Lipid-binding protein and uses thereof

Abstract

A lipid-binding protein comprising an amino acid sequence having at least 85% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the glycine residue at position 12 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with an amino acid residue selected from the group consisting of cysteine, valine, leucine, isoleucine, alanine, and methionine, and wherein the lipid-binding protein has a higher binding affinity for PI(3,5)P₂ than a protein consisting of the amino acid sequence of SEQ ID NO: 1.

Description

Lipid-binding proteins and their uses

[0001] This invention relates to a lipid-binding protein and its use. This application claims priority based on Japanese Patent Application No. 2024-195307, filed in Japan on November 7, 2024, the contents of which are incorporated herein by reference.

[0002] Cell membranes and organelle membranes are composed of specific lipid compositions, but these compositions can change during cell death or under certain physiological and pathological conditions. Changes in lipid composition have been reported to be associated not only with basic cellular functions but also with physiological responses essential for maintaining homeostasis, cellular senescence, and pathological conditions such as neurodegenerative diseases.

[0003] Therefore, lipid probes, such as proteins that specifically bind to certain types of lipid components, are known. By using lipid probes, it may be possible to detect cells or extracellular vesicles (nanoparticles) that have cell membranes rich in the lipid components to which the lipid probe specifically binds, or to clarify the localization of the above-mentioned lipid components in organelle membranes present within cells.

[0004] For example, Non-Patent Document 1 contains PI(3,5)P ２ The PX domain of the SnxA protein from the slime mold Dictyostelium discoideum has been disclosed as a probe that specifically binds to (Phosphatidylinositol 3,5-bisphosphate).

[0005] Vines Jh. et al., A PI(3,5)P2 reporter reveals PIKfyve activity and dynamics on macropinosomes and phagosomes. Journal of Cell Biology, 222(9), e202209077, 2023.

[0006] However, the PX domain of the SnxA protein is phosphatidylinositol 3,5-bisphosphate PI(3,5)P ２ The binding force to PI(3,5)P was insufficient. ２It has binding specificity to and binds more strongly to PI(3,5)P than the PX domain of the SnxA protein. ２ No lipid probes that bind to it have been discovered to date.

[0007] The present invention has been made in view of these circumstances, and PI(3,5)P ２ The objective is to provide a technology related to a lipid-binding protein that can bind more strongly than the PX domain of the SnxA protein.

[0008] As a result of diligent research to achieve the above objective, the inventors have created a yeast that expresses a recombinant protein on its cell surface in which amino acid mutations have been randomly added to the PX domain (see SEQ ID NO: 1 as the amino acid sequence) corresponding to amino acid sequences 61-175 of the endogenous (wild-type) SnxA protein of Dictyostelium discoideum (NCBI Reference Sequence: XP_636026.1), and has used this yeast and PI(3,5)P ２ By mixing liposomes containing the above, we discovered that a common feature of the amino acid sequences of recombinant proteins expressed by yeast groups strongly bound to the liposomes is that the 12th amino acid, glycine, in the sequence shown in Sequence ID No. 1 is substituted with cysteine ​​or a small-bulk hydrophobic amino acid, thus completing the present invention. That is, one aspect of the present invention that solves the above problems includes the following aspects.

[0009] [1] The amino acid sequence has 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the 12th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, which is glycine, is substituted with any amino acid residue selected from the group consisting of cysteine, valine, leucine, isoleucine, alanine, and methionine, and the PI(3,5)P protein is more identical to the amino acid sequence shown in SEQ ID NO: 1. ２ A lipid-binding protein with high binding affinity to lipids.

[0010] [2] The lipid-binding protein according to [1], wherein the glycine is substituted with any amino acid residue selected from the group consisting of cysteine, valine, leucine, isoleucine, and methionine.

[0011] [3] The lipid-binding protein according to any one of [1] to [3], comprising any amino acid sequence of SEQ ID NO: 2 to 19.

[0012] [4] A labeled complex comprising the lipid-binding protein according to any one of [1] to [3] and a labeling substance.

[0013] [5] The labeled complex according to [4], wherein the labeling substance is a fluorescent substance or a fluorescent dye.

[0014] [6] Magnetic beads to which the lipid-binding protein according to any one of [1] to [3] is bound.

[0015] [7] A kit for isolating or detecting a PI(3,5)P ２ -containing substance, comprising the magnetic beads according to [6] and a magnet.

[0016] [8] A polynucleotide encoding the lipid-binding protein according to any one of [1] to [3].

[0017] [9] An expression vector containing the polynucleotide according to [8].

[0018]

[10] A transformant having the polynucleotide according to [8] or the expression vector according to [9].

[0019] According to the present invention, by substituting glycine, which is the 12th amino acid of the PX domain of the Snx A protein, with cysteine or a small hydrophobic amino acid, it is possible to provide a lipid-binding protein that can bind to PI(3,5)P ２ more strongly than the PX domain of the Snx A protein.

