JP2010115136A - Method for creating membrane protein with labelled n-terminal end, and cell having membrane protein with labelled n-terminal end in its outer layer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for specifically labelling only a target protein by modifying a specific probe to the target protein, a method for labelling a membrane protein in which its N-terminal end exists extracellularly, a cell having a membrane protein specifically modified by the method, and a method for screening using the cell. <P>SOLUTION: There is disclosed a method for creating a membrane protein with a labelled N-terminal end, wherein the method is featured by using a labelled molecular probe in which a membrane protein having LPX<SP>1</SP>X<SP>2</SP>(G)<SB>n</SB>sequence (X<SP>1</SP>is an optional amino acid, and X<SP>2</SP>represents threonine or alanine. n is any one of an integer from 2 to 8.) in a region of N-terminal side and having an N-terminal end extracellularly existing, a peptide transferring enzyme Sortase A, and LPX<SP>1</SP>X<SP>2</SP>(G)<SB>m</SB>sequence (X<SP>1</SP>is an optional amino acid, and X<SP>2</SP>represents threonine or alanine. m is 1 or 2.) are added. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a bioimaging technique in the field of molecular biology and a protein modification technique in the field of protein engineering. More specifically, the present invention relates to a bioimaging technique for directly observing the function and dynamics of a specific protein while a cell is alive, and a protein labeling technique therefor.

  In research and development in the field of molecular cell biology such as elucidation of cell function and disease onset mechanism, it is essential to know the function of a specific protein, localization in the cell and changes in activity. For this purpose, a bioimaging technique for visualizing and directly observing a target protein is widely used. There are the following three methods as representative methods of current bioimaging.

(1) A method of fluorescently labeling a protein by fusing a fluorescent protein such as green fluorescent protein (GFP).
In this technique, since the fluorescent protein and the target protein are fused by genetic engineering, it is possible to express only the target protein in a state of being specifically fluorescently labeled.
However, this method has the following problems: 1) The size of the fluorescent protein is so large that the expression / function of the target protein may be inhibited with the labeling. 2) The target protein is always fluorescently labeled. Therefore, it is difficult to analyze changes in intracellular localization (pulse chase labeling) with different expression times.

(2) A method of fluorescently labeling a protein by modifying a fluorescent synthetic molecular probe by an organic chemical reaction.
In this method, a fluorescent synthetic molecular probe having a small size can be modified by an organic chemical reaction at an arbitrary timing to be observed, and the target protein can be fluorescently labeled. Therefore, it can be said that the problem of the above-mentioned fluorescent protein fusion method is compensated. In addition to fluorescent synthetic molecular probes, various functional synthetic molecular probes such as photoreactive probes and pH sensitive probes can be modified to target proteins.
However, since organic chemical reactions are functional group-specific reactions, that is, “amino acid” -specific reactions, it is very difficult to specifically fluorescently label only the target protein in cells and on the cell surface where various proteins exist. It is difficult.

(3) A technique of fluorescently labeling proteins using fluorescently modified antibodies.
In this technique, the high specificity of the antigen-antibody reaction is utilized, and only the target protein can be specifically fluorescently labeled with the fluorescently modified antibody. It is also possible to perform pulse chase analysis.
However, also in this method, 1) the size of the antibody itself is very large, and there is a possibility that the function of the target protein may be inhibited by the labeling. 2) The antibody that is fluorescently modified for each protein to be labeled (very 3) Although the interaction between the antigen and antibody is strong, it is a non-covalent interaction. It is possible to mention problems such as the possibility of losing.

In protein labeling techniques, a technique using a biological translation system is known as a technique for specifically modifying a target protein. For example, the termination codon under the control of the promoter region is deleted in the presence of a labeling reagent composed of a label part consisting of a labeling substance and an acceptor part consisting of a compound capable of binding to the C-terminus of the protein. A method of performing protein synthesis in a cell-free translation system or living cells using a product transcribed from DNA consisting of the coding region as a template is known (Patent Documents 1 and 2).
However, this technique is a C-terminal specific reaction using a compound having the ability to bind to the C-terminus, and cannot be applied to modification of the N-terminus.

  A technique for modifying a fluorescent molecular probe specifically for a target protein using a high substrate specificity of an enzyme reaction is known. The inventors have already developed a technique for fluorescently labeling a target membrane protein by specifically modifying a fluorescent molecular probe at the C-terminal site of the target membrane protein present on the cell surface using the peptide transferase sortase A (SrtA). (Non-Patent Document 1).

  SrtA is an enzyme that catalyzes a reaction of cleaving between T and G of the recognition sequence LPXTG and ligating another peptide or protein having a GG terminal thereto (see FIG. 1). The above-mentioned method of the inventors utilizes this substrate specificity and binds a membrane protein having an LPTG sequence to the C-terminal region and a fluorescent molecular probe to which a GG sequence has been added by an enzyme reaction of SrtA, thereby producing a specific membrane. The C-terminal of the protein is fluorescently labeled.

  However, many membrane proteins including G protein-coupled receptors (GPCRs), which are major targets for drug development, have an N-terminal site located outside the cell and a C-terminal site located inside the cell. There are only a few membrane proteins whose sites are presented on the cell surface. Therefore, the above-mentioned method, which can be applied only to the membrane protein whose C-terminal site is presented on the cell surface, has a problem in versatility.

