CQ (50 mM) Zn ^{2 +} (10 mM) with PC3 cells expressing HIF-1α irrespective of oxygen concentration, with p300 C-ECFP, HIF -1 &lt; / RTI &gt;&lt; RTI ID = 0.0 &gt; C-EYFP. &Lt; / RTI &gt; Fluorescence energy efficiency was about 10% similar to that of HeLa cells, and the ratio was maintained regardless of treatment with CQ and Zn ^{2 +} . Therefore, it is considered that the treatment of CQ, Zn ^{2 +} does not affect the binding of HIF-1α and p300.

< Example  4> HIF -1α and p300  Of compounds which inhibit the degree of binding between The degree of inhibition  Real-time measurement in HeLa cells

Finding compounds that inhibit the binding of HIF-1α to p300 by screening small compounds is likely to be helpful in the treatment of many future diseases. Recently, researchers including Kung et al. Have reported that chetomin inhibits the binding of two proteins, and may act as an inhibitor, particularly inhibiting cancer growth (Kung, AL et al. Cancer Cell 6: 33-43, 2004). In order to discover another inhibitor, a compound that inhibits the binding of HIF-1α and p300 was examined using a compound library (NINS custom collection II, Microsource discovery system, USA). Representative compounds exhibiting their effects are shown in FIG. FIG. 5 is a graph showing the fluorescence resonance energy transfer efficiency as 1 as a control group using DMSO (0.5%) added to dissolve the compound in HeLa cells expressing p300 C-ECFP and HIF-1α C-EYFP, The fluorescence resonance energy transfer efficiency was measured after treating chetomin (600 nM), menadionine (13 mM) and 2-mercaptopyrithione (150 mM). The fluorescence resonance energy transfer efficiency of chetomin, a known inhibitor, was reduced by more than 50%, while the efficiency of menadionine and 2-mercaptopyrithione decreased by 30% and 10%, respectively. As a result, the two compounds selected through screening were not as strong as chetomin, but they were found to inhibit the binding of HIF-1α and p300 to some extent.

<110> KOREA INSTITUTE OF SCIENCE AND TECHNOLOGY <120> METHOD FOR QUANTITATIVE ANALYSIS OF REAL TIME INTERACTIONS          BETWEEN HIF1 ALPHA AND p300 PROTEINS <130> DPP20110612KR <160> 4 <170> Kopatentin 1.71 <210> 1 <211> 826 <212> PRT <213> Artificial Sequence <220> Amino acid sequence of full-length human HIF-1 alpha (AAC50152) <400> 1 Met Glu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu   1 5 10 15 Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys              20 25 30 Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu Pro His          35 40 45 Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg Leu Thr Ile      50 55 60 Ser Tyr Leu Arg Val Lys Leu Leu Asp Ala Gly Asp Leu Asp Ile  65 70 75 80 Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys Ala Leu                  85 90 95 Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile             100 105 110 Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr         115 120 125 Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Met     130 135 140 Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys Gly Lys Glu 145 150 155 160 Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr                 165 170 175 Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr Trp Lys Val Leu             180 185 190 His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn Ser Asn Gln Pro         195 200 205 Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile Cys     210 215 220 Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys 225 230 235 240 Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp                 245 250 255 Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly             260 265 270 Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr         275 280 285 Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr Thr Gly Gln     290 295 300 Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp Val Glu Thr Gln 305 310 315 320 Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro Gln Cys Ile Val                 325 330 335 Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His Asp Leu Ile Phe             340 345 350 Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp         355 360 365 Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser     370 375 380 Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu 385 390 395 400 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn                 405 410 415 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn             420 425 430 Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu         435 440 445 Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser     450 455 460 Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro 465 470 475 480 Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp                 485 490 495 Gln Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser G             500 505 510 Pro Asn Ser Ser Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val         515 520 525 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr     530 535 540 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser                 