[0020] Figure 1 is a schematic diagram showing the presence or absence of binding when yeast cells expressing a recombinant protein of the PX domain of the SnxA protein on the cell membrane surface are mixed with liposome composition A. Figure 2 shows the fluorescently labeled PI(3,5)P obtained by flow cytometry analysis. ２ This graph shows the binding amount (fluorescence intensity derived from the fluorescent dye contained in the liposome) of recombinant proteins of SEQ ID NOs. 2-8 expressed on the surface of yeast cells to the contained liposome A composition, for each recombinant protein with amino acid mutations. The PX domain of the SnxA protein, which has the same amino acid sequence as the endogenous protein of Dictyostelium discoideum (see SEQ ID NO. 1), is shown as a thin line. Figure 3 shows PI(3,5)P contained in the lipid membrane of the labeled liposome. ２ Figure 4 is a graph showing the amount of labeled liposomes bound to recombinant proteins consisting of the amino acid sequences shown in SEQ ID NOs: 1-4 (fluorescence intensity derived from the fluorescent dye contained in the liposomes) under different quantity conditions. ２ The amount of labeled liposome composition A bound to recombinant proteins consisting of amino acid sequences shown in SEQ ID NOs. 5-8, and PI(3,5)P when the quantity conditions are varied. ２ This graph shows the Kd values ​​of the interaction between the endogenous SnxA protein or recombinant protein and PI(3,5)P. Figure 5 shows the interaction between the endogenous SnxA protein or recombinant protein and PI(3,5)P. ２ This graph shows the Kd values ​​of the interaction between the two. Figure 6 is a graph showing the amount of liposomes containing each phospholipid bound to the PX domain of the endogenous SnxA protein and each recombinant protein with amino acid mutations. Figure 7 is a fluorescence microscope image of yeast that expresses in cells a fusion protein with GFP, which is a dimer of a recombinant protein consisting of the PX domain of the endogenous SnxA protein or any of the amino acid sequences of SEQ ID NOs. 2 to 8. Figure 8 is a fluorescence-labeled PI(3,5)P obtained by flow cytometry analysis. ２This graph shows the binding amount of recombinant proteins SEQ ID NOs. 9-14 expressed on the surface of yeast cells to the liposome composition A, for each recombinant protein. The PX domain of the SnxA protein, which has the same amino acid sequence as the endogenous protein of *Dictyostelium* (see SEQ ID NO. 1), is shown as a thin line. Figure 9 shows the fluorescently labeled PI(3,5)P obtained by flow cytometry analysis. ２ This graph shows the binding amount of recombinant proteins SEQ ID NOs. 15-19 expressed on the surface of yeast cells to the contained liposome composition A, for each recombinant protein. The PX domain of the SnxA protein, which has the same amino acid sequence as the endogenous protein of Dictyostelium discoideum (see SEQ ID NO. 1), is shown as a thin line. Figure 10 is a diagram comparing the amino acid sequences of each recombinant protein with amino acid mutations added to the PX domain of the endogenous SnxA protein shown in Figure 2. Figure 11 shows recombinant proteins consisting of the amino acid sequences of SEQ ID NOs. 1, 5, or 15 and PI(3,5)P, as measured by surface plasmon resonance. ２ This is a graph showing the Kd value of the interaction between the two. Figure 12 is a confocal microscopy green fluorescence image of RAW264.7 cells, in which a recombinant protein consisting of the PX domain of the endogenous SnxA protein or the amino acid sequence of SEQ ID NO: 5 is expressed intracellularly as a fusion protein with GFP. Figure 13 is a graph showing the number of ring structures per cell shown in Figure 12. Figure 14 is a graph of PX-SnxA containing the amino acid sequence shown in SEQ ID NO: 5. ＧＶ - These are confocal microscopy images of animal cells expressing GCC-GFP, before and after treatment with apilimod.

[0021] Preferred embodiments of the present invention will be described in detail below, with reference to the drawings as appropriate. However, the present invention is not limited to the following embodiments.

[0022] [Lipid-binding protein] A lipid-binding protein according to a preferred embodiment of the present invention contains an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the 12th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, which is glycine, is substituted with cysteine ​​or a small-bulk hydrophobic amino acid residue, and has a higher PI(3,5)P than the protein consisting of the amino acid sequence shown in SEQ ID NO: 1. ２ It has high binding affinity to [the specified amino acid]. The small, hydrophobic amino acid residues mentioned above are selected from the group consisting of valine, leucine, isoleucine, alanine, and methionine. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the PX domain of the endogenous SnxA protein expressed in the aforementioned Dictyostemma scoparium. SEQ ID NO: 1: EIYITVPRKIQGEEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN

[0023] PI(3,5)P ２ Phosphatidylinositol 3,5-bisphosphate is a phospholipid that has phosphate groups at the 3rd and 5th positions of the inositol ring in the polar head.

[0024] As will be described in detail in the examples below, the lipid-binding protein of this embodiment (hereinafter also simply referred to as "lipid-binding protein") has glycine, which is the 12th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1, replaced with the corresponding amino acid, thereby forming PI(3,5)P ２ It exhibits specific and excellent binding affinity to lipids. For this reason, lipid-binding proteins are expected to be used, for example, for the visualization of membranes such as organelle membranes and cell membranes, the isolation and analysis of extracellular microparticles such as exosomes, the detection of lipid metabolic disorders, and ELISA diagnosis. However, the applications of lipid-binding proteins are not limited to these.

[0025] The amino acid sequence of the lipid-binding protein preferably includes an amino acid sequence having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, more preferably includes an amino acid sequence having 95% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, particularly preferably includes an amino acid sequence having 97% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and most preferably includes an amino acid sequence having 98% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. The lipid-binding protein may consist only of these amino acid sequences.

[0026] In this specification, the sequence identity of a target amino acid sequence with respect to a reference amino acid sequence (for example, the amino acid sequence shown in Sequence ID No. 1 above) can be determined as follows: First, the reference amino acid sequence and the target amino acid sequence are aligned. Each amino acid sequence is given a gap to maximize sequence identity. Next, the number of matching positions between the reference amino acid sequence and the target amino acid sequence and the total number of positions within the comparison region are calculated, and the sequence identity can be calculated according to the following formula (1). Positions containing gaps in either of the two sequences being compared are also added to the total number in the denominator. Sequence Identity (%) = Number of Matching Positions / Total Number of Positions within the Comparison Region × 100 ... (1)

[0027] The "target amino acid sequence" mentioned above is a sequence that aligns with a reference amino acid sequence among the amino acid sequences that make up a lipid-binding protein (in other words, it is the sequence of the region of the amino acid sequence that corresponds to the reference amino acid sequence among the amino acid sequences that make up a lipid-binding protein, and it is the sequence for which sequence identity is sought). The N-terminal and C-terminal positions of the target amino acid sequence correspond (align) to the N-terminal and C-terminal positions of the reference amino acid sequence. The "sequence that aligns with the reference amino acid sequence" and the "N-terminal and C-terminal positions of the target amino acid sequence" can be easily identified by someone skilled in the art, for example, using commercially available amino acid sequence analysis software (for example, SnapGen).

[0028] Lipid-binding proteins may also contain additional amino acid residues at the N-terminus and / or C-terminus of the amino acid sequence having the sequence identity described above.

[0029] The number of amino acid residues in a lipid-binding protein is not particularly limited, but may be 1000 or less, 500 or less, 300 or less, 250 or less, 200 or less, 150 or less, 130 or less, 120 or less, 118 or less, or 116 or less.

