JP2003294736A - Screening method using fluorescent protein fusion probe - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply performing a fusion screening between a probe protein and a target substance. <P>SOLUTION: The method for screening target substances for a probe protein to fuse from a plurality of candidate target substances immobilized on a carrier, by which a fluorescent protein fusion probe is prepared which is a fusion protein between a probe protein and a fluorescent protein, to identify a target substance that combines with the probe protein by detecting a fluorescent light signal of the fluorescent protein after the fluorescent protein fusion probe into contact with all of the candidate target substances. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The invention of this application relates to a method for screening a target substance using a fluorescent protein fusion probe, which can be used for investigating the interaction between a probe protein and various substances.

[0002]

Many new genes have been discovered in the genome project. In order to investigate the functions of the proteins encoded by these genes and to develop new drugs using these proteins, it is necessary to find substances that bind to these proteins. Therefore, various assay methods for that have been developed (EM Phizicky a
nd S. Fields, Microbiol. Rev. 59: 94-123, 1995; A.
R. Mendelsohn and R. Brent, Science 284: 1948-195
0, 1999). The most general method is a method in which a plurality of target candidate substances are fixedly arranged on a carrier, and a labeled probe is allowed to act on them to examine whether or not they bind.

When the probe is a protein, it is first necessary to prepare a protein to be the probe. In many cases, a recombinant protein is expressed by introducing an expression vector of a cDNA encoding the protein into various cells. The expressed protein is isolated and purified, labeled with a radioisotope, a fluorescent dye, an enzyme, etc., and then used as a probe. Labels with fluorescent dyes are often used because of their ease of detection.

On the other hand, fluorescent proteins derived from luminescent jellyfish are known as fluorescent labeling substances for proteins. When this fluorescent protein is expressed intracellularly, a fluorescent signal is emitted inside the cell. Therefore, a fluorescent protein fused to an intracellular protein is expressed inside the cell, and the localization site of the protein is examined from the fluorescent emission site. Is possible (R. Rizzuto et al., Cur
r. Biol. 5: 635-642, 1995). In addition, a fusion protein of a fluorescent protein and an arbitrary protein was used as an E. coli (JC Lewis and
S. Daunert, Anal. Chem. 71: 4321-4327, 1999) and in vitro transcription / translation (TW Kahn et al., Curr. Biol.
7: R207-R208, 1997) has been reported.
However, there is no example in which such a fluorescent protein fusion protein is used as a probe for screening a target substance.

[0005]

Recently, it has become possible to use a DNA chip, a protein chip, a compound chip or the like in which a target candidate substance is fixedly arranged in a microarray in a minute area on a carrier. Accordingly, it is possible to screen a large number of target candidate substances with a trace amount of probe. However, in the conventional preparation of a labeled probe protein, it is necessary to perform a great deal of labor and time for covalent binding, ionic binding, hydrophobic binding, or binding using a crosslinking agent such as a maleimide compound between the protein and the labeling substance. was there. Since such a great amount of labor and time are required regardless of the amount of probe, it cannot be ignored that the benefit of reducing the amount of probe due to the spread of DNA chips and the like is slight.

The invention of this application has been made in view of the above circumstances, and an object thereof is to provide a method for screening a target substance using a fluorescence-labeled protein probe that can be easily prepared. .

[0007]

The invention of this application, as an invention for solving the above-mentioned problems, screens a target substance to which a probe protein binds from a plurality of target candidate substances fixedly arranged on a carrier. A method of preparing a fluorescent protein fusion probe, which is a fusion protein of a probe protein and a fluorescent protein, contacting this fluorescent protein fusion probe with all target candidate substances, and then detecting the fluorescent signal of the fluorescent protein. Provides a screening method characterized by identifying a target substance bound with a probe protein.

In a preferred embodiment of the method of the present invention, a fluorescent protein fusion probe is prepared as an in vitro transcription / translation product of a fusion polynucleotide of a polynucleotide encoding a probe protein and a polynucleotide encoding a fluorescent protein. .

In the method of the present invention, another preferred embodiment is that the target candidate substance immobilized on the carrier is a cell population containing the target protein.