JP-A-11-322781 JP 2000-139468 A T. Tanaka et al. ChemBioChem, 9 (2008) pp.802-807 (February 22,2008)

  The problem of the present invention is that, as a new protein labeling method that compensates for the shortcomings of conventional bioimaging techniques, only the target protein is modified by specifically modifying the probe with the target protein using the high substrate specificity of the enzyme reaction. Method for specifically labeling, in particular, a method for specifically labeling the N-terminal site of a membrane protein present outside the cell, a cell having a membrane protein specifically modified by the method, and a screening using the cell It is to provide a method.

  The inventors target a cell surface membrane protein that plays a very important role in life phenomena such as signal transduction and substance transport, and use the peptide transferase sortase A (SrtA) at the N-terminal site of the target membrane protein. While proceeding with a study on a method for specifically modifying a fluorescent molecular probe, using a labeled protein having both a sortase A recognition sequence and a sortase A transfer sequence, and a probe to which a sortase A recognition sequence has been added, It was found that the N-terminus of the membrane protein can be specifically labeled, and the present invention has been completed.

That is, the present invention
[1] LPX ¹ X ² (G) n sequence in the N-terminal region (X ¹ represents any amino acid, X ² represents threonine or alanine, n represents any integer of 2 to 8) A membrane protein having an N-terminal extracellularly,
Peptide transferase Sortase A and
A labeled molecular probe to which an LPX ¹ X ² (G) m sequence (X ¹ represents an arbitrary amino acid, X ² represents threonine or alanine, m represents 1 or 2),
A method for producing an N-terminally labeled membrane protein, characterized in that
[2] The present invention relates to the method for producing an N-terminal labeled membrane protein according to [1] above, which comprises the following steps.
(I) LPX ¹ X ² (G) n sequence in the N-terminal region (X ¹ represents any amino acid, X ² represents threonine or alanine, and n represents any integer of 2 to 8) A process in which peptide transferase sortase A is allowed to act on a membrane protein having an N-terminus outside the cell, in the presence of a peptide containing polyglycine having 2 or more residues at the N-terminus;
(Ii) LPX ¹ X ² (G) m sequence (X ¹ represents any amino acid, X ² represents threonine or alanine) to the membrane protein whose N-terminus has become a polyglycine sequence in step (i). Represents 1 or 2. The step of allowing peptidease sortase A to act in the presence of a labeled molecular probe to which is added.

The present invention also provides
[3] A cell having a membrane protein whose N-terminus is labeled with a labeled molecular probe on the surface, wherein the label is an LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid and X ² represents threonine or Alanine, n represents any integer of 2 to 8), and a cell that is added to a membrane protein via a polypeptide comprising
[4] LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, n represents any integer of 2 to 8) Cells having a membrane protein on the surface layer in the side region,
[5] LPX ¹ X ² (G) n sequence (X ¹ represents an arbitrary amino acid, X ² represents threonine or alanine; n represents ² in the N-terminal region of a membrane protein having an N-terminal extracellularly. LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine. N represents 2 to 8). A plasmid vector comprising a DNA fragment encoding
[6] A labeled molecular probe to which an LPX ¹ X ² (G) m sequence (X ¹ represents an arbitrary amino acid, X ² represents threonine or alanine, m represents 1 or 2),
[7] LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine; n represents ² in the N-terminal region of the membrane protein whose N-terminal is present outside the cell. LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine. N represents any of 2 to 8) Or a plasmid vector comprising a DNA fragment encoding LPX ¹ X ² (G) m sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, m is 1 or 2) A cell surface membrane protein, comprising: a labeled molecular probe to which a peptide containing a peptide containing a glycan is added; a peptide transferase Sortase A; and a peptide containing a polyglycine having 2 or more residues at the N-terminus. Signs On the door.

Furthermore, the present invention provides
[8] A cell having an N-terminal labeled membrane protein on the surface, wherein the label is an LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, n represents 2 The present invention relates to a screening method for drug discovery, characterized in that cells added to a membrane protein via a polypeptide containing -8) are represented.

According to the present invention, it is possible to specifically label only a specific membrane protein of interest among a wide variety of membrane proteins present on the cell surface.
Moreover, since the molecular size added with labeling is very small, expression inhibition and function inhibition of the target membrane protein accompanying labeling can be avoided. As for labeling substances, not only fluorescent synthetic molecular probes but also various functional synthetic molecular probes such as photoreactive probes and pH sensitive probes can be modified to membrane proteins, so membrane proteins are simply labeled. In addition, it is possible to perform functional analysis of membrane proteins by modifying membrane proteins with various functional synthetic molecular probes. The synthetic molecular probe used in this method can be designed with a very high degree of freedom, and can be synthesized very easily by a combination of general peptide solid-phase synthesis and a simple chemical reaction.
Furthermore, the peptide transfer reaction catalyzed by Sortase A proceeds in a short time in a serum-containing medium and does not require cofactors such as ATP. Therefore, a technique for modifying membrane proteins while maintaining their functions on living cells As very useful.
And since the probe is modified by “retrofitting” to the membrane protein expressed on the cell surface layer by an enzyme reaction, the membrane protein can be labeled at any timing desired to be observed, and pulse chase analysis can be performed.
The method of the present invention and the cell of the present invention are considered to be powerful tools in fields such as drug discovery and biological basic research.

