JP5283105B2 - BRET expression system with high energy transfer efficiency - Google Patents

Description

The present invention relates to green fluorescent protein (GFP) by resonance energy transfer using luciferin.
As with the system observed in natural marine luminescent organisms, which emit light efficiently, any fluorescent protein can be emitted close to the original fluorescence without the need for an external excitation light source and with a brightness that can be recognized by the naked eye To do.

  The luminescence mechanism of natural GFP has recently attracted attention as “Bioluminescence Resonance Energy Transfer (BRET)”. In order for resonance energy transfer to occur, the distance between the energy donor (luciferase) and the energy acceptor (fluorescent protein) needs to be close to 1-10 nm. This distance of 1-10 nm corresponds to the physical distance at which protein-protein interactions occur in the formation of protein complexes, and is therefore applied exclusively as a technique for detecting protein-protein interactions. Since the current high-performance and high-sensitivity detection devices can detect even a slight change, the present situation is that protein-protein interactions are evaluated with low-efficiency resonance energy transfer. In extreme terms, conventional methodologies do not assume high-efficiency resonance energy transfer comparable to natural BRET.

  The present inventor returned to the origin of BRET “fluorescent fluorescent protein” that occurs in natural organisms, which was a blind spot in the conventional BRET technology, and established a new application technology by fusion of luciferase and fluorescent protein. At the same time, we tried to develop a technology to illuminate fluorescent proteins with their original color.

Regarding BRET, Patent Document 1 discloses a chimeric protein in which Cypridina luciferase (Vluc) and yellow fluorescent protein (EYFP) are linked by a linker peptide, but its energy transfer efficiency is low and there is room for improvement.
WO2004 / 22600 WO2004 / 052934 WO01 / 46694 Special table 2006-518209 Special table 2005-525111

  An object of the present invention is to provide a chimeric protein capable of realizing highly efficient resonance energy transfer comparable to natural BRET.

  Another object of the present invention is to provide a fluorescent label with higher sensitivity than conventional luciferases.

In the conventional BRET system in which luciferase and fluorescent protein (GFP, YFP, etc.) are linked, luciferase is at the N-terminal and fluorescent protein is at the C-terminal, and these are linked by a linker peptide. In the BRET system having such a configuration, the peak intensity ratio between the maximum absorption wavelength of luciferase and the maximum absorption wavelength of fluorescent protein is limited to about 1: 1. In the case of this level of energy transfer efficiency, fluorescence can be detected with a highly sensitive measuring instrument, but cannot be observed with the naked eye.

  The inventor arranges the Renilla luciferase and the fluorescent protein in reverse, and arranges the fluorescent protein at the N-terminal and the luciferase at the C-terminal, so that the maximum absorption wavelength of the fluorescent protein relative to the peak intensity of the maximum absorption wavelength of the luciferase By increasing the peak intensity ratio to 3 times or more and further optimizing the linker peptide, the peak intensity ratio was successfully increased to 4.38 times.

  The present inventor has confirmed that highly efficient resonance energy transfer occurs in AcGFP, EGFP, RFP and EYFP using the chimeric protein of the present invention. Thus, it is clear that the system of the present invention works with a combination of Renilla luciferase and any fluorescent protein.

BAF using the present linker (B RET-based A uto- illuminated F luorescent-protein) technique is to present the utilization of a fluorescent protein which has not been used so far.

The present invention provides the following chimeric protein, a gene encoding the chimeric protein, an expression vector containing the gene, and a transformant introduced with the vector.
1. A chimeric protein comprising an energy accepting protein and an energy generating protein linked via a linker, wherein a visible energy transfer can occur between the energy accepting protein and the energy generating protein, wherein the energy accepting protein is a fluorescent protein. The energy generating protein is Renilla luciferase, the linker is composed of 8 to 26 amino acids, the energy accepting protein is on the N-terminal side, the energy generating protein is on the C-terminal side, and the BRET ratio (energy accepting) A chimeric protein having a luminescence maximum wavelength intensity of protein / (intensity of luminescence maximum wavelength of energy generating protein) of 3 or more.
2. Item 1 wherein the fluorescent protein is GFP, YFP, BFP, CFP, DsRED, or RFP
A chimeric protein according to 1.
3. Item 2. The chimeric protein according to Item 1, wherein the fluorescent protein is YFP.
4). Item 2. The chimeric protein according to Item 1, wherein the linker is composed of 8 to 16 amino acids.
5. Item 2. The chimeric protein according to Item 1, wherein the linker is composed of 10 to 14 amino acids.
6). Item 2. The chimeric protein according to Item 1, wherein the linker is composed of 12 amino acids.
7). Item 7. A polynucleotide encoding the secretory or membrane-bound chimeric protein according to any one of Items 1 to 6, or a complementary strand thereof.
8). Item 8. A vector comprising the polynucleotide according to Item 7.
9. Item 9. A transformant transformed with the vector according to Item 8.
10. Item 7. Use of the chimeric protein according to any one of Items 1 to 6 as a label.
11. Item 11. The use according to Item 10, for labeling a substance such as an antigen, antibody, enzyme, DNA, RNA, protein, sugar chain, cell or ligand capable of recognizing a receptor.
12 Item 8. A transformant comprising introducing a vector according to Item 8 into a host to produce a transformant, applying Renilla luciferin to the transformant, and detecting luminescence by BRET from the chimeric protein. Body imaging method.
13. Item 13. The method according to Item 12, wherein the transformant is a prokaryotic cell, eukaryotic cell, tissue, organ, animal or plant.
14 Item 13. The method according to Item 12, wherein the transformant is a mammalian cell.

  The self-contained chimeric protein according to the present invention can realize luminescence close to the fluorescence inherent in a fluorescent protein with a visible level of brightness using only luciferin without using external excitation light.

In addition, the chimeric protein of the present invention can be expressed in cultured cells, animals or plants and can be illuminated in the presence of luciferin.

  The chimeric protein according to the present invention can change the BRET spectrum not only by the linker sequence but also by fusing any protein to the N-terminus or C-terminus thereof. Fused additional protein that negatively affects the three-dimensional structure of the chimeric protein of the present invention (ie, lowers BRET efficiency) and cleaves the second linker sequence between them to restore the damaged BRET It is also possible.

  The chimeric protein of the present invention is a more excellent reporter protein that exhibits stronger luminescence than Renilla luciferase alone. Therefore, a superior enzyme label can be provided by using the chimeric protein of the present invention instead of the conventional Renilla luciferase label. For example, by linking the chimeric protein of the present invention with an antibody (for example, a secondary antibody), it is possible to measure with higher sensitivity in an enzyme immunoassay such as ELISA. In addition, it is possible to clearly image the movement of each intracellular element such as the nucleus, endoplasmic reticulum, ribosome, or a labeling substance such as a protein between these elements.

The chimeric protein of the present invention is characterized in that a visible level of fluorescence can be obtained by intramolecular energy transfer. Intramolecular energy transfer means that energy transfer occurs between the light energy released from the energy generating protein (Renilla luciferase) and the energy accepting protein (fluorescent protein). It is characterized by the fact that it emits fluorescence at a level that can be seen without giving light, whereby the degree of energy transfer can be quantified.

  If the chimeric protein of the present invention is secretable, its presence can be easily detected by fluorescence by providing luciferin outside the cell. When the chimeric protein is localized in the cell, it can be similarly detected by permeating luciferin into the cell or using a cell disruption solution.