[0030] Lipid-binding proteins may include amino acid sequences in which one or more amino acids (amino acid residues) are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 1. In this specification, "a nucleotide sequence in which one or more amino acids are deleted, substituted, added, or inserted" means an amino acid sequence in which one or more and ten or fewer amino acids, preferably one or more and eight or fewer, more preferably one or more and five or fewer, and even more preferably one or more and three or fewer amino acids are deleted, substituted, added, or inserted.

[0031] The lipid-binding protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, i.e., PI(3,5)P, which is more PX domain than the PX domain of the SnxA protein. ２ Whether or not it has high binding affinity to the PX domain or lipid-binding protein and PI(3,5)P ２ The interaction between lipid-binding proteins and PI(3,5)P can be judged by comparing their Kd values ​​(equilibrium dissociation constant, unit: M). ２ The Kd value of the interaction between the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and PI(3,5)P ２ If the Kd value of the interaction between the two is significantly lower than the PI(3,5)P domain, then the lipid-binding protein is more likely to have PI(3,5)P than the PX domain. ２ It has been observed to have high binding affinity to [the substance].

[0032] The Kd value is a well-known parameter that indicates the strength of the binding. When protein P binds to ligand L to form a complex called PL, the equilibrium constant Kd for the binding reaction shown in the following chemical formula (i) is expressed by the following formula (2), where [P] is the concentration of protein P, [L] is the concentration of ligand L, and [PL] is the concentration of the PL complex.

[0033]

[0034] Kd = [P][L] / [PL]... (2) In equation (2), Kd is also called the equilibrium dissociation constant, and its unit is M.

[0035] The method for measuring the Kd value involves lipid-binding protein and PI(3,5)P ２ The Kd value of the interaction between and the PX domain of the SnxA protein and PI(3,5)P ２ The Kd value of the interaction between the two is not particularly limited, as long as it is measured under the same conditions and using the same method.

[0036] For example, as shown in the example described later, yeast (yeast display) in which a recombinant protein in which an amino acid mutation has been added to the PX domain of the endogenous SnxA protein is displayed on the cell membrane surface, and PI(3,5)P ２ Liposomes containing a lipid membrane are mixed with recombinant protein PI(3,5)P ２ The amount of liposomes contained will be measured by flow cytometry. For information on yeast displays that present recombinant proteins on the yeast surface, please refer to the following literature, for example: VanAntwerp et al., Fine Affinity Discrimination by Yeast Surface Display and Flow Cytometry. Biotechnol Prog 16:31-37, 2000.

[0037] After measurement by flow cytometry analysis, PI(3,5)P in the liposome membrane ２ From the data plotting the relationship between liposome binding amount for each content, commercially available statistical analysis software (e.g., GraphPad Prism9) was used to determine PI(3,5)P２ It is possible to easily calculate the Kd value of the interaction between the two. The Kd value can be measured, for example, under conditions of pH 7.5 and temperature 25°C.

[0038] Furthermore, as shown in the examples described later, PI(3,5)P was obtained by surface plasmon resonance (SPR). ２ It is also possible to measure the Kd value of the interaction between them.

[0039] In this specification, "significant" means statistically significant, and a result can be judged as statistically significant if p (significance probability) < 0.05, p < 0.01, or p < 0.001. The test method can be any known method that can determine the presence or absence of significance, and the type is not particularly limited, but examples include Student's t-test and Tukey's test for multiple comparisons.

[0040] The amino acid in which glycine, the 12th amino acid from the N-terminus of the amino acid sequence shown in Sequence ID No. 1, is substituted (in other words, the amino acid in the amino acid sequence of the lipid-binding protein corresponding to the position of glycine, the 12th amino acid in the amino acid sequence shown in Sequence ID No. 1 (cysteine, or a small-bulb hydrophobic amino acid) may be any one amino acid selected from the group consisting of cysteine, valine, leucine, isoleucine, alanine, and methionine, or any one amino acid selected from the group consisting of cysteine, valine, leucine, isoleucine, and methionine. The amino acid in the amino acid sequence of the lipid-binding protein corresponding to the position of glycine can be easily identified by a person skilled in the art, for example, by using known alignment software.

[0041] Lipid-binding proteins may contain the amino acid sequence shown in any one of SEQ ID NOs: 2-19. In the amino acid sequences of SEQ ID NOs: 2-19 below, the amino acid mutation sites from the endogenous PX domain are highlighted in parentheses. Lipid-binding proteins containing any of these amino acid sequences may further have an initiating methionine at the N-terminus. The initiating methionine may be, for example, one N-terminal position to the glutamic acid located at the N-terminus of the amino acid sequences of SEQ ID NOs: 2-19. Sequence ID 2: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENK[S]YQVTRRY[R]QFVLLHTQLVRVFGEH[V]LPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRD[D]GQN Sequence ID 3: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLP[P]LPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 4: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQ[S] Sequence ID 5: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 6: EIYITVPRKIQ[C]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 7: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRV[S]LQEYLQNLAKNPAILNSPVFY[R]FLKRDEGQNSequence ID 8: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSE[Y]KRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQ[S]LAKNPAILNSPVFYHFLKRDEGQN Sequence ID 9: EIYITVPRKIQ[V][R]EGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 10: EIYITVPRKIQ[V]EEGL[F]RKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 11: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVE[T]SENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 12: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKR[W]QVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 13: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQV[L]RRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 14: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQVTRRY[S]QFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 15:EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRV[A]LQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 16: EIYITVPRKIQ[V]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRV[L]LQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 17: EIYITVPRKIQ[I]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN Sequence ID 18: EIYITVPRKIQ[L]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN SEQ ID NO: 19: EIYITVPRKIQ[M]EEGLMRKYTAYVIEVEGSENKRYQVTRRYKQFVLLHTQLVRVFGEHDLPSLPAKANGLYFSKDDHTEKRRVNLQEYLQNLAKNPAILNSPVFYHFLKRDEGQN

[0042] Lipid-binding proteins may be dimers, trimers, or polymers having two or more monomers having the amino acid sequence shown in, for example, SEQ ID NOs: 2 to 19. The same applies to the labeling complexes, magnetic beads, kits, etc., described in detail below.