  The membrane protein labeled in the method for producing an N-terminal labeled membrane protein of the present invention is a membrane protein having an N-terminus present outside the cell. For example, a type I cytokine receptor family, a type II cytokine receptor family, an immunoglobulin Specific examples include proteins belonging to the superfamily (IgSF), the G protein-coupled receptor family (GPCR), and the like.

  Examples of proteins of the type I cytokine receptor family include interleukin-2 receptor, interleukin-3 receptor, interleukin-4 receptor, interleukin-5 receptor, interleukin-6 receptor, and interleukin. -7 receptor, interleukin-9 receptor, interleukin-11 receptor, interleukin-12 receptor, interleukin-13 receptor, interleukin-15 receptor, granulocyte macrophage colony stimulating factor receptor, hair Examples include a neurotrophic factor receptor, an erythropoietin receptor, a leukemia inhibitory factor receptor, an OSM receptor, or a granulocyte colony stimulating factor receptor.

  Examples of the protein of the type II cytokine receptor family include an interferon-α receptor, an interferon-β receptor, an interferon-γ receptor, and an interleukin-10 receptor.

  Examples of the immunoglobulin superfamily protein include interleukin-1 receptor and interleukin-18 receptor.

Examples of proteins of the G protein coupled receptor family include rhodopsin receptor, catecholamine receptor [β ₁ , β ₂ , β ₃ , α ₁ , α ₂ ], dopamine receptor [D1, D5, D2], muscarinic Receptor [m1, m3, m2, m4, m5], adenosine receptor [A1, A3, A2], PGE receptor [EP2, EP4, EP3, EP1], leukotriene receptor [BLT, Cys-LT1], sphingosine 1-phosphate receptor [S1P ₁ , S1P ₂ , S1P ₃ ], lysophosphatidic acid receptor [LPA ₁ , LPA ₂ , LPA ₃ ], nociceptin receptor, hGPCR44, hGPCR4, hGPCR10, hGPCR17, hGPCR19, hGPCR39, or hGPCR48 etc. can be mentioned.

In the production method of the present invention, the LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine in the N-terminal region thereof, and n is any one of 2 to 8 “N-terminal region” in a membrane protein having an integer) (hereinafter sometimes referred to as “LPX ¹ X ² (G) n sequence fusion membrane protein”) means a cell in the vicinity of the N-terminus of the membrane protein. It means an outer region and is preferably a site that does not affect post-translational modification or transport of the protein synthesized in the cytoplasm. For example, it is preferable to have the amino acid sequence downstream of the N-terminal signal sequence.

^{LPX 1 X 2 (G) X} 1 in n represents any amino acid, alanine (A), arginine (R), asparagine (N), consisting of aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), valine (V), and the like. Among these, glutamic acid, methionine, tyrosine, leucine, glutamine, asparagine, and phenylalanine are more preferable.
X ² is threonine (T) or alanine (A), and threonine is more preferable.
n represents an integer of 2 to 8, and is more preferably 4 to 6.

The above-mentioned LPX ¹ X ² (G) n sequence fusion membrane protein can be obtained in a form expressed on the surface layer of any cell. A method for preparing a cell in which the LPX ¹ X ² (G) n sequence fusion membrane protein is expressed on the surface layer of the cell is not particularly limited, including known methods. For example, the N-terminal side of the DNA encoding the target membrane protein is used. A plasmid vector containing a DNA encoding the LPETGGGGGG sequence fusion membrane protein, into which a DNA fragment encoding the LPETGGGGGG sequence (SEQ ID NO: 1) has been inserted, is constructed using a known gene introduction method, For example, the LPETGGGGGG sequence fusion membrane protein can be expressed by introduction into cells using any of the calcium phosphate method, lipofection method, and electroporation method. Further, ^LPX 1 ^X 2 (G) upstream and / or a further sequence downstream of the n sequences such LPETGGGGG sequence, for ^example, a tag sequence such as to verify the insertion of the LPX ¹ X 2 (G) n sequence It can also be included.

The cells that express the LPX ¹ X ² (G) n sequence fusion membrane protein can be selected according to the purpose and are not particularly limited. For example, known mammalian cells or insect cells can be used. Examples of known mammalian cells include HEK293 cells, Vero cells, Hela cells, CV1 cells, COS1 cells, CHO cells, Namalva cells and the like. Examples of known insect cells include Sf9 cells and MG1 cells.

  Peptide transfer enzyme Sortase A used in the present invention is an enzyme present in Staphylococcus sp., Etc., which cuts between T and G of recognition sequence LPXTG and binds another peptide or protein having a GG terminal thereto. To catalyze the reaction (see FIG. 1). Sortase A is expressed in microorganisms by a known genetic engineering method (for example, the method described in T. Tanaka et al. ChemBioChem, 9 (2008) pp.802-807 (February 22,2008)), purified and purified. What was obtained can be used.

As a labeled molecular probe used in the production method of the present invention, LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine. N is any one of 2 to 8. Is not particularly limited as long as it can bind to a membrane protein via, for example, a fluorescent synthetic molecular probe, a luminescent probe, a chromogenic probe, a biotin-labeled probe, a radioisotope probe, a receptor Examples include detection probes such as probes. Of these, fluorescent synthetic molecular probes are preferable from the viewpoint of ease of detection in bioimaging. Moreover, not only a detection probe but functional probes, such as a photoreactive probe and a pH sensitive probe, can also be used.