Qualitative analysis by fluorescence is possible only with a fluorescent protein such as GFP, but it cannot be quantified because it emits fluorescence by external excitation light. On the other hand, since the chimeric protein of the present invention emits fluorescence from the fluorescent protein by the excitation light from Renilla luciferase, each modified product of the chimeric protein (cleavage in the linker peptide, modification by BTB domain, ATF6, etc.) has the fluorescence wavelength. Each can be quantified based on the shift.
In the present invention, the linker is not particularly limited as long as it does not hinder the transfer of light energy from Renilla luciferase to the fluorescent protein. The number of amino acids in the linker is usually 8 to 26, preferably 8 to 16, more preferably 10 to 14, especially 12. When the number of amino acids in the linker is 7 or less or 27 or more, the energy transfer efficiency is greatly reduced.
In a linker, the number of amino acids is particularly important, and the amino acid sequence is less important.
The amino acid sequences of 18 kinds of linkers used by the present inventors are listed below. These linkers are useful for obtaining chimeric proteins that emit visible fluorescence.
As apparent from Table 1 below, the number of amino acids of the linker is preferably 8 to 12, and 12 is particularly preferable.

As shown in Table 1 above, all of the chimeric proteins of the present invention have a conventional ratio (ie, (light intensity at the maximum emission wavelength of the acceptor) / (light intensity at the maximum emission wavelength of the donor)) = maximum value = about The value is higher than 1). For the first time, the present inventors have found that when a fluorescent protein and Rluc are connected in this direction with a linker consisting of 8-26 amino acid residues, BRET with significantly higher efficiency than before occurs.

  In the present specification, natural Renilla luciferase (e.g., Rluc) may be used as the Renilla luciferase, and Renilla luciferase (e.g., Rluc8, Rluc8 / A123S / D162E) having improved properties such as stability and luminescence properties. / I163L) may be used. In the present specification, “Renilla luciferase” includes both a natural Renilla luciferase and any modified Renilla luciferase in which the properties of luciferase are changed.

Examples of the fluorescent protein include green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), DsRED, and red fluorescent protein (RFP). GFP includes various GFP derivatives such as EGFP and AcGFP. As for YFP, amino acid-substituted mutants such as EYFP, Topaz, Venus, and Citrine are widely included. Similarly, other fluorescent proteins include naturally occurring fluorescent proteins and mutants in which the amino acid sequence is modified (substitution, addition, deletion, insertion, etc.). If a fluorescent protein whose RLU or wavelength changes depending on pH (for example, YFP) can be used, the pH of the fluorescent protein can be measured. The chimeric protein of the present invention is a pH indicator. Can be used as In addition, substances such as GFP, whose RLU or wavelength does not change much depending on pH, can be used to quantify substances such as the chimeric protein of the present invention or proteins labeled thereby without depending on pH.

  The fluorescent protein of the present invention is selected so that the light emitted from Renilla luciferase becomes the excitation wavelength of the fluorescent protein. Examples of such combinations include the following.

The polynucleotide of the present invention is a polynucleotide encoding the chimeric protein of the present invention. When NLS (nuclear translocation signal) is linked to the polynucleotide, the chimeric protein translocates to the nucleus, and imaging of the nucleus can be performed.

  The protein of the present invention can be obtained by incorporating the gene of the present invention described later into an expression vector and expressing it in an appropriate host cell. Examples of host cells include animal cells including mammalian cells, plant cells, eukaryotic cells such as yeast, prokaryotic cells such as Escherichia coli, Bacillus subtilis, algae, and fungi, and plant cells. Good. As preferred host cells, mammalian cultured cells and the like can be used.

  As shown in the data of the example (FIG. 3b), the self-contained fluorescent protein according to the present invention is luciferin alone without using external excitation light, and the light emission close to the fluorescence of the fluorescent protein can be visually recognized. Realize. So far, there has been no successful example of using BRET to illuminate a fluorescent protein and show a change in color tone. In that sense, luciferin has made it possible to illuminate the fluorescent protein to an extent comparable to the original fluorescence. There are features.

The luminescence reaction using coelenterazine, the luciferin of Renilla luciferase, is different from firefly luciferase and the like, and ATP is unnecessary and can emit light if there is only luciferase and oxygen molecules. Coelenterazine is a compound that is also included in natural organisms and itself has an antioxidant action, and does not have strong cytotoxicity. In other words, if there is luciferin, it can be lit freely in a normal environment.

  One of the features of the chimeric protein of the present invention is that it is stable and easy to handle. The chimeric protein of the present invention comprises only amino acids, can be expressed in cultured cells, animals or plants, and can be illuminated in the presence of luciferin.

  The chimeric protein according to the present invention can change the BRET spectrum not only by the linker sequence but also by fusing any protein to the N-terminus or C-terminus thereof (DRmmBTB-BAF-Y in FIG. 7). , See NLS-BAF-G-ATF6C in Figure 8). It is spoiled by fusing additional proteins that negatively affect the three-dimensional structure of the self-contained fluorescent protein according to the present invention (ie, lowering BRET efficiency) and cleaving the second linker sequence between them. It is also possible to recover BRET.

Since the chimeric protein of the present invention emits light stronger than luciferase or fluorescent protein, it can be used in place of conventional luciferase or fluorescent protein. For example, a promoter assay can be performed by linking a gene encoding the chimeric protein of the present invention to a target promoter. Moreover, the chimeric protein of the present invention can label any substance by linking to the labeling target substance. Examples of substances to be labeled include antigens, antibodies, enzymes, DNA, RNA, proteins (for example, proteins capable of recognizing biological materials such as receptors, hormones, cytokines), sugar chains (for example, sialyl Lewis X), cells or receptors. Substances such as recognizable ligands (for example, hormones, cytokines, lymphokines, prostaglandins, etc.) can be mentioned. When the labeling substance is protein / peptide,
The chimeric protein of the present invention can be bound to the protein / peptide to be labeled directly or via an appropriate spacer. In the case of a sugar chain, a method such as biotinylation of the chimeric protein and the sugar chain and binding via streptavidin can be employed. The chimeric protein of the present invention can also be used for cell imaging by introducing a gene encoding the chimeric protein into a host, expressing the gene, and emitting light in the presence of coelenterazine.

EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, it cannot be overemphasized that this invention is not limited to these Examples.
Example 1
(1) Preparation of plasmid
In order to construct a BAF-Y expression vector, the gene encoding hRluc was amplified by PCR. The primers used for PCR are as follows:
hRL-F1, 5'-GAGCAGCTGTACAAGCATATGGCTTCCAAGGTGTACGACCCCGAG-3 '(SEQ ID NO: 1);
hRL-R1, 5'-GCTCTAGATCACTGCTCGTTCTTCAGCACGCGCTCCAC-3 '(SEQ ID NO: 2). The PCR fragment was digested with BsrGI and XbaI and incorporated into the BsrGI / XbaI site of pEYFP to construct pEYFP-Rluc. In this plasmid, any oligo DNA can be inserted as a linker sequence into the BsrGI and NdeI sites. For construction of pBAF-Y, the following oligo DNA fragment was inserted into the BsrGI / NdeI site of pEYFP-Rluc:
BRET-tag12 aa sense, 5'- GTACAAGTCGGGGCTGCGGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 3); BRET-tag 12 aa antisense 5'- TAGTCGCCAACGCCTGCGCCCTGCTCCGCAGCCCCGACTT
-3 '(SEQ ID NO: 4). Several oligo DNA fragments were used to generate single amino acid substitution variants to the linker sequence of BAF-Y:
S239A-F, 5'-GTACAAGGCGGGGCTGCGGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 5);
S239A-R, 5'-TAGTCGCCAACGCCTGCGCCCTGCTCCGCAGCCCCGCCTT3 '(SEQ ID NO: 6);
G240A-F, 5'-GTACAAGTCGGCGCTGCGGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 7);
G240A-R, 5'-TAGTCGCCAACGCCTGCGCCCTGCTCCGCAGCGCCGACTT-3 '(SEQ ID NO: 8); L241A-F, 5'-GTACAAGTCGGGGGCGCGGAGCAGGGCGCAGGCGTTGGCGAC-3' (SEQ ID NO: 9); L241A-R, 5'-TAGTCGCCAACGCGTGTGCCCCCTCG R242A-F, 5'-GTACAAGTCGGGGCTGGCGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 11); R242A-R, 5'-TAGTCGCCAACGCCTGCGCCCTGCTCGCCAGCCCCGACTT-3' (SEQ ID NO: 12); S243A-F, 5'-GTACAAGTCGGGGCTGCAGGCGGGGG ); S243A-R, 5'-TAGTCGCCAACGCCTGCGCCCTGGCCCGCAGCCCCGACTT-3 '(SEQ ID NO: 14); R244A-F, 5'-GTACAAGTCGGGGCTGCGGAGCGCGGCGCAGGCGTTGGCGAC-3' (SEQ ID NO: 15); R244A-R, 5'-TAGTCGCGCGCGCTCGCGCGCGCGCGCGCGCGCGCGC 16); Q246A-F, 5'-GTACAAGTCGGGGCTGCGGAGCAGGGCGGCGGCGTTGGCGAC-3 '(SEQ ID NO: 17); Q246A-R, 5'-TAGTCGCCAACGCCGCCGCCCTGCTCCGCAGCCCCGACTT-3' (SEQ ID NO: 18);
L248A-F, 5'-GTACAAGTCGGGGCTGCGGAGCAGGGCGCAGGCGGCGGCGAC-3 '(SEQ ID NO: 19); L248A-R, 5'-TAGTCGCCGCCGCCTGCGCCCTGCTCCGCAGCCCCGACTT-3' (SEQ ID NO: 20); H250A-F, 5'-GTACAAGTCGGGGCTGCGGACGGGCGGCTGCGGACGGGGG H250A-R, 5'-TAGCCGCCAACGCCTGCGCCCTGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 22).
Each oligo DNA pair was annealed and inserted into the BsrGI / NdeI site of pEYFP-Rluc.

In order to prepare an expression plasmid of E. coli, genes encoding Rluc, BAF-Y and BAF-Y mutants were amplified by PCR using the primers shown below. Individual PCR fragments were subcloned into the NdeI-XbaI site of pCold-TF DNA, pCT-hRL, pCT-BAF-Y and pCT-BAF-YR244
A got.
The primers used for PCR are as follows:
TFEGFP-F, GTCGCCCATATGGTGAGCAAGGGCGAGGAGC-3 '(SEQ ID NO: 23);
TFhRL-F, 5'-CCGCTCGAGGCTAGCATGGCTTCCAAGGTGTACGACCCCGAG-3 '(SEQ ID NO: 24); TFhRL-R, 5'-GTTATCTAGAATTACTGCTCGTTCTTCAGCACGC-3' (SEQ ID NO: 25).

(2) Preparation of Recombinant Protein Recombinant Rluc, BAF-Y _R244A , and derived mutant proteins were synthesized as a TF tag fusion protein by a cold shock inducible promoter system in E. coli BL21 strain. Recombinant protein was purified with Ni-NTA affinity column and quantified with BCA protein quantification kit. The purified protein was limitedly digested with HRV-3C protease to obtain a mature recombinant protein.

(3) Cell culture and transfection
HeLa cells and NIH3T3 cells were maintained at 37 ° C. in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum. Cells were seeded 4-6 × 10 ⁵ cells per well in 6-well plates one day prior to transfection. Transient transfection was performed using Lipofectamine 2000 reagent for HeLa cells and Lipofectamine plus reagent for NIH3T3 cells. 30 hours after transfection, the cells were washed with 2 ml PBS, and then the cells were extracted with passive lysis buffer.

(4) Spectral measurement Spectral measurement was performed using AB1850 spectrophotometer (Ato). The measurement was performed by adding 1 μl of 100 μM coelenterazine as a substrate to 40 μl of the transfected cell extract or recombinant protein. Immediately after the addition of the substrate, a bioluminescence spectrum was measured under the following conditions. That is, for a cell extract, measurement is performed for 3 seconds with a slit width of 0.5 mm, and for a recombinant protein, measurement is performed for 20 seconds with a slit width of 0.25 mm. All spectra were standardized with a maximum wavelength value of 1 after correcting the photosensitivity of the CCD camera.

(5) Quantitative bioluminescence measurement Equimolar amounts of recombinant protein were measured using Rluc assay buffer (500 mM NaCl, 50 mM
Tris-HCl (pH 8.0), 1.0 mM EDTA, 1 mg / ml bovine gelatin, 0.02% Azide) was used for dilution. The amount of luminescence was calculated by integrating for 10 seconds using Luminescencer-MCA (Ato).

(6) Confocal microscope observation
HeLa cells were transfected with an expression vector encoding Rluc-EYFP or BAF-Y, 9 hours later, detached by trypsinization and reseeded on a glass-based dish.
30 hours after transfection, fluorescent images of live cells were taken using a confocal microscope (RT2000; Bio-Rad Laboratories).

(7) In vivo BRET spectrum measurement
BAF-Y _R244A expression plasmid was transfected into NIH3T3 cells and after 30 hours the cells were washed twice with PBS. The coelenterazine derivative ViviRen (Promega) having a final concentration of 60 μM was added, and immediately the spectrum in the living cells of each culture dish was measured with an AB1850 spectrophotometer (Ato).

(8) In vivo 1-cell imaging using BAF protein
Expression plasmids encoding BAF-Y _R244A , NLS-BAF-Y and NLS-BAF-Y-NES were introduced into NIH3T3 cells. Ten hours after transfection, the cells were detached by trypsinization and replated on a glass-based dish. 30 hours after the transfection, the medium was changed to a phenol red-free medium, and a coelenterazine derivative ViviRen (Promega) having a final concentration of 60 μM was added. The cells were then observed using AB3000B Cellgraph (Ato).

(9) Creation of BAF-Y (BRET-based Auto-illuminated Fluorescent Protein on EYFP) by high-efficiency bioluminescence resonance energy transfer (BRET) (FIG. 1).
a. Schematic diagram of an artificial fusion protein consisting of Rluc (Renilla luciferase) and EYFP. SGLRSRAQALAT is a linker peptide.
b. Emission spectrum when Rluc, Rluc-EYFP, and BAF-Y are expressed in HeLa cells and coelenterazine, Rluc luciferin, is added to the resulting cell extract. The spectrum of Rluc (blue line) changes to a spectrum close to the emission spectrum of EYFP due to the resonance energy transfer that occurs between Rluc and EYFP in the BAF-Y molecule. In contrast, Rluc-EYFP is a linker peptide sequence consisting of 12 amino acid residues for highly efficient resonance energy transfer.
It is important to link them in this order. As an index to evaluate the efficiency of BRET, the BRET ratio is expressed as (value of 525 nm (maximum emission wavelength of EYFP) of spectrum) / (value of spectrum 480 nm (maximum emission wavelength of Rluc)) (conventional maximum value) Is 1, Otsuji et al.), Rluc-EYFP is 1.06, and BAF-Y is 3.32.
c. Intracellular localization of Rluc-EYFP and BAF-Y. Focusing on the fact that proteins of 9 nm or less or 60 kD or less are distributed in the nucleus by passive diffusion in the cell, cells of both proteins (Rluc-EYFP and BAF-Y have the same amino acid composition and an estimated molecular weight of 64 kDa) Internal localization was observed with a confocal microscope. Since both are also distributed in the nucleus, it seems that the apparent size is smaller than expected. The relative orientation of EYFP and Rluc is important for highly efficient resonance energy transfer at the same time.

