Nanopore protein

Abstract

To provide a novel nanopore protein suitable for nanopore sensing.SOLUTION: The present invention provides a nanopore protein that is a mutant protein of a cholesterol-dependent pore-forming cytolysin (CDC) family protein containing a specific mutation.SELECTED DRAWING: Figure 4A

Description

[Technical field]

[0001] The present invention relates to nanopore proteins. [Background technology]

[0002] Nanopore sensing, which uses membrane proteins that form nanopores (nanopore proteins), is a method for electrically detecting biomolecules (especially DNA) that pass through nanopores, and in recent years, highly sensitive nanopore DNA sequencers using this method have been put to practical use. Biomolecules are detected by constructing nanopore proteins on an artificial cell membrane, and utilizing the fact that the nanopores on the membrane obstruct the flow of ions when the biomolecules pass through the artificial cell membrane. Each type of nanopore protein has different functions and pore sizes. OmpG and OmpA, which are outer membrane proteins that form nanopores, are known as nanopore proteins.

[0003] On the other hand, intermedilysin (ILY) is a type of membrane protein belonging to the cholesterol-dependent pore-forming cytolytic toxin family (CDC family) (Non-Patent Document 1). The CDC family, including ILY, is known to form relatively large pores of several tens of nm (Non-Patent Document 1). The CDC family is known to exist in three three-dimensional structures, namely, a structure when it exists as a free monomer in an aqueous solution (Figure 1(a)), a prepore structure that can interact with other monomers (Figure 1(b)), and a pore structure in which a part of the monomer penetrates the lipid bilayer membrane (Figure 1(c)) (Non-Patent Documents 2 and 3). As a step of forming a pore in the CDC family, first, the monomer takes on a prepore structure, which enables the monomers to interact with each other, and the monomer oligomerizes to form a prepore on the membrane surface. Next, the monomer that forms the prepore structure changes its conformation to a pore structure, and a part of the monomer penetrates the lipid bilayer membrane to form a pore.

[0004] In nanopore sensing, it is necessary to use an appropriate nanopore protein depending on the size or shape of the molecule to be detected. Non-Patent Document 4 discloses that the pore size of OmpG was modified by increasing or decreasing the number of β-strands constituting OmpG that form a barrel-shaped pore. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] PeraroM. D. and Goot FG Nature Reviews, 2016, 14, 77-92 [Non-Patent Document 2] JohnsonS., et al., Cell Reports, 2013, 3, 1369-1377 [Non-Patent Document 3] Shah NR et al., Nature Comm., 2020, 11, 5818 [Non-Patent Document 4] Tosaka T. and Kamiya K. ACS Appl. Nano Mater., 2022, 5,6149-6158 [Non-Patent Document 5] KawanoR.et al., PLOS ONE, 2014, 9, e102427-e102427 [Non-Patent Document 6] Ohara,M., et al., ACS Synth. Biol., 2017,6, 1427-1432 [Non-Patent Document 7] Saigo, N., et al., ACS Omega, 2019, 4, 13124-13130 [Non-Patent Document 8] YANG, Wayne, et al., Detection of CRISPR-dCas9 on DNA with solid-state nanopores, Nanoletters, 2018, 18.10: 6469-6474. [Non-Patent Document 9] Shoji K., et al., Anal Chem, 2020, 92, 10856-10862 [Non-Patent Document 10] Kawano R., et al., Sci Rep. 2013, 3, 1995 [Non-Patent Document 11] Varongchayakul, N., et al., Chem Soc Rev. 2018, 26, 8512-8524 [Non-Patent Document 12] Moon CP and Fleming KG Proc. Natl. Acad. Sci. USA, 2011, 108, 10174-10177 [Non-Patent Document 13] Farrand, AJ et al., Proc. Natl. Acad. Sci. USA, 2010, 107, 4341-4346 [Non-Patent Document 14] Dowd,KJet al., PLos pathogens, 2012, 8, e1002787 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a novel nanopore protein that can be used for nanopore sensing. [Means for solving the problem]

[0007] As a result of extensive research, the inventors have discovered that mutant proteins of intermedilysin containing specific mutations have improved stability or a higher probability of opening a desired large pore size than wild-type intermedilysin, and have also discovered that such mutant proteins can actually be used to detect proteins, thereby completing the present invention.