[0043] [Labeled Complex] In another preferred embodiment, the present invention provides a labeled complex comprising a lipid-binding protein and a labeling substance. The lipid-binding protein is the same as the lipid-binding protein detailed in the above embodiment of [Lipid-Binding Protein].

[0044] The labeling substance can be any substance capable of labeling lipid-binding proteins, and its type is not particularly limited. For example, it may be a fluorescent substance such as a fluorescent protein, a fluorescent dye such as FITC or Alexa dye, a radioactive substance, luciferase, or alkaline phosphatase. When luciferase or alkaline phosphatase is used as the labeling substance, the labeling complex is brought into contact with the luminescent substrate to produce PI(3,5)P ２ This can be visualized.

[0045] When the labeling substance is a fluorescent substance, it may be, for example, green fluorescent protein (GFP) derived from the jellyfish Aequorea victorea, or EGFP (Enhanced-humanized GFP) or rsGFP (red-shift GFP) which are modified green fluorescent protein, or blue fluorescent protein (BFP), or yellow fluorescent protein (YFP), or cyan fluorescent protein (CFP) ligand-dependent fluorescent protein halo tag, or ligand-dependent fluorescent protein SNAP tag, or ligand-dependent fluorescent protein CLIP tag, but the type of fluorescent substance used as the labeling substance is not limited to these.

[0046] When the labeling substance is a fluorescent dye, the labeling substance may be, for example, violetFluor450 or Pacific Blue, FITC (Fluorescein isothiocyanate), Alexa Fluor 488, PE (Phycoerythrin), Texas Red, Cy3, Cy5, PE-Cy5, PerCP (Peridinin Chlorophyll Protein), PerCP-Cy5.5, PE-Cy7, APC (Allophycocyanin), AlexaFluor647, redFluor710, Alexa Fluor700, or APC-Cy7, but the type of fluorescent substance used as the labeling substance is not limited to these.

[0047] When the labeling substance is a protein such as a fluorescent protein or an enzyme, the labeling complex may be used in the form of a fusion protein of the labeling substance and the lipid-binding protein described above, or it may be in the form of a separate labeling substance bound to a lipid-binding protein. When the labeling complex is in the form of a labeling substance bound to a lipid-binding protein, the labeling substance and the lipid-binding protein may be bound via a linker.

[0048] [Magnetic Beads] In another preferred embodiment, the present invention provides magnetic beads to which a lipid-binding protein is bound. The lipid-binding protein is the same as the lipid-binding protein detailed in the above embodiment of [Lipid-Binding Protein]. Preferably, the lipid-binding protein is bound to the surface of the magnetic beads.

[0049] In this specification, "magnetic beads" means beads containing a magnetic material. Examples of magnetic materials include, but are not limited to, iron oxides such as ferrite, cobalt, and chromium oxide.

[0050] The lipid-binding protein is bound to the magnetic beads, PI(3,5)P ２Magnetic beads with lipid-binding proteins that have captured their contents can be recovered and / or purified using a magnet, offering high convenience. PI(3,5)P ２ Examples of contained substances include PI(3,5)P ２ Extracellular microparticles containing (such as exosomes and other nanoparticles), and PI(3,5)P ２ Examples include, but are not limited to, cell-derived membrane components and organelle membranes.

[0051] [PI(3,5)P ２ [Kit for isolation or detection of contained substances] In another preferred embodiment, the present invention provides a PI(3,5)P kit comprising magnetic beads to which lipid-binding proteins are bound and a magnet. ２ A kit for isolating or detecting the contents is provided. The lipid-binding protein is the same as the lipid-binding protein detailed in the [Lipid-Binding Protein] embodiment above, and the magnetic beads are the same as the magnetic beads detailed in the [Magnetic Beads] embodiment above.

[0052] According to the isolation kit of this embodiment, which further includes a magnet, PI(3,5)P ２ PI(3,5)P, including vesicles and cellular components. ２ The contents can be captured by magnetic beads to which lipid-binding proteins are bound, and then recovered and purified with a magnet, hence PI(3,5)P ２ It allows for easy isolation or detection of contained substances, making it highly convenient.

[0053] [Polynucleotides] In another preferred embodiment, the present invention provides polynucleotides encoding lipid-binding proteins. The lipid-binding proteins are similar to the lipid-binding proteins detailed in the [Lipid-Binding Proteins] embodiment above.

[0054] By inserting a polynucleotide encoding a lipid-binding protein into a cell, for example, a viral vector, and introducing it into the cell, the lipid-binding protein is expressed, and PI(3,5)P is introduced into the cell. ２The localization of the polynucleotide can be determined. The polynucleotide may be synthesized DNA, cDNA, mRNA, or cRNA. The polynucleotide may be in single-stranded or double-stranded form.

[0055] The base sequence of the polynucleotide is not particularly limited as long as it is a sequence that encodes the lipid-binding protein described above, and the type of codon is not specified. The codon may be appropriately optimized depending on the cell type. Examples of base sequences that encode lipid-binding proteins consisting of the amino acid sequences of SEQ ID NOs. 20 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 21 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 22 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 4 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 5 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 6 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 7 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 8 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 9 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 10 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 11 encodes a protein consisting of the amino acid sequence of SEQ ID NOs. 12 Sequence ID 31 encodes a protein consisting of the amino acid sequence of Sequence ID 13. Sequence ID 32 encodes a protein consisting of the amino acid sequence of Sequence ID 14. Sequence ID 33 encodes a protein consisting of the amino acid sequence of Sequence ID 15. Sequence ID 34 encodes a protein consisting of the amino acid sequence of Sequence ID 16. Sequence ID 35 encodes a protein consisting of the amino acid sequence of Sequence ID 17. Sequence ID 36 encodes a protein consisting of the amino acid sequence of Sequence ID 18. Sequence ID 37 encodes a protein consisting of the amino acid sequence of Sequence ID 19.

[0056] Furthermore, the polynucleotide may encode a fusion protein of a lipid-binding protein and a fluorescent protein. By introducing the polynucleotide encoding the fusion protein into cells, PI(3,5)P ２ The localization of the fluorescent protein can be easily visualized. The type of fluorescent protein is not particularly limited, but examples include the fluorescent proteins detailed in the embodiment of the labeling complex.