(10) Search for more efficient BAF-Y mutants by Ala scanning (Figure 2)
a. The result of measuring the spectrum in the same manner as in FIG. 1 by introducing a mutation into the linker peptide by Ala substitution. When the linker peptide was SGLRSAAQALAT (BAF-Y _R244A ), the BRET ratio showed a maximum of 4.38 (N = 5).
b. Emission spectrum of BAF-Y _R244A . The shoulder at 480 nm is lowered, and the emission spectrum of EYFP at 525 nm is dominant.

(11) Characteristics of BAF-Y _R244A when producing recombinant protein in E. coli (FIG. 3).
a. Recombinant protein was produced using an expression system with a cold-inducible promoter. In order to enhance protein folding, it was prepared as a fusion protein with an E. coli chaperone (Trigger-Factor (TF)), purified, and cleaved with a sequence-specific protease to obtain a mature protein. The examples were performed with a mixed solution of TF tags (TF tag portion was used for stabilization of mature protein and protein amount correction described later).
b. The luminescence after adding coelenterazine to the mature recombinant protein solution of Rluc, BAF-G _R244A and BAF-Y _R244A was photographed with a digital camera. Rluc alone emitted blue light, but BAF-G _R244A emitted light close to the original green color of EGFP, and BAF-Y _R244A emitted light close to the original yellow green color of EYFP. This is the first example in the world where fluorescent protein is emitted in a color close to the original emission color using coelenterazine instead of external excitation light and an optical filter for blocking excitation light.
c. Quantitative correction was performed on the TF-tag part (common to each recombinant protein). In calculation, 4 pmoles of protein was separated by SDS-PAGE and then subjected to CBB staining. Lane 1; Rluc, Lane 2; BAF-Y _R244A .
d. 100 pmoles coelenterazine was added to 4 pmoles recombinant protein, and the emission spectrum was measured under quantitative measurement conditions. When the amount of luminescence was evaluated as the integrated value of the spectrum, that is, the area surrounded by each spectrum, BAF-Y _R244A was about 4 times brighter than Rluc (N = 5).

(12) Quantitative linearity of BAF-Y _R244A compared with Rluc (FIG. 4).
An equimolar amount of the recombinant protein was serially diluted every 10-fold and measured with a multicolor luminometer (ATTO) by the amount of luminescence (integration for 10 seconds).
a. Measurement results without filter (10-second integration).
b. Measurement results when using a long-pass filter that transmits light in the wavelength range longer than 560 nm (integration for 10 seconds). In both cases, the number of measurements was three, the average value of each measurement point was plotted, and the standard deviation was shown as an error bar, but it was hidden behind the symbol.
In all measurement points, BAF-Y _R244A showed higher luminescence than Rluc, and showed quantitative linearity in the concentration range of at least 6 digits. In particular, near the limit of quantification of Rluc, it showed a more reliable quantification by being bright.

(13) In vivo BRET imaging at the 1 cell level using BAF-Y (FIG. 5).
a. _Confirmation that resonance energy transfer within the BAF-Y _R244A molecule occurs in the physiological environment of living cells. BAF-Y _R244A was introduced into NIH3T3 cells and allowed to fully express, then a coelenterazine derivative (ViviRen, Promega) was added, and resonance energy transfer occurring in living cells was measured as a BRET spectrum. High efficiency BRET similar to that in the test tube was confirmed in the living cells.
b. Single-cell BRET-imaging of NIH3T3 cells expressing BAF-Y mutant after addition of ViviRen. Three types of BAF-Y mutants with different intracellular localization were introduced into NIH3T3 cells, ViviRen was added, and fluorescent protein imaging using the luciferin luciferase reaction was attempted. When using a long-pass filter that transmits light in the wavelength range longer than 515 nm, we succeeded in cell imaging, comparable to the case without a filter. This is also the world's first successful case of imaging at the single cell level by using fluorescent protein only with luciferin without using external excitation light.
(14) A BRET spectrum between Renilla luciferase and Renilla GFP is shown (FIG. 6).
(15) Recovery of high-efficiency BRET, additional data 1 (FIG. 7)
By fusing a domain that forms a dimer (in this case, a BTB domain) to the N-terminus of BAF-Y, steric hindrance stress of the BAF-Y protein occurs, and a decrease in BRET efficiency is observed.
By inserting an appropriate protease cleavage sequence or the like into the second linker portion, recovery of high-efficiency BRET can be expected when cleaved with a protease (In vitro & In vivo).

(16) Recovery of high-efficiency BRET, additional data 2 (Fig. 8)
When the C-terminal part of ATF6 (ATF6-C) is fused to the C-terminal of NLS-BAF-G, a marked decrease in BRET efficiency is observed in cultured cells. Probably due to conformational stress due to the incorporation of ATF6 into the endoplasmic reticulum membrane. When cleavage at the second linker site (for example, Caspase) occurs, high-efficiency BRET is restored, and the emission color changes from blue to green, and the NLS-BAF-G moiety moves from the endoplasmic reticulum membrane to the nucleus. Is expected (In vivo).

Example 2
In the following, “BAF-Y _R244A ” is simply referred to as “BAF-Y”. (1) pH-dependent spectrum measurement of BAF-Y The measurement buffers are pH 6.3, pH 6.6, pH 6.9, pH 7.2, pH 7.5. 150 mM BIS-TRIS-propane, 500 mM NaCl, and 1 mM EDTA aqueous solution adjusted to pH 7.8 and pH 8.1 were used. 1 μl of each recombinant protein solution was added to 39 μl of the buffer adjusted to each pH to obtain a uniform solution. To this was added 1.5 μl of a 100 μM coelenterazine solution, and the spectrum was immediately measured with an AB1850 spectrophotometer (Ato) under exposure conditions of a slit width of 0.25 mm and 3 seconds. The results are shown in FIG.

(2) Spectral measurement of BAF-Y depending on salt concentration A 50 mM Tris-HCl (pH 8.0) aqueous solution containing 0 mM NaCl, 35 mM NaCl, and 300 mM NaCl was used as a measurement buffer. 1 μl of each recombinant protein solution was added to 40 μl of measurement buffer adjusted to each salt concentration to obtain a uniform solution. To this was added 1 μl of a 100 μM coelenterazine solution, and the spectrum was immediately measured with an AB1850 spectrophotometer (Ato) under exposure conditions of a slit width of 0.25 mm and 1 second. The results for Rluc are shown in FIG. 10a, and the results for BAF-Y are shown in FIG. 10b.