[0008] That is, the present invention relates to the following inventions:

[0009] [1] A nanopore protein which is a mutant protein of a CDC family protein containing at least one of the following mutations (1) to (3): (1) Any one of the following substitutions (a) to (g) in the amino acid sequence set forth in SEQ ID NO:1: (a) Substitution of the amino acid residue at the position corresponding to arginine at position 302 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (b) substitution of the amino acid residue at the position corresponding to histidine at position 242 of a CDC family protein other than the proteins having the amino acid sequences set forth in SEQ ID NOs: 48, 51, and 55 with leucine, tyrosine, tryptophan, alanine, cysteine, valine, isoleucine, methionine, or proline. (c) substitution of the amino acid residue at the position corresponding to threonine at position 334 of a CDC family protein other than the proteins having the amino acid sequences set forth in SEQ ID NOs: 49, 53, and 15 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (d) Substitution of the amino acid residue at the position corresponding to valine at position 241 with glutamic acid, arginine, histidine, lysine, aspartic acid, glycine, serine, threonine, asparagine, or glutamine. (e) Substitution of the amino acid residue at the position corresponding to threonine at position 315 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (f) Substitution of the amino acid residue at the position corresponding to asparagine 338 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (g) Substitution of the amino acid residue at the position corresponding to isoleucine at position 339 with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, glycine, threonine, asparagine, or glutamine. (2) A mutation in which 5 to 23 consecutive amino acids including Y196-H200 in N178-H200 in the amino acid sequence set forth in SEQ ID NO: 1 are deleted, or amino acid residues at positions corresponding to 2 to 13 consecutive amino acids including T517-L518 in W514-V526 are deleted. (3) A mutation in which an amino acid residue is inserted at a position corresponding to 2 to 25 consecutive amino acid residues, including T517-L518, of Q508-D532 in the amino acid sequence set forth in SEQ ID NO: 1, at the C-terminus of the amino acid sequence set forth in SEQ ID NO: 1, or a mutation in which an amino acid residue is inserted at a position corresponding to 1 to 24 consecutive amino acid residues, including R495, of R480-L503 in the amino acid sequence set forth in SEQ ID NO: 1, between amino acid residues at positions corresponding to L503 and P504 in the amino acid sequence set forth in SEQ ID NO: 1 [2] The nanopore protein according to [1], wherein the amino acid residues at positions corresponding to R302, H242, T334, V241, T315, N338 or I339 in the amino acid sequence set forth in SEQ ID NO:1 are mutated by substitution with R302F, R302M, R302L, R302I, H242L, H242I, H242Y, T334V, T334I, V241T, V241S, V241K, V241E, T315V, T315I, N338L, N338F, I339T, I339S or I339K. [3] The nanopore protein according to [2], wherein the amino acid residues at positions corresponding to R302, H242, T334, V241, T315 or I339 in the amino acid sequence set forth in SEQ ID NO:1 are mutated by substitution with R302F, H242L, H242I, H242Y, T334V, T334I, V241E, T315V, T315I or I339S. [4] A nanopore sensor comprising the nanopore protein according to any one of [1] to [3]. [5] A nanopore sensing method using the nanopore protein according to any one of [1] to [2], or the nanopore sensor according to [4]. [6] A nanopore protein suspension comprising a nanopore protein and a surfactant in an amount sufficient to stabilize the nanopore protein. Effect of the Invention