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser             580 585 590 Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln         595 600 605 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp Glu Leu     610 615 620 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile Leu Ile Ala 625 630 635 640 Ser Pro Ser Pro Thr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser                 645 650 655 Ser Pro Tyr Arg Asp Thr Gln Ser Arg Thr Ala Ser Pro Asn Arg Ala             660 665 670 Gly Lys Gly Val Ile Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro         675 680 685 Asn Val Leu Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu     690 695 700 Glu Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg 705 710 715 720 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly Thr                 725 730 735 Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser Leu Ser Trp             740 745 750 Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln Asn Gly Met Glu Gln         755 760 765 Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly     770 775 780 Gln Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys 785 790 795 800 Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu                 805 810 815 Glu Leu Leu Arg Ala Leu Asp Gln Val Asn             820 825 <210> 2 <211> 2414 <212> PRT <213> Artificial Sequence <220> Amino acid sequence of full-length human p300 protein (AAA18639) <400> 2 Met Ala Glu Asn Val Val Glu Pro Gly Pro Pro Ser Ala Lys Arg Pro   1 5 10 15 Lys Leu Ser Ser Ala Leu Ser Ala Ser Ala Ser Asp Gly Thr Asp              20 25 30 Phe Gly Ser Leu Phe Asp Leu Glu His Asp Leu Pro Asp Glu Leu Ile          35 40 45 Asn Ser Thr Glu Leu Gly Leu Thr Asn Gly Gly Asp Ile Asn Gln Leu      50 55 60 Gln Thr Ser Leu Gly Met Val Gln Asp Ala Ala Ser Lys His Lys Gln  65 70 75 80 Leu Ser Glu Leu Leu Arg Ser Gly Ser Ser Pro Asn Leu Asn Met Gly                  85 90 95 Val Gly Gly Pro Gly Gln Val Met Ala Ser Gln Ala Gln Gln Ser Ser             100 105 110 Pro Gly Leu Gly Leu Ile Asn Ser Met Val Lys Ser Pro Met Thr Gln         115 120 125 Ala Gly Leu Thr Ser Pro Asn Met Gly Met Gly Thr Ser Gly Pro Asn     130 135 140 Gln Gly Pro Thr Gln Ser Thr Gly Met Met Asn Ser Pro Val Asn Gln 145 150 155 160 Pro Ala Met Gly Met Asn Thr Gly Thr Asn Ala Gly Met Asn Pro Gly                 165 170 175 Met Leu Ala Ala Gly Asn Gly Gln Gly Ile Met Pro Asn Gln Val Met             180 185 190 Asn Gly Ser Ile Gly Ala Gly Arg Gly Arg Gln Asp Met Gln Tyr Pro         195 200 205 Asn Pro Gly Met Gly Ser Ala Gly Asn Leu Leu Thr Glu Pro Leu Gln     210 215 220 Gln Gly Ser Pro Gln Met Gly Gly Gln Thr Gly Leu Arg Gly Pro Gln 225 230 235 240 Pro Leu Lys Met Gly Met Met Asn Asn Pro Asn Pro Tyr Gly Ser Pro                 245 250 255 Tyr Thr Gln Asn Pro Gly Gln Gln Ile Gly Ala Ser Gly Leu Gly Leu             260 265 270 Gln Ile Gln Thr Lys Thr Val Leu Ser Asn Asn Leu Ser Pro Phe Ala         275 280 285 Met Asp Lys Lys Ala Val Pro Gly Gly Gly Met Pro Asn Met Gly Gln     290 295 300 Gln Pro Ala Pro Gln Val Gln Gln Pro Gly Leu Val Thr Pro Val Ala 305 310 315 320 Gln Gly Met Gly Ser Gly Ala His Thr Ala Asp Pro Glu Lys Arg Lys                 325 330 335 Leu Ile Gln Gln Gln Leu Val Leu Leu Leu His Ala His Lys Cys Gln             340 345 350 Arg Arg Glu Gln Ala Asn Gly Glu Val Arg Gln Cys Asn Leu Pro His         355 360 365 Cys Arg Thr Met Lys Asn Val Leu Asn His Met Thr His Cys Gln Ser     370 375 380 Gly Lys Ser Cys Gln Val Ala His Cys Ala Ser Ser Arg Gln Ile Ile 385 390 395 400 Ser His Trp Lys Asn Cys Thr Arg His Asp Cys Pro Val Cys Leu Pro                 405 410 415 Leu Lys Asn Ala Gly Asp Lys Arg Asn Gln Gln Pro Ile Leu Thr Gly             420 425 430 Ala Pro Val Gly Leu Gly Asn Pro Ser Ser Leu Gly Val Gly Gln Gln         435 440 445 Ser Ala Pro Asn Leu Ser Ser Val Ser Ser Gln Ile Asp Pro Ser Ser Ile     450 455 460 Glu Arg Ala Tyr Ala Ala Leu Gly Leu Pro Tyr Gln Val Asn Gln Met 465 470 475 480 Pro Thr Gln Pro Gln Val Gln Ala Lys Asn Gln Gln Asn Gln Gln Pro                 485 490 495 Gly Gln Ser Pro Gln Gly Met Arg Pro Met Ser Asn Met Ser Ala Ser             500 505 510 Pro Met Gly Val Asn Gly Gly Val Gly Val Gln Thr Pro Ser Leu Leu         515 520 525 Ser Asp Ser Met Leu His Ser Ala Ile Asn Ser Gln Asn Pro Met Met     530 535 540 Ser Glu Asn Ala Ser Val Pro Ser Leu Gly Pro Met Pro Thr Ala Ala 545 550 555 560 Gln Pro Ser Thr Thr Gly Ile Arg Lys Gln Trp His Glu Asp Ile Thr                 565 570 575 Gln Asp Leu Arg Asn His Leu Val His Lys Leu Val Gln Ala Ile Phe             580 585 590 Pro Thr Pro Asp Pro Ala Ala Leu Lys Asp Arg Arg Met Glu Asn Leu         595 600 605 Val Ala Tyr Ala Arg Lys Val Glu Gly Asp Met Tyr Glu Ser Ala Asn     610 615 620 Asn Arg Ala Glu Tyr Tyr His Leu Leu Ala Glu Lys Ile Tyr Lys Ile 625 630 635 640 Gln Lys Glu Leu Glu Glu Lys Arg Arg Thr Arg Leu Gln Lys Gln Asn                 645 650 655 Met Leu Pro Asn Ala Ala Gly Met Val Val Ser Val Met Asn Pro Gly             660 665 670 Pro Asn Met Gly Gln Pro Gln Pro Gly Met Thr Ser Asn Gly Pro Leu         675 680 685 Pro Asp Pro Ser