Hereinafter, each of the above inventions will be described in detail with reference to embodiments.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the “probe protein” is a protein or peptide for screening a target substance of interest from a plurality of target substance candidates by its specific binding property with the target substance. . The probe protein can be any protein derived from any biological species including human. The function may be known or the function may be unknown. The amino acid sequence of the probe protein and the DNA sequence encoding it may be unknown, but are preferably known. The amino acid sequence may be a naturally-occurring protein-derived sequence or an artificially designed sequence. Furthermore, this probe protein may be a polypeptide or oligopeptide consisting of a partial continuous sequence in the amino acid sequence of the natural protein.

The “plurality of target candidate substances” are a group of substances that may contain “target substances” that specifically bind to a probe protein, and such substances are nucleic acids (DNA, R
NA, etc.), saccharides, proteins, synthetic peptides, cells, natural compounds such as cells and organic compounds, or synthetic substances, etc. In this invention, the target substance is a protein,
A preferred embodiment uses a cell population containing such a target protein as a target candidate substance.

The "specific bond" between the target substance and the probe protein is, for example, a bond based on a hydrogen bond, a hydrophobic bond, an ionic bond, a coordinate bond or the like between a plurality of molecules constituting the both.

A plurality of target candidate substances are fixedly arranged on a carrier. As the "carrier", a transparent plate such as a slide glass used for a usual DNA chip or protein chip can be used. The “fixed arrangement” means that the target candidate substances are aligned and arranged at a predetermined position on the carrier by a spotting method or the like, and the type of each target candidate substance can be specified by the position. In order to reduce the probe amount as much as possible, it is desirable to immobilize the target candidate substance in the narrowest possible area on the carrier.

In the method of the present invention, a fluorescent protein fusion probe which is a fusion protein of a probe protein and a fluorescent protein is prepared. "Fluorescent protein" is a protein that emits fluorescence when irradiated with excitation light, for example, green fluorescent protein (GFP) derived from luminescent jellyfish, and its mutants EGFP and E.
YFP (yellow fluorescence), ECFP (blue fluorescence), DsRed1 (red fluorescence), DsRed2, green fluorescent protein hrGF derived from Renilla
P etc. can be illustrated.

The fusion protein of the probe protein and the fluorescent protein is prepared by a genetic engineering technique. That is,
A fusion polynucleotide prepared by fusing a polynucleotide encoding a probe protein with a polynucleotide encoding a luminescent protein is incorporated into an appropriate expression vector to produce an expression vector for a fluorescent protein fusion probe. "Polynucleotide" is DNA that encodes a protein
It is a fragment or an RNA fragment, and a protein cDNA or a DNA fragment of its coding region is particularly preferable. The fluorescent protein fusion probe expressed from such an expression vector has the order of the probe protein and the fluorescent protein as long as the binding site on the probe protein is not shielded by the fusion with the fluorescent protein.
Either may be on the N-terminal side. In addition, a spacer peptide may be interposed between the probe protein and the fluorescent protein.

Expression of the fusion polynucleotide is preferably carried out by in vitro transcription / translation, but it can also be carried out using an appropriate host cell.

When the photoprotein fusion probe is prepared as an in vitro transcription / translation product, the fusion polynucleotide is recombined into an expression vector having an RNA polymerase promoter, and a rabbit reticulocyte lysate containing an RNA polymerase corresponding to the promoter. The fluorescent protein fusion probe can be prepared in vitro by adding it to an in vitro transcription / translation system such as wheat germ extract or E. coli lysate. In vitro transcription and in vitro translation may be performed separately. Examples of RNA polymerase promoters include T7, T3, SP6 and the like. Vectors containing these RNA polymerase promoters include pKA
1, pCDM8, pT3 / T7 18, pT7 / 3 19, pBluescript II, pIV
Examples include EX series.

When the fluorescent protein fusion probe is prepared using a microorganism such as Escherichia coli, an origin capable of replicating in the microorganism, a promoter, a ribosome binding site, and a DNA.
An expression vector having a cloning site, a terminator, etc., is prepared by recombining a fusion polynucleotide with an expression vector, and a host cell is transformed with this expression vector. A fusion protein encoded by nucleotides can be produced in large quantities in a microorganism. Examples of the expression vector for E. coli include pUC system, pBluescript II, pET expression system, pGEX expression system and the like.