[0010] The nanopore protein of the present invention is likely to form oligomers and / or large-sized pores, and therefore can be suitably used in nanopore sensing systems. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows the three-dimensional structure of ILY. [Diagram 2] The first half of the figure shows the results of an alignment of CDC family proteins, with amino acids conserved among the CDC family members shown in white letters on a black background. [Diagram 3] The second half of the figure shows the results of an alignment of CDC family proteins, with amino acids conserved among the CDC family members shown in white letters on a black background. [Figure 4A] FIG. 1 shows the design of various stabilized mutants of ILY. [Figure 4B] FIG. 1 shows the design of various ILY mutants that improve the probability of opening large pores. [Diagram 5] FIG. 1 shows the results of evaluating the stability of various ILY mutants (lysates) by electrophoresis. [Figure 6] FIG. 1 shows the results of evaluating the stability of various ILY mutants (purified proteins) by electrophoresis. [Figure 7] FIG. 1 shows the results of comparing pore stability between WT and mut9. [Figure 8] FIG. 1 shows the results of investigating the promotion of ILY oligomerization by surfactants. [Figure 9] FIG. 1 shows the results of comparing the pore stability of WT and an ILY mutant directed toward the formation of large diameter pores. [Figure 10A] FIG. 1 shows the probability of opening pores larger than or smaller than 10 nm for WT and ILY mutants directed towards the formation of large diameter pores. [Figure 10B] FIG. 1 shows the results of comparing the diameter of pores formed between WT and mut32. [Figure 11] This figure shows the electrical signals observed in a nanopore sensing system using mut32 when dCas9 / sgRNA / cesA was not added (A) and was added (B). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] (Nanopore Protein) Nanopore protein means a protein capable of forming a nanopore. Wild-type nanopore proteins exist on cell membranes and are responsible for transporting substances in and out of cells by forming small holes (nanopores) through which water-soluble molecules can pass. The nanopore protein of the present invention is a nanopore protein that is a mutant protein of a cholesterol-dependent pore-forming cytolytic toxin (CDC) family protein that contains at least one of the following mutations (1) to (3). (1) Any one of the following substitutions (a) to (g) in the amino acid sequence set forth in SEQ ID NO:1: (a) Substitution of the amino acid residue at the position corresponding to arginine at position 302 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (b) substitution of the amino acid residue at the position corresponding to histidine at position 242 of a CDC family protein other than the proteins having the amino acid sequences set forth in SEQ ID NOs: 48, 51, and 55 with leucine, phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, isoleucine, methionine, or proline. (c) substitution of the amino acid residue at the position corresponding to threonine at position 334 of a CDC family protein other than the proteins having the amino acid sequences set forth in SEQ ID NOs: 49, 53, and 15 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (d) Substitution of the amino acid residue at the position corresponding to valine at position 241 with glutamic acid, arginine, histidine, lysine, aspartic acid, glycine, serine, threonine, asparagine, or glutamine. (e) Substitution of the amino acid residue at the position corresponding to threonine at position 315 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (f) Substitution of the amino acid residue at the position corresponding to asparagine 338 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (g) Substitution of the amino acid residue at the position corresponding to isoleucine at position 339 with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, glycine, threonine, asparagine, or glutamine. (2) A mutation in which 5 to 23 consecutive amino acids including Y196-H200 in N178-H200 in the amino acid sequence set forth in SEQ ID NO: 1 are deleted, or amino acid residues at positions corresponding to 2 to 13 consecutive amino acids including T517-L518 in W514-V526 are deleted. (3) A mutation in which an amino acid residue is inserted at a position corresponding to 2 to 25 consecutive amino acid residues, including T517-L518, of Q508-D532 in the amino acid sequence set forth in SEQ ID NO: 1, at the C-terminus of the amino acid sequence set forth in SEQ ID NO: 1, or a mutation in which an amino acid residue is inserted at a position corresponding to 1 to 24 consecutive amino acid residues, including R495, of R480-L503 in the amino acid sequence set forth in SEQ ID NO: 1, between amino acid residues at positions corresponding to L503 and P504 in the amino acid sequence set forth in SEQ ID NO: 1

[0013] Regarding the mutation (1) above, the amino acid residues at the positions corresponding to R302, H242, T334, V241, T315, N338, or I339 in the amino acid sequence set forth in SEQ ID NO:1 are R302F, R302M, R302L, R302I, H242L, H242I, H242Y, T334V, T334I, V241T, V241S, V241K, V241E, T315V, T315I, N338L, N33 The mutation may be due to substitution with 8F, I339T, I339S or I339K, and it is preferable that the amino acid residue at the position corresponding to R302, H242, T334, V241, T315 or I339 in the amino acid sequence set forth in SEQ ID NO: 1 is mutated by substitution with R302F, H242L, H242I, H242F, T334V, T334I, V241E, T315V, T315I or I339S.

[0014] The nanopore protein means a membrane protein that forms a nanopore on a cell membrane. The nanopore means a hole on the nano-order, and the diameter (diameter or major axis) of the pore may be, for example, 1 nm to 100 nm, 1 nm to 50 nm, or 1 nm to 30 nm.

[0015] Nanopore proteins include outer membrane proteins OmpG and OmpA, and also include membrane proteins belonging to the cholesterol-dependent pore-forming cytolysin family (CDC family). The nanopore protein of the present invention includes mutant proteins of CDC family proteins. Examples of CDC family proteins include, but are not limited to, Intermedilysin, Perfringolysin O, Listeriolysin O, Lectinolysin, Anthrolysin O, Streptolysin O, Suilysin, Vaginolysin, Alveolysin, Ivanolysin, Seeligeriolysin, Pneumolysin, and the like.