LPX ¹ X ² (G) m sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, m represents 1 or 2) is added to the labeled molecular probe. In the LPX ¹ X ² (G) m sequence, X ¹ and X ² are the same as those exemplified above, and m is 1 or 2, and more preferably 2.
The amino acid sequence LPX ¹ X ² (G) m may be added to the probe by a chemical method, for example, in T. Tanaka et al. ChemBioChem, 9 (2008) pp.802-807 (February 22,2008). It can be added by the method described.
The amino acid sequence may be added directly to the probe, or may be added via a spacer composed of another peptide and / or polymer between the LPX ¹ X ² (G) m sequence and the probe. . Furthermore, you may have another peptide and / or a polymer downstream of the said amino acid sequence.

The method of the present invention uses the LPX ¹ X ² (G) n sequence fusion membrane protein, peptide transferase Sortase A, and a labeled molecular probe detailed above to label the N-terminus of the target membrane protein, The strategy is
(1) Sortase recognition sequence site of LPX ¹ X ² (G) n sequence fusion membrane protein is cleaved by Sortase A, and GG sequence is presented at the N-terminus;
(2) The sortase A binds the GG sequence to the sortase A recognition sequence of the labeled molecular probe;
According to the two-step reaction.

The addition amount of Sortase A and the labeled molecular probe is not particularly limited as long as the reaction proceeds, but for example, a culture solution containing 1 to 1000 μM Sortase A and 1 to 1000 μM labeled probe, preferably 5 to 500 μM Sortase A. A culture solution containing 2 to 500 μM of the labeled probe can be used.
The reaction temperature is not particularly limited as long as the cells can survive. For example, the reaction can be performed at 20 ° C. to 40 ° C., preferably in a 37 ° C. incubator.
The reaction time is 5 minutes to 10 hours, preferably 5 minutes to 2 hours.

The reaction may be started by adding Sortase A and a labeled molecular probe to a culture solution of cells expressing LPX ¹ X ² (G) n sequence fusion membrane protein, or by previously sorting Sortase A and a labeled molecular probe. A culture solution containing the medium may be prepared in advance, and the medium may be replaced with the culture solution.
The reaction can be completed by washing the cells with PBS or the like after the lapse of a desired time, and removing Sortase A, extra probes, free N-terminal fragments, and the like.

The fact that an N-terminal labeled membrane protein has been obtained by the above reaction can be confirmed by a known method. For example, when the probe is fluorescent, it can be confirmed by a confocal laser microscope.
As described above, in the N-terminal labeled membrane protein, the labeled molecular probe has an LPX ¹ X ² (G) n sequence (X ¹ represents an arbitrary amino acid, X ² represents threonine or alanine. N is 2 to 8). It is a membrane protein added to the N-terminus via

In addition to the above-mentioned LPX ¹ X ² (G) n sequence fusion membrane protein, peptide transferase Sortase A and labeled molecular probe, in addition to the above-mentioned LPX ¹ X ² (G) n sequence fusion membrane protein, the number of residues of 2 or more at the N-terminus A method using a peptide containing polyglycine is also a method of the present invention.
The number of residues of the peptide containing polyglycine having 2 or more residues at the N-terminus is not particularly limited as long as the target reaction is not inhibited, but 2 to 50 is preferable and 2 to 20 is more preferable.
The number of residues of the N-terminal polyglycine is not particularly limited as long as it is 2 or more, but is preferably 2 to 12, and more preferably 3 to 8.
The peptide may be composed only of polyglycine, and may contain any amino acid other than polyglycine, but is more preferably composed only of polyglycine, specifically, the number of residues is 3 to 3. Eight polyglycines can be exemplified. The sequence other than polyglycine is not particularly limited as long as the peptide is dissolved in the reaction system and does not inhibit the reaction.

When using a peptide containing polyglycine having 2 or more residues at the N-terminal, an N-terminal labeled membrane protein is produced by a method including the following steps (see FIG. 3).
(I) LPX ¹ X ² (G) n sequence in the N-terminal region (X ¹ represents any amino acid, X ² represents threonine or alanine, and n represents any integer of 2 to 8) A step of allowing a peptide transferase, sortase A, to act on a membrane protein having an N terminus outside the cell, in the presence of a peptide containing polyglycine having 2 or more residues at the N terminus;
(Ii) LPX ¹ X ² (G) m sequence (X ¹ represents any amino acid, X ² represents threonine or alanine) to the membrane protein whose N-terminus has become a polyglycine sequence in step (i). Represents 1 or 2. The step of allowing peptidease sortase A to act in the presence of a labeled molecular probe to which is added.

First, in step (i), when the action of sortase A to ^LPX 1 ^X 2 (G) n sequence fusion membrane protein having a sortase A recognition ^sequence, LPX ¹ X 2 (G) n sequence fusion membrane protein recognition sequences portion And the N-terminal fragment is released, and the polyglycine sequence is presented at the N-terminus of the remaining membrane protein. The released N-terminal fragment binds to a peptide containing polyglycine at the free N-terminus in the reaction system.

  It is desirable to provide a washing step after step (i) to remove the polypeptide in which the released N-terminal fragment and polyglycine are bound, excess Sortase A, a peptide containing polyglycine at the N-terminus, and the like. . Washing can be performed by a general method such as PBS washing.

  Next, in step (ii), a labeled molecular probe having Sortase A and Sortase A recognition sequence is added to the reaction system, and labeled with a membrane protein presenting a polyglycine sequence at the N-terminus by the action of Sortase A. Molecular probes can be coupled via polyglycine sequences.