(3) Preparation of plasmid Rluc mutant (Rluc8) (AMLoening et al., Protein) into which 8 point mutations already reported (A55T, C124A, S130A, K136R, A143M, M185V, M253L, S287L) were introduced Engineering, Design and Selection vol.19 no.9 pp.391-400 (2006)), 8 single amino acid substitution mutations were introduced into Rluc. Site-specific substitution mutagenesis is QuickChange II XL Kit
(STRATAGENE) was used. The primers used for introducing site-specific substitution mutations at 8 sites are as follows.
(1) A55T amino acid substitution primer set
RlucA55T sence, CTGCATGGTAACGCTACCTCCAGCTACCTGT (SEQ ID NO: 43) and
RlucA55T antisense, ACAGGTAGCTGGAGGTAGCGTTACCATGCAG (SEQ ID NO: 44);
(2) C124A amino acid substitution primer set
RlucC124A sense, GTGGGCCACGACTGGGGGGCTGCTCTGGCCTTTCACTACTCCTA (SEQ ID NO: 45) and
RlucC124A antisense, TAGGAGTAGTGAAAGGCCAGAGCAGCCCCCCAGTCGTGGCCCAC (SEQ ID NO: 46);
(3) S130A amino acid substitution primer set
RlucS130A sense, CTGGCCTTTCACTACACCTACGAGCACCAAG (SEQ ID NO: 47) and
RlucS130A antisense, CTTGGTGCTCGTAGGTGTAGTGAAAGGCCAG (SEQ ID NO: 48);
(4) K136R amino acid substitution primer set
RlucK136R sense, ACGAGCACCAAGACAGGATCAAGGCCATCGT (SEQ ID NO: 49) and
RlucK136R antisense, ACGATGGCCTTGATCCTGTCTTGGTGCTCGT (SEQ ID NO: 50);
(5) A143M amino acid substitution primer set
RlucA143Msense, AGGATCAAGGCCATCGTCCACATGGAGAGTGTCGTGGACGTGATC (SEQ ID NO: 51)
RlucA143M antisense, GATCACGTCCACGACACTCTCCATGTGGACGATGGCCTTGATCCT (SEQ ID NO: 52); (6) M185V amino acid substitution primer set
RlucM185V sense, CTTCTTCGTCGAGACCGTGCTCCCAAGCAAGAT (SEQ ID NO: 53) and
RlucM185V antisense, ATCTTGCTTGGGAGCACGGTCTCGACGAAGAAG (SEQ ID NO: 54);
(7) M253L amino acid substitution primer set
RlucM253L sense, GACGATCTGCCTAAGCTGTTCATCGAGTCCG (SEQ ID NO: 55) and
RlucM253L antisense, CGGACTCGATGAACAGCTTAGGCAGATCGTC (SEQ ID NO: 56);
(8) S287L amino acid substitution primer set
RlucS287L sense, AAGGTGAAGGGCCTCCACTTCCTCCAGGAGGACGCTCCAGATGA (SEQ ID NO: 57)
RlucS287L antisense, TCATCTGGAGCGTCCTCCTGGAGGAAGTGGAGGCCCTTCACCTT (SEQ ID NO: 58).

  The clones into which each one amino acid substitution mutation was introduced were confirmed by sequencing. By repeating this operation continuously, pCMV-EYFP-Rluc8 having 1 amino acid substitution mutation at 8 sites was constructed. pCMV-EYFP-Rluc and pCMV-EYFP-Rluc8 were digested with AgeI and NdeI to remove the region encoding EYFP, and after blunting the DNA ends, pCMV-Rluc and pCMV-Rluc8 were obtained by self-ligation. PCMV-EYFP-R244A oligo DNA pair (DNA fragment annealed R244A-F, 5'-GTACAAGTCGGGGCTGCGGAGCGCGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 15) and R244A-R, 5'-TAGTCGCCAACGCCTGCGCCGCGCTCCGCAGCCCCGACTT-3' (SEQ ID NO: 16)) PCMV-eBAF-Y (eBAF-Y, abbreviated enhanced BAF-Y) was obtained by insertion into the BsrGI / NdeI site.

In order to construct a histone H2AX-eBAF-Y expression plasmid, histone H2AX cDNA (GenBank
Accession number: NM_002105) was amplified by PCR reaction. The primers used in the PCR reaction are as follows. N-H2AX-F-Xh, 5'-CCGACTCGAATGTCGGGCCGCGGCAAGAC-3 '(SEQ ID NO: 59); and N-H2AX-R-Hd3, GCCCAAGCTTCGTACTCCTGG GAGGCCTGGG-3' (SEQ ID NO: 60). The amplified PCR fragment was digested with XhoI and HindIII and inserted into XhoI / HindIII-digested pCMV-eBAF-Y to construct pCMV-H2AX-eBAF-Y. In order to construct eBAF-Y-H2AX expression plasmid, histone H2AX cDNA was amplified by PCR reaction. The primers used in the PCR reaction are as follows. C-H2AX-F-Kpn, 5′-CCGAGGTACCATGTCGGGCCGCGGCAAGAC-3 ′ (SEQ ID NO: 61) and C-H2AX-R-Bam, 5′-GGCGGAATCCTTAGTACTCCTGGGAGGCCT-3 ′ (SEQ ID NO: 62). The amplified PCR fragment was digested with KpnI and BamHI HindIII and inserted into KpnI / BamHI-digested pCMV-eBAF-Y to construct pCMV-eBAF-Y-H2AX.

(4) Cell culture and transfection
HeLa cells (luminescence intensity comparison experiment) or NIH3T3 cells (short-term stability evaluation experiment of luminescence) were maintained at 37 ° C. in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum. Cells were seeded 4-6 × 10 ⁵ cells per well in 6-well plates one day prior to transfection. Transient transfection was performed using Lipofectamine 2000 reagent for HeLa cells and Lipofectamine plus reagent for NIH3T3 cells. Each expression plasmid used for transfection was adjusted to equimolarity with the Rluc (or Rluc8) cDNA portion, and cotransfected with the North American firefly luciferase expression plasmid as an internal standard to correct transfection efficiency. It was done. 30 hours after transfection (luminescence intensity comparison experiment) At 24, 48 and 72 hours time points (short-term stability evaluation experiment of luminescence), cells were washed with 2 ml PBS and then passive lysis Extracted with buffer.
-Luminescence intensity comparison experiment Each extract was measured for the luminescence activity of firefly luciferase with a luminometer, and then the spectrum of each extract was measured using an AB1850 spectrophotometer (Ato). The measurement was performed by adding 1 μl of 100 μM coelenterazine as a substrate to 40 μl of the transfected cell extract. Immediately after the addition of the substrate, a bioluminescence spectrum was measured under the following conditions. That is, for the cell extract, the measurement is performed for 3 seconds with a slit width of 0.25 mm, and for the recombinant protein, the measurement is performed for 20 seconds with a slit width of 0.25 mm. All spectra were corrected for the light-sensitive characteristics of the CCD camera. Four independent measurements were made for each sample. Each data was corrected for transfection efficiency by the activity of firefly luciferase.
-Short-term stability evaluation experiment of luminescence Each extract was measured by measuring the luminescence activity of firefly luciferase with a luminometer and then adding a coelenterazine luminescence solution. The experiment was performed three times independently. Each data was corrected for transfection efficiency by the activity of firefly luciferase.

  The results are shown in FIG.

(5) Measurement of in vivo BRET spectrum
Expression plasmids for eBAF-Y-H2AX and H2AX-eBAF-Y were transfected into NIH3T3 cells, and after 30 hours the cells were washed twice with PBS. ViviRen, a coelenterazine derivative with a final concentration of 60 μM
(Promega) was added, and the spectrum in the living cells immediately after the culture dish was measured with an AB1850 spectrophotometer (Ato).