Met Ile Arg Gly Ser Val Pro Asn Gln Met Met Pro     690 695 700 Arg Ile Thr Pro Gln Ser Gly Leu Asn Gln Phe Gly Gln Met Ser Met 705 710 715 720 Ala Gln Pro Pro Ile Val Pro Arg Gln Thr Pro Pro Leu Gln His His                 725 730 735 Gly Gln Leu Ala Gln Pro Gly Ala Leu Asn Pro Pro Gly Tyr Gly             740 745 750 Pro Arg Met Gln Gln Pro Ser Asn Gln Gly Gln Phe Leu Pro Gln Thr         755 760 765 Gln Phe Pro Ser Gln Gly Met Asn Val Thr Asn Ile Pro Leu Ala Pro     770 775 780 Ser Ser Gly Gln Ala Pro Val Ser Gln Ala Gln Met Ser Ser Ser Ser 785 790 795 800 Cys Pro Val Asn Ser Pro Ile Met Pro Pro Gly Ser Gln Gly Ser His                 805 810 815 Ile His Cys Pro Gln Leu Pro Gln Pro Ala Leu His Gln Asn Ser Pro             820 825 830 Ser Pro Val Pro Ser Arg Thr Pro Thr Pro His His Thr Pro Pro Ser         835 840 845 Ile Gly Ala Gln Gln Pro Pro Ala Thr Ile Pro Ala Pro Val Pro     850 855 860 Thr Pro Pro Ala Met Pro Pro Gly Pro Gln Ser Gln Ala Leu His Pro 865 870 875 880 Pro Pro Arg Gln Thr Pro Thr Pro Pro Thr Thr Gln Leu Pro Gln Gln                 885 890 895 Val Gln Pro Ser Leu Pro Ala Ala Pro Ser Ala Asp Gln Pro Gln Gln             900 905 910 Gln Pro Arg Ser Gln Gln Ser Thr Ala Ala Ser Val Pro Thr Pro Asn         915 920 925 Ala Pro Leu Leu Pro Pro Gln Pro Ala Thr Pro Leu Ser Gln Pro Ala     930 935 940 Val Ser Ile Glu Gly Gln Val Ser Asn Pro Pro Ser Thr Ser Ser Thr 945 950 955 960 Glu Val Asn Ser Gln Ala Ile Ala Glu Lys Gln Pro Ser Gln Glu Val                 965 970 975 Lys Met Glu Ala Lys Met Glu Val Asp Gln Pro Glu Pro Ala Asp Thr             980 985 990 Gln Pro Glu Asp Ile Ser Glu Ser Lys Val Glu Asp Cys Lys Met Glu         995 1000 1005 Ser Thr Glu Thr Glu Glu Arg Ser Thr Glu Leu Lys Thr Glu Ile Lys    1010 1015 1020 Glu Glu Glu Asp Gln Pro Ser Thr Ser Ala Thr Gln Ser Ser Ala 1025 1030 1035 1040 Pro Gly Gln Ser Lys Lys Lys Ile Phe Lys Pro Glu Glu Leu Arg Gln                1045 1050 1055 Ala Leu Met Pro Thr Leu Glu Ala Leu Tyr Arg Gln Asp Pro Glu Ser            1060 1065 1070 Leu Pro Phe Arg Gln Pro Val Asp Pro Gln Leu Leu Gly Ile Pro Asp        1075 1080 1085 Tyr Phe Asp Ile Val Lys Ser Pro Met Asp Leu Ser Thr Ile Lys Arg    1090 1095 1100 Lys Leu Asp Thr Gly Gln Tyr Gln Glu Pro Trp Gln Tyr Val Asp Asp 1105 1110 1115 1120 Ile Trp Leu Met Phe Asn Asn Ala Trp Leu Tyr Asn Arg Lys Thr Ser                1125 1130 1135 Arg Val Tyr Lys Tyr Cys Ser Lys Leu Ser Glu Val Phe Glu Gln Glu            1140 1145 1150 Ile Asp Pro Val Met Gln Ser Leu Gly Tyr Cys Cys Gly Arg Lys Leu        1155 1160 1165 Glu Phe Ser Pro Gln Thr Leu Cys Cys Tyr Gly Lys Gln Leu Cys Thr    1170 1175 1180 Ile Pro Arg Asp Ala Thr Tyr Tyr Ser Tyr Gln Asn Arg Tyr His Phe 1185 1190 1195 1200 Cys Glu Lys Cys Phe Asn Glu Ile Gln Gly Glu Ser Val Ser Leu Gly                1205 1210 1215 Asp Asp Pro Ser Gln Pro Gln Thr Thr Ile Asn Lys Glu Gln Phe Ser            1220 1225 1230 Lys Arg Lys Asn Asp Thr Leu Asp Pro Glu Leu Phe Val Glu Cys Thr        1235 1240 1245 Glu Cys Gly Arg Lys Met His Gln Ile Cys Val Leu His His Glu Ile    1250 1255 1260 Ile Trp Pro Ala Gly Phe Val Cys Asp Gly Cys Leu Lys Lys Ser Ala 1265 1270 1275 1280 Arg Thr Arg Lys Glu Asn Lys Phe Ser Ala Lys Arg Leu Pro Ser Thr                1285 1290 1295 Arg Leu Gly Thr Phe Leu Glu Asn Arg Val Asn Asp Phe Leu Arg Arg            1300 1305 1310 Gln Asn His Pro Glu Ser Gly Glu Val Thr Val Arg Val Val His Ala        1315 1320 1325 Ser Asp Lys Thr Val Glu Val Lys Pro Gly Met Lys Ala Arg Phe Val    1330 1335 1340 Asp Ser Gly Glu Met Ala Glu Ser Phe Pro Tyr Arg Thr Lys Ala Leu 1345 1350 1355 1360 Phe Ala Phe Glu Glu Ile Asp Gly Val Asp Leu Cys Phe Phe Gly Met                1365 1370 1375 His Val Gln Glu Tyr Gly Ser Asp Cys Pro Pro Pro Asn Gln Arg Arg            1380 1385 1390 Val Tyr Ile Ser Tyr Leu Asp Ser Val His Phe Phe Arg Pro Lys Cys        1395 1400 1405 Leu Arg Thr Ala Val Tyr His Glu Ile Leu Ile Gly Tyr Leu Glu Tyr    1410 1415 1420 Val Lys Lys Leu Gly Tyr Thr Thr Gly His Ile Trp Ala Cys Pro Pro 1425 1430 1435 1440 Ser Glu Gly Asp Asp Tyr Ile Phe His Cys His Pro Pro Asp Gln Lys                1445 1450 1455 Ile Pro Lys Pro Lys Arg Leu Gln Glu Trp Tyr Lys Lys Met Leu Asp            1460 1465 1470 Lys Ala Val Ser Glu Arg Ile Val His Asp Tyr Lys Asp Ile Phe Lys        1475 1480 1485 Gln Ala Thr Glu Asp Arg Leu Thr Ser Ala Lys Glu Leu Pro Tyr Phe    1490 1495 1500 Glu Gly Asp Phe Trp Pro Asn Val Leu Glu Glu Ser Ile Lys Glu Leu 1505 1510 1515 1520 Glu Glu Glu Glu Glu Glu Arg Lys Arg Glu Glu Asn Thr Ser Asn Glu                1525 1530 1535 Ser Thr Asp Val Thr Lys Gly Asp Ser Lys Asn Ala Lys Lys Lys Asn            1540 1545 1550 Asn Lys Lys Thr Ser Lys Asn Lys Ser Ser Leu Ser Arg Gly Asn Lys        1555 1560 1565 Lys Lys Pro Gly Met Pro Asn Val Ser Asn Asp Leu Ser Gln Lys Leu    1570 1575 1580 Tyr Ala Thr Met Glu Lys His Lys Glu Val Phe Phe Val Ile Arg Leu 1585 1590 1595 1600 Ile Ala Gly Pro Ala Ala Asn Ser Leu Pro Ile Val Asp Pro Asp                1605 1610 1615 Pro Leu Ile Pro Cys Asp Leu Met Asp Gly Arg Asp Ala Phe Leu Thr            1620 1625 1630 Leu Ala Arg Asp Lys His Leu Glu Phe Ser Ser Leu Arg Arg Ala Gln        