In the above reaction, the reaction concentration and temperature can be the same as those in the above-described method not using a peptide containing polyglycine at the N-terminus.
The reaction time can be 5 minutes to 10 hours, preferably 10 minutes to 2 hours in step (i). Step (ii) is 5 minutes to 4 hours, preferably 5 minutes to 1 hour, and the labeled probe binds to the membrane protein and can be labeled in a short time even in a short time of about 5 minutes.

The cell of the present invention is a cell having a membrane protein whose N-terminus is labeled with a labeled molecular probe on the surface, and the labeled molecular probe is an LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid). , X ² represents threonine or alanine, n represents any integer of 2 to 8) and a cell added to a membrane protein via a polypeptide containing LPX ¹ X ² (G) n sequence The cells are not particularly limited as long as they have a fusion membrane protein on the surface layer. Among these cells, the former is useful in a screening method for drug discovery, and the latter can be advantageously used for the preparation of the former.

The present invention also relates to a cell surface membrane protein labeling kit, which comprises at least an LPX ¹ X ² (G) n sequence (X ¹ in the N-terminal region of a membrane protein whose N-terminal is present outside the cell). Represents any amino acid, X ² represents threonine or alanine, n represents any integer of 2 to 8) LPX ¹ X ² (G) n sequence for incorporating (X ¹ represents any amino acid) X ² represents threonine or alanine, n represents any integer of 2 to 8, and a plasmid vector comprising a DNA fragment encoding LPX ¹ X ² (G) m sequence (X ¹ is any An amino acid, X ² represents threonine or alanine, m represents 1 or 2), a labeled molecular probe to which a peptide containing peptide is added, peptide transferase Sortase A, and N-terminal residues of 2 or more Poly Including peptides, including lysine.
The plasmid vector, the labeled molecular probe, the peptide transferase Sortase A, and the peptide containing polyglycine at the N-terminus are the same as described in detail above. , A buffer solution, a gene introduction reagent, an energy supply solution, a pH adjuster, a reaction vessel, and the like may be contained.

The present invention is also a cell having an N-terminal labeled membrane protein on the surface, wherein the label is an LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid and X ² represents threonine or alanine. Is a cell added to a membrane protein through a polypeptide containing 2), and a screening method for drug discovery, characterized in that a membrane is used as a screening target. Examples include substances that act on proteins to activate their functions (for example, agonists) or inactivate substances (for example, antagonists). Examples of substances include low molecular weight organic compounds, proteins, natural peptides, Artificial peptides, polymers, hormones, carbohydrates, lipids, nucleic acids, viruses, cells, etc. can be mentioned (hereinafter referred to as “substances to be screened”) .).

  Examples of the screening method include a method using fluorescence observation, a method using FRET (fluorescence resonance energy transfer), and the like.

  As a method of using fluorescence observation, a membrane protein (for example, a receptor such as GPCR) targeted by the method of the present invention is fluorescently labeled, and the presence or absence of a desensitization phenomenon when a ligand candidate substance is added is detected from the fluorescence. The screening method of the ligand which determines and specifies a ligand substance can be mentioned.

As a method using FRET, for example, after labeling a target membrane protein by the labeling method of the present invention, a fluorescently-labeled ligand candidate substance is added, and the presence or absence of FRET on the cell surface is observed. And a method for screening a ligand for determining the presence or absence of binding to a membrane protein and identifying a ligand substance.
Alternatively, the presence or absence of changes in FRET when a fluorescently labeled ligand known to bind to the target membrane protein is added to cause FRET in advance, and when another substance that is not fluorescently labeled is added. For example, when a receptor that is a membrane protein and its agonist are known, FRET was caused by the membrane protein labeled by the method of the present invention and the labeled agonist, and various antagonist candidate substances were added. An antagonist screening method in which an antagonist is identified by a change in FRET can be mentioned.

As another example of drug screening using FRET, a part of the target membrane protein is fluorescent acceptor-labeled by the method of the present invention, the rest is fluorescent donor-labeled, and a ligand candidate substance that induces oligomerization is added thereto, Alternatively, there can be mentioned a screening method in which the presence or absence of oligomer formation is confirmed by the presence or absence of FRET when a stimulus is applied, and the ligand substance or stimulus sensitivity is specified.
Alternatively, the N-terminal of the target membrane protein is labeled with a fluorescent acceptor or donor by the method of the present invention, and another site of the membrane protein is labeled with a fluorescent donor or acceptor by another method, in the presence of various substances. Alternatively, a method for screening a ligand that identifies a ligand substance by observing a change in the structure of the target membrane protein by observing a change in FRET in the absence thereof can be mentioned.