In vivo single-cell imaging with BAF protein
Expression plasmids encoding eBAF-Y-H2AX and H2AX-eBAF-Y were introduced into NIH3T3 cells. Ten hours after transfection, the cells were detached by trypsinization and replated on a glass-based dish. 30 hours after the transfection, the medium was changed to a phenol red-free medium, and a coelenterazine derivative ViviRen (Promega) having a final concentration of 60 μM was added. Cells were then observed at 1 minute intervals with an exposure condition of 10 seconds using an AB3000B Cellgraph (Ato) fitted with a 40 × lens. The results are shown in FIG.
Example 3
Preparation of plasmid Already reported mutants of RLuc (RLuc8 / A123S / D162E / I163L and RLuc8.6-532) (AMLoening rt al., Nature Methods, vol.4 no.8 pp.641-643 (2007) 3 or 4 single amino acid substitution mutations were introduced into RLuc. Site-specific substitution mutagenesis is QuickChange
II XL Kit (STRATAGENE) was used.
・ Introduction of RLuc8 / A123S / D162E / I163L mutation into eBAF-Y
A123S and D162E / I163L mutations were further introduced in two steps into the RLuc8 part of eBAF-Y (mutant in which the RLuc part of BAF-Y was replaced with RLuc8, abbreviated as enhanced BAF-Y). The primer sets used for mutation introduction are as follows.
RLuc8A123S sense,
5'-GGCCACGACTGGGGGtCTgcTCTGGCCTTTCA-3 '(SEQ ID NO: 63);
RLuc8A123S anti,
5'-TGAAAGGCCAGAgcAGaCCCCCAGTCGTGGCC-3 '(SEQ ID NO: 64);
RLuc8AD162E_I163L sense, 5'-GGCCTGACATCGAGGAGGAacTCGCCCTGATCAAGAGCGA-3 '(SEQ ID NO: 65);
RLuc8AD162E_I163L anti, 5'-TCGCTCTTGATCAGGGCGAgtTCCTCCTCGATGTCAGGCC-3 '(SEQ ID NO: 66)
(Lower case is the mutation site).

In addition, it has been confirmed by determining the DNA sequence that no extra mutation or the like has occurred in each step. The plasmid introduced with the above mutation (pBAF-Y_m3) is BsrGI
The following oligo DNA fragment was inserted into the resulting BsrGI / NdeI site. The sequence of the introduced oligo DNA is as follows.
BRET-tag12 aa sense, 5'-GTACAAGTCGGGGCTGCGGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 67);
BRET-tag 12 aa antisense, 5'-TAGTCGCCAACGCCTGCGCCCTGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 68);
S239A-F,
5'-GTACAAGGCGGGGCTGCGGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 69);
S239A-R,
5'-TAGTCGCCAACGCCTGCGCCCTGCTCCGCAGCCCCGCCTT3 '(SEQ ID NO: 70);
G240A-F,
5'-GTACAAGTCGGCGCTGCGGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 71);
G240A-R,
5'-TAGTCGCCAACGCCTGCGCCCTGCTCCGCAGCGCCGACTT-3 '(SEQ ID NO: 72);
L241A-F,
5'-GTACAAGTCGGGGGCGCGGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 73);
L241A-R,
5'-TAGTCGCCAACGCCTGCGCCCTGCTCCGCGCCCCCGACTT-3 '(SEQ ID NO: 74);
R242A-F,
5'-GTACAAGTCGGGGCTGGCGAGCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 75);
R242A-R,
5'-TAGTCGCCAACGCCTGCGCCCTGCTCGCCAGCCCCGACTT-3 '(SEQ ID NO: 76);
S243A-F,
5'-GTACAAGTCGGGGCTGCGGGCCAGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 77);
S243A-R,
5'-TAGTCGCCAACGCCTGCGCCCTGGCCCGCAGCCCCGACTT-3 '(SEQ ID NO: 78);
R244A-F,
5'-GTACAAGTCGGGGCTGCGGAGCGCGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 79);
R244A-R,
5'-TAGTCGCCAACGCCTGCGCCGCGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 80);
Q246A-F,
5'-GTACAAGTCGGGGCTGCGGAGCAGGGCGGCGGCGTTGGCGAC-3 '(SEQ ID NO: 81);
Q246A-R,
5'-TAGTCGCCAACGCCGCCGCCCTGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 82);
L248A-F,
5'-GTACAAGTCGGGGCTGCGGAGCAGGGCGCAGGCGGCGGCGAC-3 '(SEQ ID NO: 83);
L248A-R,
5'-TAGTCGCCGCCGCCTGCGCCCTGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 84);
T250A-F,
5'-GTACAAGTCGGGGCTGCGGAGCAGGGCGCAGGCGTTGGCGGC-3 '(SEQ ID NO: 85);
T250A-R,
5'-TAGCCGCCAACGCCTGCGCCCTGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 86);
L241R244A-F,
5'-GTACAAGTCGGGGGCGCGGAGCGCGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 87);
L241R244A-R,
5'-TAGTCGCCAACGCCTGCGCCGCGCTCCGCGCCCCCGACTT-3 '(SEQ ID NO: 88);
R244Q246A-F,
5'-GTACAAGTCGGGGCTGCGGAGCGCGGCGGCGGCGTTGGCGAC-3 '(SEQ ID NO: 89);
R244Q246A-R,
5'-TAGTCGCCAACGCCGCCGCCGCGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 90);
L241R244Q246A-F,
5'-GTACAAGTCGGGGGCGCGGAGCGCGGCGGCGGCGTTGGCGAC-3 '(SEQ ID NO: 91);
L241R244Q246A-R,
5'-TAGTCGCCAACGCCGCCGCCGCGCTCCGCGCCCCCGACTT-3 '(SEQ ID NO: 92);
R244G-F,
5'-GTACAAGTCGGGGCTGCGGAGCGGGGCGCAGGCGTTGGCGAC-3 '(SEQ ID NO: 93);
R244G-R,
5'-TAGTCGCCAACGCCTGCGCCCCGCTCCGCAGCCCCGACTT-3 '(SEQ ID NO: 94);
all-AF,
5'-GTACAAGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGC-3 '(SEQ ID NO: 95);
all-AR,
5′-TAGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCGCCTT-3 ′ (SEQ ID NO: 96).

Each pair of oligo DNAs was annealed and inserted into the BsrGI / NdeI site of the above plasmid (production of an improved new BAF-Y, the example illustrated is the sequence of L241R244Q246A, which was tested above. In all linkers, the shoulder of the spectrum at 480 nm is eliminated; FIGS. 13 and 14).
・ Fabrication of eBAF-R1
In order to crush the endogenous BsrGI site without changing the amino acid sequence of TurboRFP (EVROGEN) and generate a new BsrGI site, a substitution mutation was introduced (pTurboRFP'-C). The primer sets used for mutation introduction are as follows.
TurboRFPΔBsrGI-F,
5'-CATGCACATGAAGCTcTACATGGAGGGCACC-3 '(SEQ ID NO: 97);
TurboRFPΔBsrGI-R,
5'-GGTGCCCTCCATGTAgAGCTTCATGTGCATG-3 '(SEQ ID NO: 98);
TurboRFP + BsrGI-F,
5'-GACCTCCCTAGCAAACTGGtGtACAGAGATGAATCCGGACT-3 '(SEQ ID NO: 99);
TurboRFP + BsrGI-R,
5′-AGTCCGGATTCATCTCTGTaCaCCAGTTTGCTAGGGAGGTC-3 ′ (SEQ ID NO: 100).
By introducing this substitution mutation, two amino acid substitutions of G227V / H228Y are introduced into TurboRFP. The prepared pTurboRFP'-C was digested with NheI and BsrGI to obtain TurboRFP cDNA. This was inserted into the NheI / BsrGI site of peBAF-Y, and the fluorescent protein portion was replaced from EYFP to RFP (peBAF-TgR). This was digested with BsrGI and NdeI, and the oligo-DAN pair described above was introduced into this, and the spectrum was examined. When R244Q246A is used as a linker, it has the highest BRET efficiency, and this is called peBAF-R1
(FIG. 15).