1635 1640 1645 Trp Ser Thr Met Cys Met Leu Val Glu Leu His Thr Gln Ser Gln Asp    1650 1655 1660 Arg Phe Val Tyr Thr Cys Asn Glu Cys Lys His His Val Glu Thr Arg 1665 1670 1675 1680 Trp His Cys Thr Val Cys Glu Asp Tyr Asp Leu Cys Ile Thr Cys Tyr                1685 1690 1695 Asn Thr Lys Asn His Asp His Lys Met Glu Lys Leu Gly Leu Gly Leu            1700 1705 1710 Asp Asp Glu Ser Asn Asn GIn Gln Ala Ala Ala Thr Gln Ser Pro Gly        1715 1720 1725 Asp Ser Arg Arg Leu Ser Ile Gln Arg Cys Ile Gln Ser Leu Val His    1730 1735 1740 Ala Cys Gln Cys Arg Asn Ala Asn Cys Ser Leu Pro Ser Cys Gln Lys 1745 1750 1755 1760 Met Lys Arg Val Val Gln His Thr Lys Gly Cys Lys Arg Lys Thr Asn                1765 1770 1775 Gly Gly Cys Pro Ile Cys Lys Gln Leu Ile Ala Leu Cys Cys Tyr His            1780 1785 1790 Ala Lys His Cys Gln Glu Asn Lys Cys Pro Val Pro Phe Cys Leu Asn        1795 1800 1805 Ile Lys Gln Lys Leu Arg Gln Gln Gln Leu Gln His Arg Leu Gln Gln    1810 1815 1820 Ala Gln Met Leu Arg Arg Arg Met Ala Ser Met Gln Arg Thr Gly Val 1825 1830 1835 1840 Val Gly Gln Gln Gln Gly Leu Pro Ser Pro Thr Pro Ala Thr Pro Thr                1845 1850 1855 Thr Pro Thr Gly Gln Gln Pro Thr Thr Pro Gln Thr Pro Gln Pro Thr            1860 1865 1870 Ser Gln Pro Gln Pro Thr Pro Pro Asn Ser Met Pro Pro Tyr Leu Pro        1875 1880 1885 Arg Thr Gln Ala Ala Gly Pro Val Ser Gln Gly Lys Ala Ala Gly Gln    1890 1895 1900 Val Thr Pro Pro Thr Pro Pro Gln Thr Ala Gln Pro Pro Leu Pro Gly 1905 1910 1915 1920 Pro Pro Thr Ala Val Glu Met Ala Met Gln Ile Gln Arg Ala Ala                1925 1930 1935 Glu Thr Gln Arg Gln Met Ala His Val Gln Ile Phe Gln Arg Pro Ile            1940 1945 1950 Gln His Gln Met Pro Pro Met Thr Pro Met Ala Pro Met Gly Met Asn        1955 1960 1965 Pro Pro Pro Met Thr Arg Gly Pro Ser Gly His Leu Glu Pro Gly Met    1970 1975 1980 Gly Pro Thr Gly Met Gln Gln Gln Pro Pro Trp Ser Gln Gly Gly Leu 1985 1990 1995 2000 Pro Gln Pro Gln Gln Leu Gln Ser Gly Met Pro Arg Pro Ala Met Met                2005 2010 2015 Ser Val Ala Gln His Gly Gln Pro Leu Asn Met Ala Pro Gln Pro Gly            2020 2025 2030 Leu Gly Gln Val Gly Ile Ser Pro Leu Lys Pro Gly Thr Val Ser Gln        2035 2040 2045 Gln Ala Leu Gln Asn Leu Leu Arg Thr Leu Arg Ser Ser Ser Ser Pro    2050 2055 2060 Leu Gln Gln Gln Gln Val Leu Ser Ile Leu His Ala Asn Pro Gln Leu 2065 2070 2075 2080 Leu Ala Ala Phe Ile Lys Gln Arg Ala Ala Lys Tyr Ala Asn Ser Asn                2085 2090 2095 Pro Gln Pro Ile Pro Gly Gln Pro Gly Met Pro Gln Gly Gln Pro Gly            2100 2105 2110 Leu Gln Pro Pro Thr Met Pro Gly Gln Gln Gly Val His Ser Asn Pro        2115 2120 2125 Ala Met Gln Asn Met Asn Pro Met Gln Ala Gly Val Gln Arg Ala Gly    2130 2135 2140 Leu Pro Gln Gln Gln Pro Gln Gln Gln Leu Gln Pro Pro Met Gly Gly 2145 2150 2155 2160 Met Ser Pro Gln Ala Gln Gln Met Asn Met Asn His Asn Thr Met Pro                2165 2170 2175 Ser Gln Phe Arg Asp Ile Leu Arg Arg Gln Gln Met Met Gln Gln Gln            2180 2185 2190 Gln Gln Gln Gly Ala Gly Pro Gly Ile Gly Pro Gly Met Ala Asn His        2195 2200 2205 Asn Gln Phe Gln Gln Pro Gln Gly Val Gly Tyr Pro Pro Gln Pro Gln    2210 2215 2220 Gln Arg Met Gln His Met Gln Gln Met Gln Gln Gly Asn Met Gly 2225 2230 2235 2240 Gly Ile Gly Gly Ale Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly                2245 2250 2255 Leu Gln Ala Tyr Gln Gln Arg Leu Leu Gln Gln Gln Met Gly Ser Pro            2260 2265 2270 Val Gln Pro Asn Pro Met Ser Pro Gln Gln His Met Leu Pro Asn Gln        2275 2280 2285 Ala Gln Ser Pro His Leu Gln Gly Gln Gln Ile Pro Asn Ser Leu Ser    2290 2295 2300 Asn Gln Val Arg Ser Pro Gln Pro Val Ser Pro Arg Pro Gln Ser 2305 2310 2315 2320 Gln Pro Pro His Ser Ser Pro Ser Pro Arg Met Gln Pro Gln Pro Ser                2325 2330 2335 Pro His His Val Ser Pro Gln Thr Ser Ser Pro His Gly Leu Val            2340 2345 2350 Ala Ala Gln Ala Asn Pro Met Glu Gln Gly His Phe Ala Ser Pro Asp        2355 2360 2365 Gln Asn Ser Met Leu Ser Gln Leu Ala Ser Asn Pro Gly Met Ala Asn    2370 2375 2380 Leu His Gly Ala Ser Ala Thr Asp Leu Gly Leu Ser Thr Asp Ser Ser 2385 2390 2395 2400 Asp Leu Asn Ser Asn Leu Ser Gln Ser Thr Leu Asp Ile His                2405 2410 <210> 3 <211> 3678 <212> DNA <213> Artificial Sequence <220> <223> Human HIF-1a mRNA (U22431), embedded bases of 29-2509 are the          coding sequence <400> 3 gtgaagacat cgcggggacc gattcaccat ggagggcgcc ggcggcgcga acgacaagaa 60 aaagataagt tctgaacgtc gaaaagaaaa gtctcgagat gcagccagat ctcggcgaag 120 taaagaatct gaagtttttt atgagcttgc tcatcagttg ccacttccac ataatgtgag 180 ttcgcatctt gataaggcct ctgtgatgag gcttaccatc agctatttgc gtgtgaggaa 240 acttctggat gctggtgatt tggatattga agatgacatg aaagcacaga tgaattgctt 300 ttatttgaaa gccttggatg gttttgttat ggttctcaca gatgatggtg acatgattta 360 catttctgat aatgtgaaca aatacatggg attaactcag tttgaactaa ctggacacag 420 tgtgtttgat tttactcatc catgtgacca tgaggaaatg agagaaatgc ttacacacag 480 aaatggcctt gtgaaaaagg gtaaagaaca aaacacacag cgaagctttt ttctcagaat 540 gaagtgtacc ctaactagcc gaggaagaac tatgaacata aagtctgcaa catggaaggt 600 attgcactgc acaggccaca ttcacgtata tgataccaac agtaaccaac ctcagtgtgg 660 gtataagaaa ccacctatga