When the fluorescent protein fusion probe is prepared using eukaryotic cells, the fusion polynucleotide is recombined into an eukaryotic expression vector having a promoter, a splicing region, a poly (A) addition site, etc., When introduced into a eukaryotic cell, a fluorescent protein fusion probe can be produced within the eukaryotic cell. Expression vectors include pKA1 and pC
Examples include DM8, pSVK3, pMSG, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, pYES2 and the like. Eukaryotic cells include monkey kidney cells COS7, mammalian cultivated cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast,
Silkworm cells, Xenopus egg cells, etc. are generally used, but any eukaryotic cells can be used as long as they can express a fluorescent protein fusion probe.

After in vitro transcription / translation or expression of a fluorescent protein fusion probe in prokaryotic cells or eukaryotic cells,
The obtained cell lysate can be directly used as a probe. If the desired fluorescent protein fusion probe is isolated and purified from the culture, known separation operations can be combined. For example, treatment with denaturing agents such as urea and surfactants, ultrasonic treatment, enzyme digestion, salting out and solvent precipitation methods, dialysis, centrifugation, ultrafiltration, gel filtration,
Examples include SDS-PAGE, isoelectric focusing, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, reverse phase chromatography and the like.

In the method of the present invention, after preparing the fluorescent protein fusion probe as described above, this fluorescent protein fusion probe is contacted with all target candidate substances. When the target substance of interest is present among the target substance candidates, the fluorescent protein fusion probe specifically binds to the target substance. Since each target candidate substance is arranged at a predetermined position on the carrier, the target substance bound to the probe protein can be identified by detecting the position of the fluorescent signal of the fluorescent protein.

The fluorescence signal can be detected using a fluorescence microscope or a fluorescence slide scanner. When the target candidate substance is arranged on the carrier at a high density, a method of detecting a fluorescent signal by fluorescence microscope observation is preferable.

[0024]

EXAMPLES The invention of this application will be described in more detail and specifically with reference to the following examples, but the invention of this application is not limited to these examples. For the basic operation and enzymatic reaction for DNA recombination, refer to "(Molec
ular Cloning. A laboratory manual ", Cold Spring Ha
rbor Laboratory, 1989). Unless otherwise specified, the restriction enzymes and various modifying enzymes used were those manufactured by Takara Shuzo. The buffer composition of each enzyme reaction and the reaction conditions were in accordance with the attached instructions.