・ Introduction of RLuc8 / A123S / D162E / I163L / V185L (RLuc8.6-532) mutation into eBAF-Y BAF-Y plasmid introduced with RLuc8 / A123S / D162E / I163L as described above ・ V185L mutation into pBAF-Y_m3 did. The primer sets used for mutation introduction are as follows.
RLuc8V185L sense,
5'-TTCTTCGTCGAGACCcTGCTCCCAAGCAAGA-3 '(SEQ ID NO: 101);
RLuc8V185L anti,
5′-TCTTGCTTGGGAGCAgGGTCTCGACGAAGAA-3 ′ (SEQ ID NO: 102). The plasmid into which the mutation was introduced was designated as peBAF-Y.6-532.

・ Production of eBAF-R2
peBAF-Y.6-532 was digested with NheI and BsrGI to remove the EYFP cDNA, and instead a TurboRFP '/ NheI-BsrGI fragment was inserted, which was designated as peBAF-R2 (FIG. 16).

Cell culture and transfection
HeLa cells and NIH3T3 cells were maintained at 37 ° C. in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum. Cells were seeded 4-6 × 10 ⁵ cells per well in 6-well plates one day prior to transfection. Transient transfection was performed using Lipofectamine 2000 reagent for HeLa cells and Lipofectamine plus reagent for NIH3T3 cells. 30 hours after transfection, the cells were washed with 2 ml PBS, and then the cells were extracted with passive lysis buffer.

Spectral measurements Spectral measurements were performed using an AB1850 spectrophotometer (Ato). The measurement was performed by adding 1 μl of 100 μM coelenterazine as a substrate to 40 μl of the transfected cell extract. Immediately after the addition of the substrate, a bioluminescence spectrum was measured under the following conditions. That is, for a cell extract, measurement is performed for 3 seconds or 20 seconds with a slit width of 0.25 mm. All spectra were standardized with a maximum wavelength value of 1 after correcting the photosensitivity of the CCD camera (FIGS. 13, 14, 15, and 16).

・ Preparation of pDRmmBTB-Casp8-BAF-Y
pDRmmBTB-HdS-BAF-Y was digested with HindIII and SalI, and the following oligo DNA pair was inserted into the HindIII / SalI site.
BTBHdCasp8Slsense,
5'-AGCTTCGAGCGGGCTGGAGACGGATGGGGTCGACTC-3 '(SEQ ID NO: 103);
BTBHdCasp8Slanti,
5′-AGCTGAGTCGACCCCATCCGTCTCCAGCCCGCTCGA-3 ′ (SEQ ID NO: 104).
The resulting plasmid was designated as pDRmmBTB-Casp8-BAF-Y.

-Preparation of pCII-DRmmBTB-Casp8-BAF-Y In order to prepare an E. coli expression plasmid, a gene encoding pDRmmBTB-Casp8-BAF-Y was amplified by PCR using the primers shown below. The obtained PCR fragment was subcloned into the NdeI-XbaI site of pCold-II DNA (TAKARA) to obtain pCII-DRmmBTB-Casp8-BAF-Y. The primers used for PCR are as follows:
DsRedmFwdColdTF-F,
5'-GTCGCCCATATGGACAACACCGAGGACGTCA-3 '(SEQ ID NO: 105);
TFhRL-R,
5′-GTTATCTAGAATTACTGCTCGTTCTTCAGCACGC-3 ′ (SEQ ID NO: 106).

Recombinant protein preparation Recombinant DRmmBTB-Casp8-BAF-Y was synthesized by a cold shock-inducible promoter system in E. coli BL21 strain. The recombinant protein was purified with a Ni-NTA affinity column to obtain a mature recombinant protein (FIGS. 17 and 18).

In vitro Caspase-8 digestion and spectral measurement of recombinant proteins Dilute 10-fold with recombinant protein 1x ICE standards buffer (100 mM Tris-HCl (pH 7.5), 10% glycrol, 0.1% CHAPS) purified from E. coli. Recombinant Caspase-8 (Calbiochem) was added and heated at 30 ° C. After reacting for a sufficient amount of time, a part of the reaction solution is diluted 8-fold with TE (10 mM Tris-HCl (pH 8.0), 1 mM EDTA), and 1 μl of 100 μM coelenterazine is added to 40 μl of the diluted solution. Spectrum measurement was performed. The measurement conditions of the spectrum are exposure measurement for 2 seconds at 20 ° C. with a slit width of 0.25 mm. All spectra were evaluated by standardization with the light emission characteristics of the CCD camera corrected and the relative emission intensity and the maximum wavelength value taken as 1. A part of the reaction solution was subjected to SDS-PAGE, separated by molecular size, and then subjected to Comassie staining. DRmmBTB-Casp8-BAF-Y is digested with Caspase-8 and separated into DRmmBTB and BAF-Y, so that the BRET-inhibition of BAF-Y, which was done by DRmmBTB, is released. It is thought that a relative decrease in the shoulder at 480 nm (FIG. 17) and an increase in light emission (FIG. 18) occur.

Until now, the use of fluorescent proteins has been limited to excitation light sources, and is rarely used outside the research field. Further, since the fluorescent protein is excited by a powerful excitation light source, it is necessary to use a special optical filter for fluorescence detection of the fluorescent protein. On the other hand, the chimeric protein according to the present invention does not require the use of a special optical filter because it irradiates the pinpoint with energy having an intensity suitable for exciting the fluorescent protein in the immediate vicinity of the fluorescent protein molecule. That is, it is a very simple system that can be realized only with luciferin (particularly coelenterazine) and the self-contained fluorescent protein according to the present invention. It opens the door to the application of bioluminescence through the fusion of luciferase and fluorescent proteins in fields that have never been envisaged.

  By changing the amino acid sequence of the linker region that is the core of the present invention, the BRET spectrum pattern can be changed, and the color tone from blue to yellow-green can be arbitrarily changed by the fusion of luciferase and fluorescent protein. It can be applied as a technology.