  In addition, the labeling method of the present invention can be used for drug screening and observation of membrane protein function / dynamics, not limited to drug discovery purposes.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Example 1]
(1) Synthesis of AF647-LPETGG and construction of ECFP-TM added with the LPETGGGGGG sequence Alexa Fluor 647-LPETGG (AF647-LPETGG) was used as a labeled molecule. After synthesizing peptide H-LPETGG-NH ₂ (SEQ ID NO: 2) by Fmoc solid phase synthesis method, Alexa Fluor 647 is modified at the N-terminal amino group using Alexa Fluor 647 succinimidyl ester (Invitrogen). Thus, a labeled molecule AF647-LPETGG was synthesized. Specifically, peptide H-LPETGG-NH ₂ dissolved in DMSO (dehydrated) and Alexa Fluor 647 succinimidyl ester (manufactured by Invitrogen) dissolved in DMSO (dehydrated) were added at a molar ratio of 2: 1. And reacted for 3 hours. As a base, 4 equivalents of DIEA was added. After the reaction, the product was purified by high performance liquid chromatography (HPLC) and identified by MALDI-TOF-MS.
In addition, a fusion of cyan fluorescent protein (ECFP) and the transmembrane domain (TM) of platelet-derived growth factor receptor, to which an LPETGGGGGG sequence (SEQ ID NO: 1) (hereinafter referred to as LPTGG5 sequence) is added near the N-terminus ( Hereinafter, an expression plasmid vector (FIG. 4) of LPETG5-ECFP-TM) was constructed. First, primer 5'-EcoRI-BglII-LPETGGGGG-ECFP (base sequence GCC GAA TTC GAC AGA TCT CTG CCG GAA ACT GGT GGC GGT GGC GGT TCT GGA GTG AGC AAG GGC GAG GAG CTG TTC ACC) (SEQ ID NO: 3) and 3 Corresponding gene from ECFP-C1 (Clontech) using '-ECFP-PstI-BamHI (base sequence CGT GGA TCC GAA CTG CAG GCC CTT GTA CAG CTC GTC CAT GCC GAG AGT GAT CCC GGC) (SEQ ID NO: 4) The region was cloned and incorporated into pBlueScriptII SK (-) (Stratagene) at the EcoRI / BamHI site to confirm the sequence. Then, it was recombined with pDisplay (manufactured by Invitrogen) at the BglII / PstI site to obtain an LPETG5-ECFP-TM expression plasmid vector (FIG. 4).

(2) Preparation of His6-SrtA A His6-SrtA expression vector (described in T. Tanaka et al. ChemBioChem, 9 (2008) pp. 802-807 (February 22, 2008)) is BL21 STAR (DE3) (Novagen) ). Culturing in TB medium; D. At the time of = 0.6, 0.1 mM IPTG was added to induce protein expression, and then further cultured with shaking at 27 ° C. for 18 hours. Then, the cells were collected, subjected to ultrasonic disruption, and centrifuged to collect the supernatant. Next, the supernatant was subjected to affinity purification using Ni Sepharose High Performance (GE Healthcare). After purification, it was dialyzed against a buffer of 20 mM Tris, 150 mM NaCl, pH 8.0. The protein concentration of Sortase A was determined using BCA Protein Assay Kit (PIERCE).

(3) Cleavage of LPTG5 sequence by addition of triglycine and SrtA
Triglycine (Sigma) 1 mM and His6-SrtA 30 μM were added to HEK 293T cells (culture time: 24 hours) in which LPETG5-ECFP-TM was expressed, and 15 minutes and 30 minutes in a CO ₂ incubator at 37 ° C. After performing the cleavage reaction for 1 hour and 2 hours, immunofluorescence staining using Alexa Fluor 647-modified anti-HA antibody was performed.
The breakage of the LPTGG5 sequence was evaluated by measuring the residual HA tag (see FIG. 4) in the cell surface layer upstream of the LPTGG5 sequence by observation with a confocal laser microscope. FIG. 5 shows the measurement results.

  From FIG. 5, the purple fluorescence intensity derived from Alexa Fluor 647 decreased as the cleavage reaction time was increased. It was confirmed that most of the HA tag on the cell surface disappeared in the cleavage reaction time of 30 minutes, and that many of the LPTG5 sequences present on the cell surface were cleaved.

(4) Labeling of Alexa Fluor 647 to ECFP-TM on the cell surface
To HEK 293T cells expressing LPETG5-ECFP-TM (culture time: 24 hours), triglycine 1 mM and His6-SrtA 30 μM were added together with serum-containing DMEM medium, and cleaved in a CO ₂ incubator at 37 ° C. for 30 minutes. Went. After removing His6-SrtA, excess triglycine and the like, the labeled molecules AF647-LPETGG 10 μM and His6-SrtA 30 μM were added together with serum-containing DMEM medium, and the labeling reaction was performed in a CO ₂ incubator at 37 ° C. for 15 minutes. It was.
As a comparative example, the reaction was performed in the same manner without adding His6-SrtA during the labeling reaction. Further, HEK 293T cells expressing LPETA5-ECFP-TM in which the LPTG5 sequence was replaced with the LPETA5 sequence were reacted in the same manner.
After removing SrtA and excess AF647-LPETGG, fluorescence observation was performed using a confocal laser microscope. The results are shown in FIG.

As shown in FIG. 6, purple fluorescence derived from Alexa Fluor 647 was observed only in the contour of LPETG5-ECFP-TM expressing cells (left figure). On the other hand, when SrtA was not added (middle figure), and when HEK 293T cells expressing LPETA5-ECFP-TM were used (right figure), purple fluorescence was not observed in the cell outline.
As described above, Alexa Fluor 647 was successfully labeled on ECFP-TM in a site-specific manner by a peptide transfer reaction catalyzed by SrtA.

[Example 2]
The biotin molecule to ECFP-TM present on the cell surface layer was the same as in Example 1 except that a biotin molecule to which the LPETGG sequence was added was used as the labeling molecule, the cleavage reaction time was 1 hour, and the labeling reaction time was 5 minutes. The label was made. The label was confirmed by Western blotting using streptavidin-HRP (Invitrogen). The results are shown in FIG.