In this example, the nuclear proteins Npw38 and Np
An example is shown in which binding with pwBP is selected as a model interaction and NpwBP is used as a probe protein and Npw38 is used as a target protein for screening. That is, it is known that the WW domain of Npw38 binds to the PGR motif of NpwBP (A. Komuro et al., J. Biol. Chem. 274: 36513-3651.
9, 1999). Therefore, a peptide containing the PGR motif of NpwBP was selected as a probe protein. An example of screening a cell chip containing cells expressing membrane-localized Npw38 using a fusion of this peptide with a fluorescent protein as a fluorescent protein fusion probe (see FIG. 1) is shown. Example 1: Preparation of expression vector (1) pKA1 was an EcoRI-NotI fragment containing cDNAs of fluorescent proteins (EGFP, DsRed2) prepared from fluorescent protein expression vectors pEGFP-N1 and pDsRed2-N1 (both Clontech). (Kato et al., Gene150: 243-2
50, 1994) and inserted into EcoRI-NotI to construct fluorescent protein expression vectors pKA1-EGFP-N1 and pKA1-DsRed2-N1. (2) Vector pKA1-NpwBP (P2) -GFP expressing a fusion protein of the PGR motif peptide of the fluorescent protein fusion probe expression vector NpwBP and GFP (see FIG. 2 (a))
(Described in JP-A-2001-327296) was used as a fluorescent protein fusion probe expression vector. This vector is cD
Since the T7 RNA polymerase promoter exists upstream of NA, transcription in vitro occurs when T7 RNA polymerase acts, and mRNA encoding the fusion protein can be synthesized (see FIG. 1). (3) Construction of GPCL fusion protein expression vector cDNA encoding human glycophorin C-like protein (GPCL)
PCR products were prepared using THP primer and a primer in which an SmaI site was added downstream of the stop codon, using pHP10524 (described in WO00 / 00506) having the template as a template. This P
After digesting the CR product with EcoRI and SmaI, the fluorescent protein expression vectors pKA1-EGFP-N1 and pKA1-DsRed2-N1 prepared in (1) above.
Inserted into each EcoRI-SmaI cleavage site of pKA1-GPCL-EGFP, pKA1-GPCL-Ds
Red2 was created. (4) Construction of GPCL-Npw38 fusion protein expression vector pKA1-Npw38 (A. Komuro et al., Nucl. Acids Res. 27:
1957-1965, 1999) as a template and placed upstream of the start codon.
A PCR product was prepared using a primer having an SmaI site and a T3 primer. After digesting this PCR product with SmaI and NotI, inserted into the SmaI-NotI cleavage site of the membrane-localized fluorescent protein expression vector pKA1-GPCL-EGFP prepared in (3) above,
GPCL-Npw38 fusion protein expression vector pKA1-GPCL-Npw38 was prepared. A schematic diagram of the fusion protein GPCL-Npw38 is shown in Fig. 2 (b).
Shown in. (5) Preparation of GPCL-Npw38-DsRed2 fusion protein expression vector pKA1-Npw38 as a template, a primer with an SmaI site added upstream of the start codon, and a PCR product using a primer containing an SmaI site inside Npw38 Was prepared. This PCR
After digesting the product with XmaI, inserted into the XmaI cleavage site of the membrane-localized fluorescent protein expression vector pKA1-GPCL-DsRed2 prepared in (3) above, GPCL-Npw38-DsRed2 fusion protein expression vector pKA1-GPCL-Npw38 -Created DsRed2. Fusion protein GPCL
A schematic diagram of -Npw38-DsRed2 is shown in Fig. 2 (c). Example 2: Preparation of cell chip (1) Cultured cells The human fibrosarcoma cell line HT-1080 is a Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS).
The cells were cultured at 37 ° C in the presence of 5% CO ₂ . 2 x 10 ⁵ HT-108
0 cells were planted in a 6-well multi-dish (Nunc) and cultured at 37 ° C. for 22 hours in the presence of 5% CO ₂ . After removing the medium, wash the cell surface with phosphate buffer (PBS) and
1.5 ml of DMEM containing FBS was added. (2) Introduction of expression vector into cells Expression vector prepared in Example 1 (2) and (3) (pKA1-GPCL-
Npw38, pKA1-GPCL-Npw38-DsRed2, or 1 μl of a solution of pHP10524 as a control vector expressing GPCL (1.
5 μg) was added to 100 μl of serum-free DMEM, and then 10 μl of transfection reagent PolyFect ^™ (Qiagen)
By mixing with and incubating for 10 minutes at room temperature
A DNA complex was formed. The cultured cells HT-1080 in (1) above were added to PBS.
After washing once with, 1.5 ml of DMEM containing 10% FBS was added.
600 μl of DMEM containing 10% FBS was added to the DNA complex prepared above, and the cells were added to the cells at 37 ° C. in the presence of 5% CO _2.
It was cultured for 22 hours. (3) Immobilization of fusion protein-expressing cells on a carrier The fusion protein-expressing cells prepared in (2) above were each treated with 0.0
It was detached from the culture substrate by reacting with 1 ml of 5% trypsin-EDTA solution at 37 ° C for 5 minutes. After 2 ml of DMEM containing 10% FBS was added to collect the cells, the fusion protein-expressing cells were 2 × 1.
0 ⁵ cells / were ml to be prepared and uniformly mixed with cells expressing only GPCL. 1 ml of this cell mixture suspension is spread on a collagen I culture slide (Falcon) and 5
The cells were cultured at 37 ° C. for 40 hours in the presence of CO ₂ . Cells in PBS
After washing with PBS, the cells were washed with PBS and then fixed with PBS containing 4% paraformaldehyde for 15 minutes at room temperature to prepare a cell chip. Example 3: Preparation of fluorescent protein fusion probe 1 μg of pKA1-NpwBP (P2) -GFP described in Example 1 (2) was
T _N T ^R Quick Master Mix 40 μl, 1 mM methionine 1 μ, included in the in vitro transcription / translation reaction kit (Promega)
The solution was added to a total volume of 50 μl containing 1 and reacted at 30 ° C. for 12 hours. This reaction solution was directly used as a screening probe. A part of the reaction solution was diluted 200 times with PBS and the fluorescence spectrum (excitation light: 488 nm) was measured with a fluorescence spectrophotometer. The results are shown in FIG. Fluorescence emission with a maximum at 510 nm was observed, indicating that the translation protein fusion protein NpwBP (P2) -GFP functions as GFP. Example 4: Screening using fluorescent protein fusion probe The cell chip containing GPCL-Npw38-expressing cells prepared in Example 2 was washed with PBS and then treated with 0.1% Triton X-100 on ice for 15 minutes. 20 μl of the fluorescent protein fusion probe NpwBP (P2) -GFP prepared in Example 3 was added to the cells on this chip, and the cells were encapsulated and reacted at 4 ° C. for 20 hours. P containing 0.05% Tween 20
After washing with BS three times, it was sealed and confocal fluorescence microscope (Bio-Rad M
It was observed with RC1024ES). As a result, cells emitting green fluorescence were observed in the endoplasmic reticulum around the nucleus (Fig. 4 (a),
(b)). To confirm that this fluorescence emission site matches the localization of GPCL-Npw38, we used a cell chip containing cells expressing GPCL-Npw38-DsRed2 in which GPCL-Npw38 was further fused with the red fluorescent protein DsRed2. Then, the same experiment was conducted. As a result, as in the case of GPCL-Npw38, cells that emit green fluorescence were observed in the endoplasmic reticulum in the peripheral part of the nucleus (FIG. 4 (c)). Moreover, this green fluorescence emission site is GPCL-Npw3
Since it coincided with the red fluorescence emission site indicating the localized site of 8-DsRed2 (Fig. 4 (d), (e)), fluorescent protein fusion probe N
It was confirmed that pwBP (P2) -GFP bound to GPCL-Npw38-DsRed2. The binding of the probe was not observed in cells expressing only GPCL, indicating that this binding is mediated by Npw38.