[0057] [Expression Vector] In another preferred embodiment, the present invention provides an expression vector comprising a polynucleotide encoding a lipid-binding protein. The polynucleotide is the same as the polynucleotide described in the [Polynucleotide] embodiment above. The expression vector is preferably a protein expression vector. The type of expression vector is not particularly limited, but examples include plasmids derived from Bacillus subtilis, plasmids derived from Escherichia coli, plasmids derived from yeast, bacteriophages, viral vectors, or vectors modified therefrom.

[0058] Examples of plasmids derived from Bacillus subtilis include pTP5, pC194, and pUB110. Examples of plasmids derived from Escherichia coli include pUC12, pUC13, pBR322, and pBR325. Examples of plasmids derived from yeast include pSH15 and pSH19. Examples of bacteriophages include lambda phage. Examples of viruses used as viral vectors include adeno-associated viruses, adenoviruses, lentiviruses, baculoviruses, and retroviruses.

[0059] The expression vector of this embodiment may have a promoter for expression using animal cells as a host, a promoter for expression using plant cells as a host, or a promoter for expression using insect cells as a host.

[0060] Examples of promoters used for expression in animal cells include, but are not limited to, the EF1α promoter, SRα promoter, LTR promoter, SV40 promoter, CMV (cytomegalovirus) promoter, HSV-tk promoter, and CAG promoter.

[0061] Examples of promoters used for expression in plant cells include, but are not limited to, the 35S promoter of cauliflower mosaic virus (CaMV) and the REF (rubber elongation factor) promoter.

[0062] Examples of promoters used for expression in insect cells include, but are not limited to, the polyhedrin promoter and the p10 promoter.

[0063] The expression vector may further include a multicloning site, enhancers, splicing signals, poly-A addition signals, origins of replication, selection markers, and the like.

[0064] By introducing the expression vector of this embodiment into host cells, lipid-binding proteins are expressed, and PI(3,5)P is expressed in the host cells. ２ It is possible to grasp the localization of [the phenomenon].

[0065] The expression vector may also contain a polynucleotide encoding a fusion protein of a lipid-binding protein and a fluorescent protein. By introducing the polynucleotide encoding the fusion protein into cells, PI(3,5)P ２ The localization of the fluorescent protein can be easily visualized. The type of fluorescent protein is not particularly limited, but examples include the fluorescent proteins detailed in the embodiment of the labeling complex.

[0066] The method for introducing expression vectors into host cells is not particularly limited, but known methods include the competent cell method, heat shock method, electroporation method, lipofection method, Agrobacterium method, and methods using viruses.

[0067] [Transformed] In another preferred embodiment, the present invention provides a transformed having a polynucleotide encoding a lipid-binding protein and / or an expression vector containing the polynucleotide. When the transformed in this embodiment has the polynucleotide, the polynucleotide is the same as the polynucleotide detailed in the [Polynucleotide] embodiment. When the transformed in this embodiment has an expression vector, the expression vector is the same as the expression vector detailed in the [Expression Vector] embodiment.

[0068] The transformant of this embodiment is an organism transformed to express the lipid-binding protein detailed in the [Lipid-binding protein] embodiment, and may be, for example, a host into which the expression vector detailed in the [Expression vector] embodiment has been introduced. The host may be, for example, an animal cell, a plant cell, or a microorganism such as yeast or E. coli, but is not limited to these.

[0069] The expression vector only needs to be expressible within the transformed organism or on the cell surface; specifically, it only needs to be expressible under the control of a promoter. The types of promoters corresponding to the types of transformants are as described above.

[0070] The polynucleotides and / or expression vectors present in the transformant may be located within the transformant's cells, for example, in the cytoplasm, or they may be introduced into the genome.

[0071] According to the transformant of this embodiment, the lipid-binding protein detailed in the [lipid-binding protein] embodiment can be expressed and produced.

[0072] Although preferred embodiments have been described in detail above, the present invention is not limited to these embodiments, and various modifications are possible within the scope of the invention as described in the claims.

[0073] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0074] [Experimental Example 1] In this experiment, among a number of recombinant proteins in which amino acid mutations were randomly added to the amino acid residues of the PX domain of the endogenous SnxA protein of the mold Dictyostelium, PI(3,5)P was selected. ２ Recombinant proteins with higher binding affinity to the endogenous PX domain were selected. A detailed explanation follows.

[0075] (PI(3,5)P ２ Preparation of liposomes containing: First, 2 mol% PI(3,5)P ２ A liposome composition A was prepared containing 0.5 mol% of the fluorescent dye 18:1 Liss Rhod PE (810150C, Avanti®).

[0076] (Preparation of yeast cells displaying recombinant protein on the cell surface) Figure 1 is a schematic diagram showing whether or not binding occurs when yeast cells expressing recombinant protein of the PX domain of the SnxA protein on the cell membrane surface are mixed with liposome composition A. Yeast cells expressing recombinant protein of the PX domain on the cell membrane surface via the linker region were prepared. The linker region is tagged with an HA tag.

[0077] (FACS analysis) Liposome composition A was mixed with yeast cells expressing recombinant protein on the cell membrane surface. Then, in order to label only the yeast cells expressing the protein on the cell membrane surface, the precipitate obtained by centrifugation was stained with Alexa647 anti-HA ab.

[0078] The obtained yeast cells were analyzed using Cell analyzer FACSymphonyA1 to measure the fluorescence intensity emitted by Rhod-PE in the Alexa647-positive cell fraction (in other words, the amount of labeled liposome composition A bound to the cells).

[0079] Furthermore, PI(3,5)P contained in labeled liposome composition A ２ The conditions for the amount are varied, and each PI(3,5)P ２ The fluorescence intensity (amount of liposome binding to recombinant protein) was measured. Furthermore, the obtained fluorescence intensity was compared with PI(3,5)P ２Based on the data plotting the relationship with quantity (concentration) (see Figure 3), the commercially available medical statistical analysis software GraphPad Prism9 was used to analyze each recombinant protein and PI(3,5)P ２ The Kd value of the interaction between the two was calculated.