  Fig. 7 confirms a band of streptavidin-HRP around the same molecular weight (36 kDa) as the anti-c-myc antibody (Bethyl Laboratories) against the c-myc tag (see Fig. 4) inserted into LPETG5-ECFP-TM It was confirmed that the biotin molecule was labeled on LPETG5-ECFP-TM (leftmost lane). In addition, no band was confirmed at other positions, and it was confirmed that the biotin molecule was specifically labeled only with LPETG5-ECFP-TM in the presence of various membrane proteins on the cell surface layer. On the other hand, when SrtA was not used (second lane from the left) and when HEK 293T cells expressing LPETA5-ECFP-TM were used (right two lanes), no band was confirmed at the same position. It was confirmed that the biotin molecule was not labeled.

[Example 3]
In the same manner as in Example 1, 1 mM triglycine and 30 μM His6-SrtA were added to HEK 293T cells expressing LPETG5-ECFP-TM together with serum-containing DMEM medium, and a cleavage reaction was performed in a CO ₂ incubator at 37 ° C. for 1 hour. went. After removing SrtA, excess triglycine, etc., the labeled molecules AF647-LPETGG and SrtA were added together with serum-containing DMEM medium, and the labeling reaction was performed for 5 minutes, 15 minutes, and 30 minutes in a CO ₂ incubator. went. After removing SrtA and excess AF647-LPETGG, fluorescence observation was performed using a confocal laser microscope. The results are shown in FIG.

  From FIG. 8, even when the labeling reaction time was set to 5 minutes, Alexa Fluor 647-derived purple fluorescence was observed only on the contour of the ECFP-TM-expressing cells, confirming the labeling of Alexa Fluor 647 on ECFP-TM. It was confirmed that the intensity of purple fluorescence derived from Alexa Fluor 647 increased as the reaction time was increased and gradually saturated.

[Example 4]
Labeling was performed in the same manner as in Example 3 except that the reaction time of the cleavage reaction was 30 minutes. The results are shown in FIG.

  From FIG. 9, even when the labeling reaction was performed for 5 minutes, Alexa Fluor 647-derived purple fluorescence was observed only on the contour of the ECFP-TM-expressing cells, confirming the labeling of Alexa Fluor 647 on ECFP-TM. It was confirmed that Alexa Fluor 647-derived purple fluorescence intensity observed according to the reaction time increased and gradually became saturated.

[Example 5]
Labeling was performed in the same manner as in Example 3 except that the reaction time for the cleavage reaction was 15 minutes. The results are shown in FIG.

  From FIG. 10, even when the labeling reaction was performed for 5 minutes, purple fluorescence derived from Alexa Fluor 647 was observed only in the contour of the ECFP-TM expressing cells, and the label of Alexa Fluor 647 on ECFP-TM was confirmed. It was confirmed that Alexa Fluor 647-derived purple fluorescence intensity observed according to the reaction time increased and gradually became saturated.

[Example 6]
The labeling reaction time was fixed at 5 minutes and the reaction time for the cleavage reaction was set to 0 minutes, 15 minutes, 30 minutes, and 1 hour, and then Alexa Fluor 647 was labeled in the same procedure as in Example 1. The results are shown in FIG.

  As shown in FIG. 11, the purple fluorescence intensity derived from Alexa Fluor 647 observed in the contour of ECFP-TM-expressing cells increased with increasing cleavage reaction time. It has been suggested that the subsequent labeling reaction efficiency is increased by adding triglycine and SrtA and performing the cleavage reaction in advance.

[Example 7]
In the same manner as in Example 1, HEK 293T cells expressing LPETG5-ECFP-TM were obtained. Without using triglycine, 10 μM of labeled molecules AF647-LPETGG and 30 μM of His6-SrtA were added, and a labeling reaction was performed at 37 ° C. for 30 minutes. The results are shown in FIG.

  From FIG. 12, it was confirmed that Alexa Fluor 647 was labeled on ECFP-TM (right figure). Although it was not as much as in the case where the cleavage reaction was carried out for 15 minutes in the presence of triglycine (left figure), it was confirmed that labeling was possible even in the absence of triglycine.

[Example 8]
Labeling was performed in the same manner as in Example 3 except that the concentration of AF647-LPETGG was changed to 30 μM. The results are shown in FIG.

  From FIG. 13, even when the concentration of AF647-LPETGG was set to 30 μM, purple fluorescence derived from Alexa Fluor 647 was observed only in the contour of the ECFP-TM expressing cells, and the label of Alexa Fluor 647 on ECFP-TM was confirmed. It was confirmed that Alexa Fluor 647-derived purple fluorescence intensity observed according to the reaction time increased and gradually became saturated.

[Reference Example 1]
In the same manner as in Example 1, HEK 293T cells expressing LPETG5-ECFP-TM were obtained.
To HEK 293T cells expressing LPETG5-ECFP-TM (cultured for 24 hours), triglycine (100 μM) was added and in a CO ₂ incubator at 37 ° C., 0 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours. After performing the cleavage reaction for each time, immunofluorescence staining using Alexa Fluor 647-labeled anti-HA antibody was performed.
The cutting efficiency of the LPTGG5 sequence was evaluated by measuring the residual HA tag located in the cell surface layer upstream of the LPTGG5 sequence by confocal laser microscope observation. FIG. 14 shows the measurement results.