cctgcttggt gctgatttgt gaacccattc ctcacccatc 720 aaatattgaa attcctttag atagcaagac tttcctcagt cgacacagcc tggatatgaa 780 attttcttat tgtgatgaaa gaattaccga attgatggga tatgagccag aagaactttt 840 aggccgctca atttatgaat attatcatgc tttggactct gatcatctga ccaaaactca 900 tcatgatatg tttactaaag gacaagtcac cacaggacag tacaggatgc ttgccaaaag 960 aggtggatat gtctgggttg aaactcaagc aactgtcata tataacacca agaattctca 1020 accacagtgc attgtatgtg tgaattacgt tgtgagtggt attattcagc acgacttgat 1080 tttctccctt caacaaacag aatgtgtcct taaaccggtt gaatcttcag atatgaaaat 1140 gactcagcta ttcaccaaag ttgaatcaga agatacaagt agcctctttg acaaacttaa 1200 gaaggaacct gatgctttaa ctttgctggc cccagccgct ggagacacaa tcatatcttt 1260 agattttggc agcaacgaca cagaaactga tgaccagcaa cttgaggaag taccattata 1320 taatgatgta atgctcccct cacccaacga aaaattacag aatataaatt tggcaatgtc 1380 tccattaccc accgctgaaa cgccaaagcc acttcgaagt agtgctgacc ctgcactcaa 1440 tcaagaagtt gcattaaaat tagaaccaaa tccagagtca ctggaacttt cttttaccat 1500 gccccagatt caggatcaga cacctagtcc ttccgatgga agcactagac aaagttcacc 1560 tgagcctaat agtcccagtg aatattgttt ttatgtggat agtgatatgg tcaatgaatt 1620 caagttggaa ttggtagaaa aactttttgc tgaagacaca gaagcaaaga acccattttc 1680 tactcaggac acagatttag acttggagat gttagctccc tatatcccaa tggatgatga 1740 cttccagtta cgttccttcg atcagttgtc accattagaa agcagttccg caagccctga 1800 aagcgcaagt cctcaaagca cagttacagt attccagcag actcaaatac aagaacctac 1860 tgctaatgcc accactacca ctgccaccac tgatgaatta aaaacagtga caaaagaccg 1920 tatggaagac attaaaatat tgattgcatc tccatctcct acccacatac ataaagaaac 1980 tactagtgcc acatcatcac catatagaga tactcaaagt cggacagcct caccaaacag 2040 agcaggaaaa ggagtcatag aacagacaga aaaatctcat ccaagaagcc ctaacgtgtt 2100 atctgtcgct ttgagtcaaa gaactacagt tcctgaggaa gaactaaatc caaagatact 2160 agctttgcag aatgctcaga gaaagcgaaa aatggaacat gatggttcac tttttcaagc 2220 agtaggaatt ggaacattat tacagcagcc agacgatcat gcagctacta catcactttc 2280 ttggaaacgt gtaaaaggat gcaaatctag tgaacagaat ggaatggagc aaaagacaat 2340 tattttaata ccctctgatt tagcatgtag actgctgggg caatcaatgg atgaaagtgg 2400 attaccacag ctgaccagtt atgattgtga agttaatgct cctatacaag gcagcagaaa 2460 cctactgcag ggtgaagaat tactcagagc tttggatcaa gttaactgag ctttttctta 2520 atttcattcc tttttttgga cactggtggc tcactaccta aagcagtcta tttatatttt 2580 ctacatctaa ttttagaagc ctggctacaa tactgcacaa acttggttag ttcaattttt 2640 gatccccttt ctacttaatt tacattaatg ctctttttta gtatgttctt taatgctgga 2700 tcacagacag ctcattttct cagttttttg gtatttaaac cattgcattg cagtagcatc 2760 attttaaaaa atgcaccttt ttatttattt atttttggct agggagttta tccctttttc 2820 gaattatttt taagaagatg ccaatataat ttttgtaaga aggcagtaac ctttcatcat 2880 gatcataggc agttgaaaaa tttttacacc ttttttttca cattttacat aaataataat 2940 gctttgccag cagtacgtgg tagccacaat tgcacaatat attttcttaa aaaataccag 3000 cagttactca tggaatatat tctgcgttta taaaactagt ttttaagaag aaattttttt 3060 tggcctatga aattgttaaa cctggaacat gacattgtta atcatataat aatgattctt 3120 aaatgctgta tggtttatta tttaaatggg taaagccatt tacataatat agaaagatat 3180 gcatatatct agaaggtatg tggcatttat ttggataaaa ttctcaattc agagaaatca 3240 tctgatgttt ctatagtcac tttgccagct caaaagaaaa caatacccta tgtagttgtg 3300 gaagtttatg ctaatattgt gtaactgata ttaaacctaa atgttctgcc taccctgttg 3360 gtataaagat attttgagca gactgtaaac aagaaaaaaa aaatcatgca ttcttagcaa 3420 aattgcctag tatgttaatt tgctcaaaat acaatgtttg attttatgca ctttgtcgct 3480 attaacatcc tttttttcat gtagatttca ataattgagt aattttagaa gcattatttt 3540 aggaatatat agttgtcaca gtaaatatct tgttttttct atgtacattg tacaaatttt 3600 tcattccttt tgctctttgt ggttggatct aacactaact gtattgtttt gttacatcaa 3660 ataaacatct tctgtgga 3678 <210> 4 <211> 9046 <212> DNA <213> Artificial Sequence <220> <223> Human p300 protein mRNA, embedded bases of 1200-8444 are the          coding sequence <400> 4 ccttgtttgt gtgctaggct gggggggaga gagggcgaga gagagcgggc gagagtgggc 60 aagcaggacg ccgggctgag tgctaactgc gggacgcaga gagtgcggag gggagtcggg 120 tcggagagag gcggcagggg ccagaacagt ggcagggggc ccggggcgca cgggctgagg 180 cgacccccag ccccctcccg tccgcacaca cccccaccgc ggtccagcag ccgggccggc 240 gtcgacgcta ggggggacca ttacataacc cgcgccccgg ccgtcttctc ccgccgccgc 300 ggcgcccgaa ctgagcccgg ggcgggcgct ccagcactgg ccgccggcgt ggggcgtagc 360 agcggccgta ttattatttc gcggaaagga aggcgaagga ggggagcgcc ggcgcgagga 420 ggggccgcct gcgcccgccg ccggagcggg gcctcctcgg tgggctccgc gtcggcgcgg 480 gcgtgcgggc ggcgctgctc ggcccggccc cctcggccct ctggtccggc cagctccgct 540 cccggcgtcc ttgccgcgcc tccgccggcc gccgcgcgat gtgaggcggc ggcgccagcc 600 tggctctcgg ctcgggcgag ttctctgcgg ccattagggg ccggtgcggc ggcggcgcgg 660 agcgcggcgg caggaggagg gttcggaggg tgggggcgca ggcccgggag ggggcaccgg 720 gaggaggtga gtgtctcttg tcgcctcctc ctctcccccc ttttcgcccc cgcctccttg 780 tggcgatgag aaggaggagg acagcgccga ggaggaagag gttgatggcg gcggcggagc 840 tccgagagac ctcggctggg caggggccgg ccgtggcggg ccggggactg cgcctctaga 900 gccgcgagtt ctcgggaatt cgccgcagcg gaccggcctc ggcgaatttg tgctcttgtg 960 ccctcctccg ggcttgggcc aggccggccc ctcgcacttg cccttacctt ttctatcgag 1020 tccgcatccc tctccagcca ctgcgacccg gcgaagagaa aaaggaactt cccccacccc 1080 ctcgggtgcc gtcggagccc cccagcccac ccctgggtgc ggcgcgggga ccccgggccg 1140 aagaagagat ttcctgagga ttctggtttt cctcgcttgt atctccgaaa gaattaaaaa 1200 tggccgagaa tgtggtggaa