[0080] Figure 2 shows the fluorescently labeled PI(3,5)P obtained by flow cytometry analysis. ２ This graph shows a histogram of the amount of recombinant proteins (fluorescence intensity derived from the fluorescent dye contained in the liposomes) bound to liposome composition A and recombinant proteins of sequence numbers 1-8 expressed on the surface of yeast cells, for each recombinant protein with amino acid mutations. In Figure 2, recombinant proteins identified by their amino acid sequence are shown with thick lines, and the PX domain of the SnxA protein, which has the same amino acid sequence as the endogenous protein of the slime mold (see sequence number 1), is shown with a thin line.

[0081] As shown in Figure 2, PI(3,5)P to each protein ２ The amount of binding was significantly greater in the recombinant protein consisting of the amino acid sequences shown in SEQ ID NOs: 2-8 than in the PX domain of the endogenous SnxA protein. This suggests that amino acid mutation from the amino acid sequence of the endogenous PX domain shown in SEQ ID NO: 1 to the amino acid sequences of SEQ ID NOs: 2-8 results in PI(3,5)P ２ This suggests that the bonding affinity with [the substance] has improved.

[0082] Figure 3 shows the PI(3,5)P contained in the lipid membrane of labeled liposome composition A. ２ When the conditions for the amount are varied, the amount of labeled liposome composition A bound to recombinant proteins consisting of the amino acid sequences shown in SEQ ID NOs: 1 to 4 (fluorescence intensity derived from the fluorescent dye contained in the liposome) and PI(3,5)P ２ This is a graph showing the Kd value of the interaction between the two. Figure 4 shows PI(3,5)P contained in the lipid membrane of labeled liposome composition A. ２ The amount of labeled liposome composition A bound to recombinant proteins consisting of amino acid sequences shown in SEQ ID NOs. 5-8, and PI(3,5)P when the quantity conditions are varied.２ This graph shows the Kd values ​​of the interaction between the two. Note that each recombinant protein and the endogenous PX domain shown in Figures 3 and 4 separately contains an initiation methionine residue at the N-terminus.

[0083] As shown in Figures 3 and 4, each recombinant protein consisting of the amino acid sequences shown in SEQ ID NOs: 2-8 exhibited higher fluorescence intensity from Rhod-PE compared to the endogenous PX domain (see SEQ ID NO: 1 for its amino acid sequence). In particular, PI(3,5)P ２ Even at low concentrations, the recombinant proteins showed high fluorescence intensity. From these results, each recombinant protein consisting of the amino acid sequences shown in SEQ ID NOs: 2-8 is PI(3,5)P ２ Regardless of the concentration, PI(3,5)P is more effective than the endogenous PX domain. ２ This suggests that it has high binding affinity to [the substance].

[0084] Figure 5 shows endogenous SnxA protein or recombinant protein and PI(3,5)P ２ This graph shows the Kd values ​​of the interaction between the two. As shown in Figure 5, recombinant proteins consisting of any of SEQ ID NOs: 2, 6, or 8 have significantly lower Kd values ​​than the PX domain of the endogenous SnxA protein (see SEQ ID NO: 1 as the amino acid sequence), and PI(3,5)P ２ High bonding affinity was confirmed. Tukey's test was used to determine statistical significance; "*" (asterisk) indicates p < 0.05, and "**" indicates p < 0.01.

[0085] [Experimental Example 2] In this experiment, PI(3,5)P was used in Experimental Example 1. ２ Recombinant proteins consisting of amino acid sequences of SEQ ID NOs. 2-8, which have been shown to bind strongly to PI(3,5)P, are known to bind strongly to PI(3,5)P. ２ We confirmed whether or not it specifically binds to only that target.

[0086] More specifically, in Experimental Example 1, each recombinant protein presented by yeast on the cell membrane surface was given PI(3,5)P ２In the same manner as when calculating the amount of liposomes containing PI(3,5)P, in Experimental Example 2, each recombinant protein was treated with PI(3,5)P. ２ Instead, the amount of liposomes containing other phospholipids that bind to the target liposomes was calculated.

[0087] Figure 6 is a graph showing the amount of liposomes containing each phospholipid bound to the PX domain of the endogenous SnxA protein and to each recombinant protein with amino acid mutations. In Figure 6, PS refers to phosphatidylserine, PA refers to phosphatidic acid, PI refers to phosphatidylinositol, PI3P refers to phosphatidylinositol 3-phosphate, PI4P refers to phosphatidylinositol 4-phosphate, PI5P refers to phosphatidylinositol 5-phosphate, and PI4,5P ２ This refers to phosphatidylinositol 4,5-bisphosphate, and PI3,4P ２ This refers to phosphatidylinositol 3,4-bisphosphate, and PI3,4,5P ３ This refers to phosphatidylinositol 3,4,5-trisphosphate.

[0088] As shown in Figure 6, PI(3,5)P bound to each recombinant protein ２ The binding levels to other phospholipids were all extremely low, similar to those of the PX domain of the endogenous SnxA protein.

[0089] From these results, recombinant proteins consisting of the amino acid sequences of SEQ ID NOs. 2-8, in which a mutation has been added to the 12th amino acid residue from the N-terminus, retain the PI(3,5)P amino acid even after the amino acid mutation. ２ It maintains specific binding affinity to PI(3,5)P ２ This suggests that it may be useful as a lipid probe that specifically binds to [the target molecule].

[0090] [Experimental Example 3] In this experiment, a fusion protein of recombinant protein consisting of one of the amino acid sequences of SEQ ID NOs: 1 to 8 and GFP was expressed in yeast cells, and PI(3,5)P was expressed in cells. ２The localization was confirmed using a fluorescence microscope. For an example of a fusion protein, please refer to SEQ ID NO: 38 for the amino acid sequence of a fusion protein between a recombinant protein consisting of the amino acid sequence shown in SEQ ID NO: 5 and GFP.

[0091] Figure 7 shows fluorescence microscopy images of yeast cells expressing a fusion protein of a recombinant protein consisting of the PX domain of the endogenous SnxA protein or one of the amino acid sequences of SEQ ID NOs: 2-8, and GFP. In Figure 7, the areas stained blue by the vacuolar marker are indicated by the code A1, and the green fluorescent areas (light areas) due to GFP are indicated by the code A2.