  As shown in FIG. 14, although the cleavage reaction efficiency is reduced as compared with the case where the triglycine concentration is 1 mM (FIG. 5), the purple fluorescence intensity derived from Alexa Fluor 647 also increases with time when the triglycine concentration is 100 μM. It disappeared and it was confirmed that the LPTG5 sequence was indeed cleaved.

FIG. 2 is a diagram schematically showing a reaction catalyzed by peptide transferase Sortase A used in the present invention. It is the figure which showed notionally the plasmid vector used for this invention. It is the figure which showed typically the N terminal labeling method of this invention. It is the figure which showed typically the construct of the LPETG5-ECFP-TM expression plasmid vector used in the Example. It is the figure which evaluated the cutting | disconnection efficiency of the LPTGG5 arrangement | sequence by addition of triglycine and SrtA by the immunofluorescence dyeing | staining. It is the figure which confirmed the label of Alexa Fluor 647 to ECFP-TM in a cell surface layer by fluorescence observation. It is the figure which confirmed the label | marker of the biotin molecule to ECFP-TM by Western blotting. It is the figure which confirmed the time-dependent change of the label | marker of Alexa Fluor 647 to ECFP-TM by the fluorescence observation by setting cleavage reaction as 1 hour. It is the figure which confirmed the time-dependent change of the label | marker of Alexa Fluor 647 to ECFP-TM by fluorescence observation by setting the cleavage reaction as 30 minutes. It is the figure which confirmed the time-dependent change of the label | marker of Alexa Fluor 647 to ECFP-TM by the fluorescence observation by setting cleavage reaction as 15 minutes. It is the figure which evaluated the labeling reaction efficiency of Alexa Fluor 647 to ECFP-TM when the labeling reaction time was fixed at 5 minutes and the cleavage reaction time was 0 minutes, 15 minutes, 30 minutes and 60 minutes by fluorescence observation. . It is the figure which confirmed the label | marker of Alexa Fluor 647 to ECFP-TM in a cell surface layer when a cleavage reaction and a labeling reaction were performed in one step without using triglycine by fluorescence observation. It is the figure which evaluated the labeling reaction efficiency by fluorescence observation when the density | concentration of Alexa Fluor 647 was 30 micromol. It is the figure which confirmed the cutting | disconnection of the LPTEG5 arrangement | sequence when triglycine was set to 100 micromol in cleavage reaction by immunofluorescence staining.

Claims (8)

It has an LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, and n represents any integer of 2 to 8) in the N-terminal region. A membrane protein having an N-terminal extracellularly;
Peptide transfer enzyme Sortase A and
A labeled molecular probe to which an LPX ¹ X ² (G) m sequence (X ¹ represents an arbitrary amino acid, X ² represents threonine or alanine, m represents 1 or 2),
A method for producing an N-terminally labeled membrane protein, comprising using The method for producing an N-terminal labeled membrane protein according to claim 1, comprising the following steps.
(I) LPX ¹ X ² (G) n sequence in the N-terminal region (X ¹ represents any amino acid, X ² represents threonine or alanine, and n represents any integer of 2 to 8) A step of allowing a peptide transferase, sortase A, to act on a membrane protein having an N terminus outside the cell, in the presence of a peptide containing polyglycine having 2 or more residues at the N terminus;
(Ii) LPX ¹ X ² (G) m sequence (X ¹ represents any amino acid, X ² represents threonine or alanine) to the membrane protein whose N-terminus has become a polyglycine sequence in step (i). Represents 1 or 2. The step of allowing peptidease sortase A to act in the presence of a labeled molecular probe to which is added. A cell that N-terminal has a membrane protein which is labeled with a labeling molecule probe to the surface, label LPX 1 X ^{2 (G) n} sequence (X ¹ represents any amino acid, X ² represents a threonine or alanine N represents an integer of any of 2 to 8.) A cell characterized by being added to a membrane protein via a polypeptide containing. LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, n represents any integer of 2 to 8) in the extracellular N-terminal region A cell having a membrane protein as a surface layer. N-terminal ^LPX 1 ^X 2 (G) n sequence at the N terminal region of the membrane proteins present in extracellular ^{(X 1} represents any amino acid, ^{X 2} represents a threonine or alanine .n is 2 to 8 LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, n is any of 2-8) A plasmid vector containing a DNA fragment encoding an integer. A labeled molecular probe to which an LPX ¹ X ² (G) m sequence (X ¹ represents an arbitrary amino acid, X ² represents threonine or alanine, m represents 1 or 2) is added. LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine. N represents 2 to 8 in the N-terminal region of the membrane protein whose N-terminus is present outside the cell. LPX ¹ X ² (G) n sequence (X ¹ represents any amino acid, X ² represents threonine or alanine. N represents an integer of 2 to 8) A plasmid vector comprising a DNA fragment encoding
A labeled molecular probe to which a peptide comprising an LPX ¹ X ² (G) m sequence (X ¹ represents any amino acid, X ² represents threonine or alanine, m represents 1 or 2),
Peptide containing peptide transferase Sortase A and polyglycine having 2 or more residues at the N-terminus,
A cell surface membrane protein labeling kit, comprising: A cell having an N-terminally labeled membrane protein on the surface, wherein the label is an LPX ¹ X ² (G) n sequence (X ¹ represents an arbitrary amino acid, X ² represents threonine or alanine. N is 2 to 8. A method for screening for drug discovery, comprising using a cell added to a membrane protein via a polypeptide containing any integer.

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