Creation of BAF-Y (BRET-based Auto-illuminated Fluorescent Protein on EYFP) by highly efficient bioluminescence resonance energy transfer (BRET). Search for more efficient BAF-Y mutants by Ala scanning. Characteristics of BAF-Y _R244A when recombinant protein is produced in E. coli. _Comparison of quantitative linearity of BAF-Y _R244A with Rluc. In vivo BRET imaging at the single cell level using BAF-Y. The spectrum of BRET between Renilla luciferase and Renilla GFP is shown. Recovery of high-efficiency BRET, additional data 1 Recovery of high-efficiency BRET, additional data 2 PH dependence of BRET in BAF-Y. (supplementary Fig. 3a, 3b) pH-dependent BAF-Y BRET spectrum change (standardized spectrum). When the change of BRET efficiency in the spectrum normalized by setting the emission maximum wavelength value of each spectrum to 1 is evaluated, there is no change in the standardized spectrum pattern of Rluc between pH 6.3 and pH 8.1, but BAF-Y With regard to, there are very significant changes. PH dependence of BRET in BAF-Y. (supplementary Fig. 3a, 3b) pH-dependent change of BRET spectrum of BAF-Y (evaluation of spectrum by relative emission intensity). The luminous intensity of Rluc is almost constant between pH 6.3 and pH 7.2, but the luminescence activity decreases from pH 7.5 to pH 8.1. Regarding BAF-Y, as the pH increases, the relative proportion of the Rluc emission maximum near 480 decreases, and conversely, the spectrum near the fluorescence maximum wavelength of EYFP becomes dominant. This is thought to be due to the sum of the balance between EYFP fluorescence instability in the acidic region and pH-dependent Rluc luminescence activity. The pH dependence of BRET observed in the BAF-Y molecule suggests that BAF-Y can be applied as a pH indicator. This property can also be applied as a technique for changing the emission color between blue and yellow-green depending on pH. BRET salt concentration dependence in BAF-Y. (supplementary Fig.4) With respect to Rluc, although the luminescence activity increases with increasing salt concentration, there is no effect on the standardized emission spectrum. It is thought to depend on the stabilization of protein by salt. BRET salt concentration dependence in BAF-Y. (supplementary Fig.4) Regarding BAF-Y, similar to Rluc, the increase in luminescence intensity with increasing salt concentration is observed. On the other hand, in the standardized BRET spectrum, a slight decrease in BRET efficiency is observed although it is slight. This is presumably due to the balance between the luminous activity of Rluc stabilized by salt and the fluorescence instability of EYFP sensitive to [Cl ⁻ ] (chloride ion concentration). This is an important factor in determining the buffer composition when using BAF-Y as a bioactivity indicator or bioactivity probe. Development of improved Rluc mutants _{(Rluc A55T / C124A / S130A /} K136R / A143M / M185V / M253L / S287L, Rluc8) was introduced e nhanced BAF-Y (eBAF- Y). (Fig.2) a. Increasing the emission intensity by 25 times or more than the original Rluc by introducing Rluc8, a mutant with improved emission intensity, reported by Loening AM et al. Instead of Rluc in BAF-Y BAF-Y mutant / enhanced BAF-Y (eBAF-Y) was successfully produced. This strongly suggests that further improvement of Rluc can be applied to the improvement of BAF-Y in the future. b. eBAF-Y is more stably expressed in cells than Rluc8. This is advantageous in that eBAF-Y is not only brighter than Rluc8 but also emits light stably. (e) Application of BAF-Y as a bioluminescent tag. (Fig.3) Histone H2AX and eBAF-Y fusion proteins, eBAF-Y-H2AX and H2AX-eBAF-Y, which are subtypes of histone H2A, which is a component of histone octamer constituting core histone, were prepared. By measuring the in vivo BRET spectrum, eBAF-Y can maintain high-efficiency BRET when fused to either the N-terminus or C-terminus of histone H2AX. In in vivo BRET imaging, both eBAF-Y-H2AX and H2AX-eBAF-Y are localized in the nucleus, and can reproduce the original intracellular distribution pattern of H2AX. Therefore, (e) BAF-Y can be used as a bioluminescent tag as a tool for visualizing the dynamics of proteins in living cells by bioluminescence. Comparison of new BAF-Y obtained in Example 3 and BAF-Y obtained in Example 2: new BAF-Y is a Rluc mutant (Rluc8 / A123S / D162E / I163L) instead of Rluc. BAF-Y used. The graph shows the results when the linker sequence is SGARSAAAALAT. All other linkers tried (SGLRSRAH, SGLRSRAQALAT, AGLRSRAQALAT, SALRSRAQALAT, SGARSRAQALAT, SGLASRAQALAT, SGLRARAQALAT, SGLRSAAQALAT, SGLRSRAAALAT, SGLRSRAQAAAT, SGLRSRAQALAT, SGLRSAAQAL It was. With new BAF-Y, the shoulder of the spectrum around 480nm is eliminated. The value of 527nm / 480nm of new BAF-Y is 7.46. The increase in apparent BRET efficiency coincided with the fluorescence maximum wavelength (527 nm) of EYFP. The comparison with the spectrum of new BAF-Y obtained in Example 3, original Rluc, conventional BAF-A (AcGFP-Rluc), and BAF-G (EGFP-Rluc) is shown. Results of BAF-R using red fluorescent protein (RFP): RFP (TurboRFP) and Rluc8 are linked in this order by the linker sequence SGLRSAAAALAT. Since Rluc8 is used, it is expressed as eBAF-R1. The maximum wavelength difference between the donor and acceptor is 91 nm, indicating that a high-resolution BRET assay is possible. Results of BAF-R using red fluorescent protein (RFP). : RFP (TurboRFP) and Rluc8.6-532 connected in this order with a linker sequence, SGLRSRAH. One molecule of BRET protein with the dominant fluorescence wavelength of RFP obtained by BRET was obtained. Application technology of BAF-Y (Example of recovery from BRET inhibition): A specific example of additional data 1 (FIG. 7), showing a normalized spectrum. The sequence of ASSG LETD GVDSAS was introduced into the space between the BTB domain and BAF-Y of DRmmBTBHdSBAF-Y. Underlined LETD is a specific recognition cleavage sequence of Caspase-8. The result is that recombinant protein was mixed with recombinant caspase-8 in a test tube and cleaved. By cutting the DRmmBTB part, which lowers the BRET efficiency, the BRET efficiency is restored (perhaps the distortion of the three-dimensional structure to the BAF-Y has been eliminated), and it appears in the form that the shoulder near 480 nm is lowered. Yes. In set is obtained by separating the recombinant protein with and without Caspase-8 by SDS-PAGE and staining with CBB. Digestion with Caspase-8 breaks down into BAF-Y and BRET inhibitory part (DRmmBTB). Application technology of BAF-Y (Example of recovery from BRET inhibition): Specific example of additional data 1 (Fig. 7) showing relative emission intensity spectrum. It was shown that recovery from inhibition of BRET increased the relative luminescence intensity with the same molecule. That is, it has been shown that the emission intensity increases as a reaction to be monitored occurs.

  The Rluc mutants (Rluc8 / A123S / D162E / I163L and Rluc8.6-532) used were those reported in Nature Methods, Vol. 4, No. 8, p641-643 (2007).

Claims (12)

A chimeric protein comprising an energy accepting protein and an energy generating protein linked via a linker, wherein a visible energy transfer can occur between the energy accepting protein and the energy generating protein, wherein the energy accepting protein is a fluorescent protein. Yes, the energy generating protein is Renilla luciferase, the linker is composed of 8 to 16 amino acids, the energy accepting protein is on the N-terminal side, the energy generating protein is on the C-terminal side, and the BRET ratio (energy accepting) A chimeric protein in which the intensity of the luminescence maximum wavelength of the protein / (intensity of the luminescence maximum wavelength of the energy generating protein) is 3 or more, and the fluorescent protein is GFP, YFP, BFP, CFP, DsRED or RFP The chimeric protein according to claim 1, wherein the fluorescent protein is YFP. The chimeric protein according to claim 1, wherein the linker consists of 10 to 14 amino acids. The chimeric protein according to claim 1, wherein the linker consists of 12 amino acids. A polynucleotide encoding the secretory or membrane-bound chimeric protein according to any one of claims 1 to 4, or a complementary strand thereof. A vector comprising the polynucleotide according to claim 5. A transformant transformed with the vector according to claim 6. Use of the chimeric protein according to any one of claims 1 to 4 as a label. The use according to claim 8, for labeling a substance selected from the group consisting of a ligand capable of recognizing an antigen, antibody, enzyme, DNA, RNA, protein, sugar chain, cell or receptor. Introducing a vector according to claim 6 into a host to produce a transformant (excluding human) , applying Renilla luciferin to the transformant, and detecting luminescence by BRET from the chimeric protein; A method for imaging a transformant. The method according to claim 10, wherein the transformant is a prokaryotic cell, a eukaryotic cell, a tissue, an organ, an animal (excluding human) or a plant. The method according to claim 10, wherein the transformant is a mammalian cell other than a human .

Images