[0092] Yeast cells have PI(3,5)P in their vacuolar membrane. ２ It is known that it is abundant. As shown in Figure 7, in yeast cells expressing recombinant proteins, the GFP-induced green fluorescent portion A2 is localized to the vacuolar membrane, indicating that any recombinant protein consisting of the amino acid sequences of SEQ ID NOs. 2-8, and consequently PI(3,5)P, is localized to the vacuolar membrane. ２ However, it was confirmed that it was localized to the vacuolar membrane.

[0093] In contrast, in yeast cells expressing the PX domain of the endogenous SnxA protein, the GFP-induced green fluorescent portion A2 was partially aggregated or spread throughout almost the entire cell, and no localization to the vacuolar membrane was observed.

[0094] These results suggest that recombinant proteins in which the 12th amino acid of the PX domain of the endogenous SnxA protein is replaced with cysteine ​​or a small, hydrophobic amino acid exhibit PI(3,5)P more specifically and potently than the endogenous PX domain. ２ It was suggested that it binds to PI(3,5)P. Therefore, recombinant proteins in which the 12th amino acid is replaced with cysteine ​​or a small hydrophobic amino acid are thought to bind to PI(3,5)P. ２ It has been shown to be useful as a lipid probe that specifically binds to PI(3,5)P. In addition, recombinant proteins in which the 12th amino acid is substituted with cysteine ​​or a small hydrophobic amino acid have been used to study intracellular PI(3,5)P ２This suggests that it is also detectable.

[0095] [Experimental Example 4] In this experiment, recombinant proteins consisting of the amino acid sequences shown in SEQ ID NOs: 9-19 were subjected to PI(3,5)P, similar to Experimental Example 1. ２ A labeled liposome composition A containing PI(3,5)P was mixed with the label, and flow cytometry analysis was performed. ２ The strength of the binding affinity was confirmed.

[0096] Figure 8 shows the fluorescently labeled PI(3,5)P obtained by flow cytometry analysis. ２ Figure 9 is a graph showing the binding amount of recombinant proteins SEQ ID NOs. 9-14 expressed on the surface of yeast cells to the liposome composition A, for each recombinant protein. ２ These graphs show the binding amount of recombinant proteins SEQ ID NOs. 15-19 expressed on the surface of yeast cells to the liposome composition A, for each recombinant protein. In Figures 8 and 9, recombinant proteins identified by their amino acid sequence are shown with thick lines, while the PX domain of the SnxA protein, which has the same amino acid sequence as the endogenous protein of the slime mold, is shown with a thin line. Note that each recombinant protein shown in Figures 8 and 9 contains an initiating methionine residue at its N-terminus.

[0097] As shown in Figures 8 and 9, PI(3,5)P to each protein ２ The amount of binding was significantly greater in the recombinant protein consisting of the amino acid sequences shown in SEQ ID NOs: 9-19 than in the PX domain of the endogenous SnxA protein. This suggests that amino acid mutation from the amino acid sequence of the endogenous PX domain shown in SEQ ID NO: 1 to the amino acid sequences of SEQ ID NOs: 9-19 results in PI(3,5)P ２ This suggests that the bonding affinity with [the substance] has improved.

[0098] Figure 10 is a drawing comparing the amino acid sequences of the PX domain of the endogenous SnxA protein and each recombinant protein with an amino acid mutation added to the PX domain. In Figure 10, the position of the 12th glycine from the N-terminus of the endogenous PX domain shown in SEQ ID NO: 1 and the amino acid mutation positions of each recombinant protein are emphasized in bold and underlined, respectively.

[0099] As shown in Figure 10, recombinant proteins that bind more strongly to PI(3,5)P than the endogenous PX domain commonly have the 12th amino acid residue from the N-terminus mutated (substituted) to cysteine, or a small-sized hydrophobic amino acid such as valine, leucine, isoleucine or methionine, where the 12th amino acid residue in the endogenous PX domain is glycine. ２ Also, since the recombinant protein consisting of the amino acid sequence shown in SEQ ID NO: 5 has the same amino acid sequence as the endogenous PX domain except that the 12th amino acid residue from the N-terminus is mutated to valine, it was strongly suggested that by mutating the 12th amino acid residue from the N-terminus of the endogenous PX domain to a small-sized hydrophobic amino acid, the binding affinity for PI(3,5)P can be enhanced.

[0100] [Experimental Example 5] In this experiment, for the recombinant proteins consisting of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 5 or SEQ ID NO: 15, the Kd value of the interaction with PI(3,5)P was measured by surface plasmon resonance (SPR). ２

[0101] (Materials and Methods for Measuring Kd Value) A Biacore 8K instrument (Cytiva) was used for the surface plasmon resonance method. The liposomes containing PI(3,5)P in the lipid membrane were made to contain 1 mol% of biotinylated cap PE (Avanti, 870273). By injecting a 200 μM total lipid solution into the buffer (150 mM NaCl, 20 mM HEPES pH 7.5) at a rate of 5 μL / min for 600 seconds, 2 mol% of PI(3,5)P ２

[0102] ２ ​２ Liposomes containing [substance] were immobilized on a Sensor Chip CAP (Series S, Cytiva). As a result, a coating level of ~300 response units (RU) was obtained. PI(3,5)P ２ Liposomes containing [substance] were injected onto Flow Cell 2. Each recombinant protein was passed over the CAP chip immobilized with liposomes at a flow rate of 30 μL / min for 120 seconds at five different concentrations under buffer conditions of 150 mM NaCl, 50 mM HEPES pH 7.5, followed by a dissociation phase of 600 seconds. After subtracting the binding to the control lane (Flow Cell 1), the final sensorgram was obtained. At the end of each run, a regeneration solution was pulsed (for a short time) to dissociate the bound protein and liposomes on the chip. The chip surface was regenerated regularly by injecting the regeneration solution at 10 μL / min for 60 seconds, and then newly coated with liposomes containing PI(3,5)P ２ as described above. The apparent Kd value (equilibrium dissociation constant) was calculated using Biacore(TM) Insight Evaluation Software (Cytiva). All experiments were repeated three times (n = 3).