[0026]

INDUSTRIAL APPLICABILITY As described in detail above, according to the invention of this application, binding screening between a probe protein and a target substance can be easily carried out.

[Brief description of drawings]

FIG. 1 is a diagram schematically showing a method for screening a cell chip containing cells expressing a target protein using a fluorescent protein fusion probe prepared by in vitro transcription / translation.

FIG. 2 is a view showing the structure of a fusion protein used in the invention of this application. NpwBP (P2) -EGFP (a), GPCL-Npw38
(B) and GPCL-Npw38-DsRed2 (c) are shown, respectively.

FIG. 3 is a view showing a fluorescence spectrum of a fluorescent protein fusion probe prepared by in vitro transcription / translation.

FIG. 4 is a confocal microscopic photograph observing fluorescence emission when a fluorescent protein fusion probe binds to a target protein of a cell chip, which shows green fluorescence (a in the case of using a cell chip containing GPCL-Npw38 expressing cells). ), Which is superposed with a differential interference image (b), and green fluorescence (c) and red emission (d) when using a cell chip containing cells expressing GPCL-Npw38-DsRed2. Overlaid with interference image (e)
It is the figure which displayed.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/58 G01N 33/58 Z // C07K 19/00 C07K 19/00 C12N 11/00 C12N 11/00 15/09 15/00 A (72) Inventor Fumie Fujimori 4-5-6 Hoyacho, Nishi-Tokyo, Tokyo Noguchi Heights 207 F term (reference) 2G054 AA08 CA23 CE02 EA03 GB02 4B024 AA11 BA80 CA07 GA30 HA11 HA20 4B033 NA11 NA42 NA45 NC06 ND05 ND20 NF06 NG01 NH10 NJ10 4H045 AA50 BA41 EA50 FA81 FA83

Claims (3)

[Claims] 1. A method for screening a target substance to which a probe protein binds from a plurality of target candidate substances fixedly arranged on a carrier, which comprises a fluorescent protein fusion probe which is a fusion protein of a probe protein and a fluorescent protein. A screening method, which comprises preparing and contacting this fluorescent protein fusion probe with all target candidate substances, and then detecting the fluorescent signal of the fluorescent protein to identify the target substance bound to the probe protein. 2. The screening method according to claim 1, wherein a fluorescent protein fusion probe is prepared as an in vitro transcription / translation product of a fusion polynucleotide of a polynucleotide encoding a probe protein and a polynucleotide encoding a fluorescent protein. 3. The screening method according to claim 1, wherein the target candidate substance immobilized on the carrier is a cell population containing the target protein.