ccggggccgc cttcagccaa gcggcctaaa ctctcatctc 1260 cggccctctc ggcgtccgcc agcgatggca cagattttgg ctctctattt gacttggagc 1320 acgacttacc agatgaatta atcaactcta cagaattggg actaaccaat ggtggtgata 1380 ttaatcagct tcagacaagt cttggcatgg tacaagatgc agcttctaaa cataaacagc 1440 tgtcagaatt gctgcgatct ggtagttccc ctaacctcaa tatgggagtt ggtggcccag 1500 gtcaagtcat ggccagccag gcccaacaga gcagtcctgg attaggtttg ataaatagca 1560 tggtcaaaag cccaatgaca caggcaggct tgacttctcc caacatgggg atgggcacta 1620 gtggaccaaa tcagggtcct acgcagtcaa caggtatgat gaacagtcca gtaaatcagc 1680 ctgccatggg aatgaacaca gggacgaatg cgggcatgaa tcctggaatg ttggctgcag 1740 gcaatggaca agggataatg cctaatcaag tcatgaacgg ttcaattgga gcaggccgag 1800 ggcgacagga tatgcagtac ccaaacccag gcatgggaag tgctggcaac ttactgactg 1860 agcctcttca gcagggctct ccccagatgg gaggacaaac aggattgaga ggcccccagc 1920 ctcttaagat gggaatgatg aacaacccca atccttatgg ttcaccatat actcagaatc 1980 ctggacagca gattggagcc agtggccttg gtctccagat tcagacaaaa actgtactat 2040 caaataactt atctccattt gctatggaca aaaaggcagt tcctggtgga ggaatgccca 2100 acatgggtca acagccagcc ccgcaggtcc agcagccagg tctggtgact ccagttgccc 2160 aagggatggg ttctggagca catacagctg atccagagaa gcgcaagctc atccagcagc 2220 agcttgttct ccttttgcat gctcacaagt gccagcgccg ggaacaggcc aatggggaag 2280 tgaggcagtg caaccttccc cactgtcgca caatgaagaa tgtcctaaac cacatgacac 2340 actgccagtc aggcaagtct tgccaagtgg cacactgtgc atcttctcga caaatcattt 2400 cacactggaa gaattgtaca agacatgatt gtcctgtgtg tctccccctc aaaaatgctg 2460 gtgataagag aaatcaacag ccaattttga ctggagcacc cgttggactt ggaaatccta 2520 gctctctagg ggtgggtcaa cagtctgccc ccaacctaag cactgttagt cagattgatc 2580 ccagctccat agaaagagcc tatgcagctc ttggactacc ctatcaagta aatcagatgc 2640 cgacacaacc ccaggtgcaa gcaaagaacc agcagaatca gcagcctggg cagtctcccc 2700 aaggcatgcg gcccatgagc aacatgagtg ctagtcctat gggagtaaat ggaggtgtag 2760 gagttcaaac gccgagtctt ctttctgact caatgttgca ttcagccata aattctcaaa 2820 acccaatgat gagtgaaaat gccagtgtgc cctccctggg tcctatgcca acagcagctc 2880 aaccatccac tactggaatt cggaaacagt ggcacgaaga tattactcag gatcttcgaa 2940 atcatcttgt tcacaaactc gtccaagcca tatttcctac gccggatcct gctgctttaa 3000 aagacagacg gatggaaaac ctagttgcat atgctcggaa agttgaaggg gacatgtatg 3060 aatctgcaaa caatcgagcg gaatactacc accttctagc tgagaaaatc tataagatcc 3120 agaaagaact agaagaaaaa cgaaggacca gactacagaa gcagaacatg ctaccaaatg 3180 ctgcaggcat ggttccagtt tccatgaatc cagggcctaa catgggacag ccgcaaccag 3240 gaatgacttc taatggccct ctacctgacc caagtatgat ccgtggcagt gtgccaaacc 3300 agatgatgcc tcgaataact ccacaatctg gtttgaatca atttggccag atgagcatgg 3360 cccagccccc tattgtaccc cggcaaaccc ctcctcttca gcaccatgga cagttggctc 3420 aacctggagc tctcaacccg cctatgggct atgggcctcg tatgcaacag ccttccaacc 3480 agggccagtt ccttcctcag actcagttcc catcacaggg aatgaatgta acaaatatcc 3540 ctttggctcc gtccagcggt caagctccag tgtctcaagc acaaatgtct agttcttcct 3600 gcccggtgaa ctctcctata atgcctccag ggtctcaggg gagccacatt cactgtcccc 3660 agcttcctca accagctctt catcagaatt caccctcgcc tgtacctagt cgtaccccca 3720 cccctcacca tactccccca agcatagggg ctcagcagcc accagcaaca acaattccag 3780 cccctgttcc tacaccacca gccatgccac ctgggccaca gtcccaggct ctacatcccc 3840 ctccaaggca gacacctaca ccaccaacaa cacaacttcc ccaacaagtg cagccttcac 3900 ttcctgctgc accttctgct gaccagcccc agcagcagcc tcgctcacag cagagcacag 3960 cagcgtctgt tcctacccca aacgcaccgc tgcttcctcc gcagcctgca actccacttt 4020 cccagccagc tgtaagcatt gaaggacagg tatcaaatcc tccatctact agtagcacag 4080 aagtgaattc tcaggccatt gctgagaagc agccttccca ggaagtgaag atggaggcca 4140 aaatggaagt ggatcaacca gaaccagcag atacgcagcc ggaggatatt tcagagtcta 4200 aagtggaaga ctgtaaaatg gaatctaccg aaacagaaga gagaagcact gagttaaaaa 4260 ctgaaataaa agaggaggaa gaccagccaa gtacttcagc tacccagtca tctccggctc 4320 caggacagtc aaagaaaaag attttcaaac cagaagaact acgacaggca ctgatgccaa 4380 cattggaggc actttaccgt caggatccag aatcccttcc ctttcgtcaa cctgtggacc 4440 ctcagctttt aggaatccct gattactttg atattgtgaa gagccccatg gatctttcta 4500 ccattaagag gaagttagac actggacagt atcaggagcc ctggcagtat gtcgatgata 4560 tttggcttat gttcaataat gcctggttat ataaccggaa aacatcacgg gtatacaaat 4620 actgctccaa gctctctgag gtctttgaac aagaaattga cccagtgatg caaagccttg 4680 gatactgttg tggcagaaag ttggagttct ctccacagac actgtgttgc tacggcaaac 4740 agttgtgcac aatacctcgt gatgccactt attacagtta ccagaacagg tatcatttct 4800 gtgagaagtg tttcaatgag atccaagggg agagcgtttc tttgggggat gacccttccc 4860 agcctcaaac tacaataaat aaagaacaat tttccaagag aaaaaatgac acactggatc 4920 ctgaactgtt tgttgaatgt acagagtgcg gaagaaagat gcatcagatc tgtgtccttc 4980 accatgagat catctggcct gctggattcg tctgtgatgg ctgtttaaag aaaagtgcac 5040 gaactaggaa agaaaataag ttttctgcta aaaggttgcc atctaccaga cttggcacct 5100 ttctagagaa tcgtgtgaat gactttctga ggcgacagaa tcaccctgag tcaggagagg 5160 tcactgttag agtagttcat gcttctgaca aaaccgtgga agtaaaacca ggcatgaaag 5220 caaggtttgt ggacagtgga gagatggcag aatcctttcc ataccgaacc aaagccctct 5280 ttgcctttga agaaattgat ggtgttgacc tgtgcttctt tggcatgcat gttcaagagt 5340 atggctctga ctgccctcca cccaaccaga ggagagtata catatcttac ctcgatagtg 5400 ttcatttctt ccgtcctaaa tgcttgagga ctgcagtcta tcatgaaatc ctaattggat 5460 atttagaata tgtcaagaaa ttaggttaca caacagggca tatttgggca tgtccaccaa 5520 gtgagggaga tgattatatc ttccattgcc atcctcctga ccagaagata