[0103] (Results) Figure 11 is a graph showing the Kd values of the interaction between the recombinant protein consisting of the amino acid sequence of SEQ ID NO: 1, 5 or 15 and PI(3,5)P measured by surface plasmon resonance method ２ "*" means p < 0.05, and "***" means p < 0.001. ANOVA (analysis of variance) and Tukey's multiple comparison test were used for statistical analysis. The same applies hereinafter. Also, Table 1 below shows the Kd values of the interaction between each recombinant protein and PI(3,5)P ２

[0104]

[0105] ​As shown in Figure 11, the recombinant protein consisting of the amino acid sequence shown in Sequence ID No. 5 had a significantly lower Kd value than the recombinant protein consisting of the amino acid sequence shown in Sequence ID No. 1, and the recombinant protein consisting of the amino acid sequence shown in Sequence ID No. 15 had a significantly lower Kd value than the recombinant protein consisting of the amino acid sequence shown in Sequence ID No. 5.

[0106] From these results, regardless of the interaction measurement method, recombinant proteins consisting of the amino acid sequences shown in SEQ ID NO: 5 or 15 are more PI(3,5)P than the endogenous PX domain. ２ It was revealed that it exhibits high binding affinity to [the substance].

[0107] [Experimental Example 6] In this experiment, a recombinant protein consisting of the PX domain of the endogenous SnxA protein shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 5 was dimerized and expressed in animal cells as a fusion protein with GFP, and PI(3,5)P was expressed in animal cells. ２ The localization of the substance was evaluated. RAW264.7 cells were used as the animal cells.

[0108] In the following, the fusion protein of the dimer of the PX domain of the endogenous SnxA protein and GFP will also be referred to as "PX-SnxA-GCC-GFP," and the recombinant protein of the dimer of the recombinant protein consisting of the amino acid sequence shown in SEQ ID NO: 5 and GFP will be referred to as "PX-SnxA ＧＶ It is also called "GCC-GFP".

[0109] Figure 12 is a confocal microscopy image of RAW264.7 cells expressing a recombinant protein consisting of the PX domain of the endogenous SnxA protein or the amino acid sequence of Sequence ID No. 5 as a fusion protein with GFP. In Figure 12, areas exhibiting green fluorescence are shown in lighter colors. Figure 13 is a graph showing the number of ring structures per cell shown in Figure 12. Unless otherwise specified, the scale bars in each figure from Figure 12 onward represent 2 μm. Error bars represent the mean ± standard error (SEM). The total number of cells expressing PX-SnxA-GCC-GFP is 35. ＧＶ The total number of cells expressing GCC-GFP was 40. The number of ring structures per cell was quantified using a blinding method.

[0110] As shown in Figures 12 and 13, cells expressing PX-SnxA-GCC-GFP are more efficient than cells expressing PX-SnxA ＧＶ Cells expressing GCC-GFP showed more green cyclic structures due to GFP. The cyclic structures were lysosome-related membranes and PI(3,5)P ２ It is localized at high concentrations.

[0111] The results shown in Figures 12 and 13 indicate that using a recombinant protein containing the amino acid sequence shown in Sequence ID No. 5 allows for more accurate and detailed analysis of PI(3,5)P in animal cells than when using an endogenous PX domain. ２ This suggests that the localization of [the region] can be evaluated.

[0112] On the other hand, Figure 14 shows PX-SnxA ＧＶ - These are confocal microscopy images of animal cells expressing GCC-GFP, taken when treated with apilimod (right side) and when not treated with apilimod (left side). In Figure 14, areas exhibiting green fluorescence are shown in lighter colors.

[0113] Treatment with apirimodo was performed at 100 nM for 30 minutes. Apirimodo is PI(3,5)P ２ It inhibits the synthesis of PI(3,5)P ２ It has the effect of reducing [something].

[0114] As shown in the left part of Figure 14, when not treated with apilimod, only the cyclic structure, which is the organelle membrane, exhibited green fluorescence. In contrast, as shown in the right part of Figure 14, when treated with apilimod, the cytoplasm exhibited green fluorescence. Normally, PI(3,5)P ２ It is localized to the organelle membrane structure within the cell, and when treated with apyrimod, PI(3,5)P ２ As a result of the decrease, PX-SnxA ＧＶ - It can be observed that the localization of GCC-GFP to the organelle membrane disappears, and its localization changes to the cytoplasm.

[0115] Based on the above results, PX-SnxA containing the amino acid sequence shown in Sequence ID No. 5 ＧＶ -GCC-GFP is converted into PI(3,5)P within cells. ２ It can be bound to PI(3,5)P in cells. ２ This suggests that the localization of [the region] can be visualized.

[0116] According to the present invention, a recombinant protein obtained by substituting glycine, the 12th amino acid of the PX domain of the endogenous SnxA protein, with cysteine ​​or a small-bulk hydrophobic amino acid, exhibits PI(3,5)P compared to the endogenous PX domain. ２ Because it exhibits excellent bonding properties, it can be used industrially.

[0117] A1...Blue fluorescent area indicating the location of the vacuole, A2...Green fluorescent area indicating the location of the recombinant protein

Claims

1. The amino acid sequence contains an amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1, and the 12th amino acid residue from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, which is glycine, isoleucine, alanine, and methionine, is substituted with an amino acid residue selected from the group consisting of cysteine, valine, leucine, isoleucine, alanine, and methionine, and is PI(3,5)P more than the protein consisting of the amino acid sequence shown in SEQ ID NO:

1. ２ A lipid-binding protein with high binding affinity to lipids.

2. The lipid-binding protein according to claim 1, wherein the glycine is substituted with any amino acid residue selected from the group consisting of cysteine, valine, leucine, isoleucine, and methionine.

3. The lipid-binding protein according to claim 1, comprising any of the amino acid sequences of SEQ ID NOs: 2 to 19.

4. A labeled complex comprising a lipid-binding protein according to any one of claims 1 to 3 and a labeling substance.

5. The labeling complex according to claim 4, wherein the labeling substance is a fluorescent substance or a fluorescent dye.

6. Magnetic beads to which the lipid-binding protein described in any one of claims 1 to 3 is bound.

7. A PI(3,5)P comprising the magnetic beads described in claim 6 and a magnet. ２ A kit for isolating or detecting contained substances.

8. A polynucleotide encoding a lipid-binding protein as described in claim 1.

9. An expression vector comprising the polynucleotide described in claim 8.

10. A transformant having the polynucleotide described in claim 8 or the expression vector described in claim 9.

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