cccaagccca 5580 agcgactgca ggaatggtac aaaaaaatgc ttgacaaggc tgtatcagag cgtattgtcc 5640 atgactacaa ggatattttt aaacaagcta ctgaagatag attaacaagt gcaaaggaat 5700 tgccttattt cgagggtgat ttctggccca atgttctgga agaaagcatt aaggaactgg 5760 aacaggagga agaagagaga aaacgagagg aaaacaccag caatgaaagc acagatgtga 5820 ccaagggaga cagcaaaaat gctaaaaaga agaataataa gaaaaccagc aaaaataaga 5880 gcagcctgag taggggcaac aagaagaaac ccgggatgcc caatgtatct aacgacctct 5940 cacagaaact atatgccacc atggagaagc ataaagaggt cttctttgtg atccgcctca 6000 ttgctggccc tgctgccaac tccctgcctc ccattgttga tcctgatcct ctcatcccct 6060 gcgatctgat ggatggtcgg gatgcgtttc tcacgctggc aagggacaag cacctggagt 6120 tctcttcact ccgaagagcc cagtggtcca ccatgtgcat gctggtggag ctgcacacgc 6180 agagccagga ccgctttgtc tacacctgca atgaatgcaa gcaccatgtg gagacacgct 6240 ggcactgtac tgtctgtgag gattatgact tgtgtatcac ctgctataac actaaaaacc 6300 atgaccacaa aatggagaaa ctaggccttg gcttagatga tgagagcaac aaccagcagg 6360 ctgcagccac ccagagccca ggcgattctc gccgcctgag tatccagcgc tgcatccagt 6420 ctctggtcca tgcttgccag tgtcggaatg ccaattgctc actgccatcc tgccagaaga 6480 tgaagcgggt tgtgcagcat accaagggtt gcaaacggaa aaccaatggc gggtgcccca 6540 tctgcaagca gctcattgcc ctctgctgct accatgccaa gcactgccag gagaacaaat 6600 gcccggtgcc gttctgccta aacatcaagc agaagctccg gcagcaacag ctgcagcacc 6660 gctaccca ggcccaaatg cttcgcagga ggatggccag catgcagcgg actggtgtgg 6720 ttgggcagca acagggcctc ccttccccca ctcctgccac tccaacgaca ccaactggcc 6780 aacagccaac caccccgcag acgccccagc ccacttctca gcctcagcct acccctccca 6840 atagcatgcc accctacttg cccaggactc aagctgctgg ccctgtgtcc cagggtaagg 6900 cagcaggcca ggtgacccct ccaacccctc ctcagactgc tcagccaccc cttccagggc 6960 ccccacctac agcagtggaa atggcaatgc agattcagag agcagcggag acgcagcgcc 7020 agatggccca cgtgcaaatt tttcaaaggc caatccaaca ccagatgccc ccgatgactc 7080 ccatggcccc catgggtatg aacccacctc ccatgaccag aggtcccagt gggcatttgg 7140 agccagggat gggaccgaca gggatgcagc aacagccacc ctggagccaa ggaggattgc 7200 ctcagcccca gcaactacag tctgggatgc caaggccagc catgatgtca gtggcccagc 7260 atggtcaacc tttgaacatg gctccacaac caggattggg ccaggtaggt atcagcccac 7320 tcaaaccagg cactgtgtct caacaagcct tacaaaacct tttgcggact ctcaggtctc 7380 ccagctctcc cctgcagcag caacaggtgc ttagtatcct tcacgccaac ccccagctgt 7440 tggctgcatt catcaagcag cgggctgcca agtatgccaa ctctaatcca caacccatcc 7500 ctgggcagcc tggcatgccc caggggcagc cagggctaca gccacctacc atgccaggtc 7560 agcagggggt ccactccaat ccagccatgc agaacatgaa tccaatgcag gcgggcgttc 7620 agagggctgg cctgccccag cagcaaccac agcagcaact ccagccaccc atgggaggga 7680 tgagccccca ggctcagcag atgaacatga accacaacac catgccttca caattccgag 7740 acatcttgag acgacagcaa atgatgcaac agcagcagca acagggagca gggccaggaa 7800 taggccctgg aatggccaac cataaccagt tccagcaacc ccaaggagtt ggctacccac 7860 cacagccgca gcagcggatg cagcatcaca tgcaacagat gcaacaagga aatatgggac 7920 agataggcca gcttccccag gccttgggag cagaggcagg tgccagtcta caggcctatc 7980 agcagcgact ccttcagcaa cagatggggt cccctgttca gcccaacccc atgagccccc 8040 agcagcatat gctcccaaat caggcccagt ccccacacct acaaggccag cagatcccta 8100 attctctctc caatcaagtg cgctctcccc agcctgtccc ttctccacgg ccacagtccc 8160 agccccccca ctccagtcct tccccaagga tgcagcctca gccttctcca caccacgttt 8220 ccccacagac aagttcccca catcctggac tggtagctgc ccaggccaac cccatggaac 8280 aagggcattt tgccagcccg gaccagaatt caatgctttc tcagcttgct agcaatccag 8340 gcatggcaaa cctccatggt gcaagcgcca cggacctggg actcagcacc gataactcag 8400 acttgaattc aaacctctca cagagtacac tagacataca ctagagacac cttgattatttt 8460 gggagcaaaa aaattatttt ctcttaacaa gactttttgt actgaaaaca atttttttga 8520 atctttcgta gcctaaaaga caattttcct tggaacacat aagaactgtg cagtagccgt 8580 ttgtggttta aagcaaacat gcaagatgaa cctgagggat gatagaatac aaagaatata 8640 tttttgttat gggctggtta ccaccagcct ttcttcccct ttgtgtgtgt ggttcaagtg 8700 tgcactggga ggaggctgag gcctgtgaag ccaaacaata tgctcctgcc ttgcacctcc 8760 aataggtttt attatttttt ttaaattaat gaacatatgt aatattaatg aacatatgta 8820 atattaatag ttattattta ctggtgcaga tggttgacat ttttccctat tttcctcact 8880 ttatggaaga gttaaaacat ttctaaacca gaggacaaaa ggggttaatg ttactttgaa 8940 attacattct atatatatat aaatatatat aaatatatat taaaatacca gttttttttc 9000 tctgggtgca aagatgttca ttcttttaaa aaatgtttaa aaaaaa 9046

Claims (10)

1) preparing an HIF-1? Plasmid comprising N-terminal, C-terminal or HIF-1? Labeled with a first fluorescent substance at both terminals;
2) preparing a p300 protein plasmid containing the p300 protein whose N-terminus, C-terminus or both ends are labeled with a second fluorescent substance;
3) injecting the prepared HIF-1? Plasmid and p300 protein plasmid into eukaryotic cells to transfect cells; And
4) measuring the fluorescence emitted from the transfected cells and calculating the fluorescence transmission efficiency according to the following equation:
Lt; / RTI &gt;
Wherein one of the first fluorescent material and the second fluorescent material is a fluorescent energy donor for transferring fluorescence energy and the other is a fluorescent energy acceptor for receiving fluorescence energy, Wherein the fluorescence spectrum is overlapped in whole or in part with the absorption spectrum of the fluorescent energy receiving body,
Methods for measuring the binding of HIF-1α and p300 proteins:
[expression]
FRET index = I _DA - (a _{DA D} I) - (I a _{A DA)}
FRET efficiency = FRET index / 255
Wherein,
a _D is the leakage rate of fluorescence of the fluorescent energy donor,
a &lt; _{A &gt;} is the fluorescence ratio due to the absorption of fluorescence energy, and
Do I _DA indicates the fluorescence intensity of the fluorescence energy donor fluorescent energy can then booty here in which the fluorescent energy can booty and fluorescence energy donor at the same time expressing cells. The method of claim 1, wherein the HIF-1α and p300 proteins are determined to be bound when the calculated fluorescence transfer efficiency is 10 to 20%. The method of claim 1,
The HIF-1 alpha is a polypeptide comprising 41 to 826 amino acids consecutively including amino acids 786 to 826 in the amino acid sequence of SEQ ID NO: 1,
Wherein the p300 protein is a polypeptide comprising a consecutive 101 to 2414 amino acids including an amino acid from position 323 to position 423 of the amino acid sequence of SEQ ID NO:
A method for measuring the binding of HIF-1 alpha to p300 protein. The method of claim 1,
The fluorescent energy donor is selected from the group consisting of BFP (Blue Fluorescent Protein), ECFP (Cyan Fluorescent Protein), GFP (Green Fluorescent Protein), EYFP (Yellow Fluorescent Protein)
Wherein the fluorescent energy receiving body is selected from the group consisting of ECFP (Cyan Fluorescent Protein), GFP (Green Fluorescent Protein), EYFP (Yellow Fluorescent Protein), RFP (Red fluorescent protein)
A method for measuring the binding of HIF-1 alpha to p300 protein. The method of claim 1,
Wherein the fluorescent energy donor and the fluorescent energy acceptor are bound to the same end of the p300 protein and HIF-1?, Respectively,
A method for measuring the binding of HIF-1 alpha to p300 protein. The method of claim 1,
p300 protein as a fluorescent energy donor and HIF-1? as a fluorescent energy donor.
A method for measuring the binding of HIF-1 alpha to p300 protein. 1) preparing a cell comprising HIF-1α labeled with N-terminal, C-terminal or both terminals with a first fluorescent substance and p300 protein having N-terminal, C-terminal or both terminals labeled with a second fluorescent substance; And
2) contacting the candidate compound with the cell and measuring fluorescence energy transfer efficiency after incubation; And
3) comparing the fluorescence energy transfer efficiency measured in the candidate compound-contacted cells with the fluorescence energy transfer efficiency measured in cells not contacted with the candidate compound
Lt; / RTI &gt;
One of the first fluorescent protein and the second fluorescent protein is a donor for transferring fluorescence energy and the other is an acceptor for receiving fluorescence energy and the fluorescence spectrum of the fluorescent energy donor is fluorescence The absorption spectrum of the energy receiving body is partially or completely overlapped with the absorption spectrum of the energy receiving body,
The fluorescence energy transfer efficiency is measured by the following equation,
When the fluorescence energy transfer efficiency measured in the candidate compound-contacted cells is higher than the fluorescence energy transfer efficiency measured in cells not contacted with the candidate compound, the candidate compound is judged to be a binding promoter between HIF-1α and p300 protein, When the fluorescence energy transfer efficiency measured in the candidate compound-contacted cells is lower than the fluorescence energy transfer efficiency measured in cells not contacted with the candidate compound, the candidate compound is judged to be a binding inhibitor between HIF-1 alpha and p300 protein doing,
Screening method of binding modulator between HIF-1? Peptide and p300 protein:
[expression]
FRET index = I _DA - (a _{DA D} I) - (I a _{A DA)}
FRET efficiency = FRET index / 255
Wherein,
a _D is the leakage rate of fluorescence of the fluorescent energy donor,
a &lt; _{A &gt;} is the fluorescence ratio due to the absorption of fluorescence energy, and
I also _DA indicates the fluorescence wavelength of the fluorescence energy donor fluorescent energy can then booty here in which the fluorescent energy can booty and fluorescence energy donor at the same time expressing cells. 8. The screening method according to claim 7, wherein the candidate compound is at least one selected from the group consisting of proteins, peptides, oligonucleotides, polynucleotides, and synthetic compounds. HIF-1α plasmid comprising HIF-1α labeled at the N-, C-, or both ends with a first fluorescent substance; A p300 protein plasmid comprising a p300 protein whose N-terminus, C-terminus or both ends are labeled with a second fluorescent substance; And kit for measuring binding between HIF-1α and p300 protein, comprising a fluorescence measuring means. The kit for measuring binding between HIF-1α and p300 protein according to claim 9, further comprising cells for transfecting and expressing the HIF-1α plasmid and the p300 protein plasmid.

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