[0016] The origin of the wild-type CDC family protein is not particularly limited, and examples thereof include Intermedilysin (SEQ ID NO: 1) derived from Streptococcus intermedius, Perfringolysin O (SEQ ID NO: 48) derived from Clostridium perfringens, Listeriolysin O (SEQ ID NO: 49) derived from Listeria monocytogenes, Lectinolysin (SEQ ID NO: 50) derived from Streptococcus mitis, Anthrolysin O (SEQ ID NO: 51) derived from Bacillus anthracis, Streptolysin O (SEQ ID NO: 52) derived from Streptococcus pyogenes, Suilysin (SEQ ID NO: 53) derived from Streptococcus suis, Vaginolysin (SEQ ID NO: 54) derived from Gardnerella vaginalis, Alveolysin (SEQ ID NO: 55) derived from Paenibacillus alvei (Bacillus alvei), Ivanolysin (SEQ ID NO: 56) derived from Listeria ivanovii, and the like. seeligeri-derived Seeligeriolysin (SEQ ID NO: 15), and Streptococcus pneumoniae-derived Pneumolysin (SEQ ID NO: 38). The alignment results of these CDC family proteins are shown in Figures 2 and 3. The sequence homology of these CDC family proteins is as follows: [Table 1]

[0017] The mutant proteins of the CDC family proteins may be mutant proteins of these wild-type CDC family proteins, and are preferably mutant proteins of Intermedilysin (ILY) derived from Streptococcus intermedius.

[0018] When an amino acid substitution occurs due to the introduction of a mutation into a gene encoding a nanopore protein, the substitution is preferably a substitution of an amino acid residue before substitution with an amino acid residue having different chemical / physical properties. That is, the amino acid substitution is preferably a substitution of an amino acid residue in a wild-type protein with an amino acid residue selected from a group of amino acid residues having different chemical / physical properties but similar chemical / physical properties. Examples of similar chemical / physical properties include hydrophilicity and hydrophobicity. Non-Patent Document 12 shows a hydrophobicity scale (amount of free energy change in the transition from water to a lipid bilayer) derived from an experiment on outer membrane phospholipase A, a lipid bilayer transmembrane protein. Based on this hydrophobicity scale, phenylalanine, leucine, isoleucine, proline, tyrosine, valine, methionine, tryptophan, alanine and cysteine ​​are considered hydrophobic (lipid bilayer-friendly) amino acids, while lysine, histidine, arginine, asparagine, glutamine, aspartic acid, serine, threonine, glycine and glutamic acid are considered hydrophilic (water-friendly) amino acids.

[0019] The mutation (1) is any one of the above substitutions (a) to (g) in the amino acid sequence described in SEQ ID NO: 1. (a) The substitution of the amino acid residue at the position corresponding to arginine (R) at position 302 in the amino acid sequence described in SEQ ID NO: 1 is a substitution with phenylalanine (F), tyrosine (Y), tryptophan (W), alanine (A), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M) or proline (P), preferably with phenylalanine (F), leucine (L), isoleucine (I) or methionine (M). Arginine (R) is a hydrophilic amino acid, and it is considered that by substituting it with a hydrophobic amino acid, the properties of the nanopore protein change and it becomes easier to form oligomers.

[0020] (b) The substitution of the amino acid residue at the position corresponding to histidine (H) at position 242 in the amino acid sequence set forth in SEQ ID NO: 1 is with leucine (L), phenylalanine (F), tyrosine (Y), tryptophan (W), alanine (A), cysteine ​​(C), valine (V), isoleucine (I), methionine (M) or proline (P), preferably with leucine (L), tyrosine (Y) or isoleucine (I). Histidine (H) is a hydrophilic amino acid, and it is believed that by substituting it with a hydrophobic amino acid, the properties of the nanopore protein change, making it easier to form oligomers.

[0021] (c) The substitution of the amino acid residue at the position corresponding to threonine (T) at position T334 in the amino acid sequence set forth in SEQ ID NO: 1 is with phenylalanine (F), tyrosine (Y), tryptophan (W), alanine (A), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M) or proline (P), preferably with valine (V) or isoleucine (I). Threonine (T) is a hydrophilic amino acid, and it is believed that by substituting it with a hydrophobic amino acid, the properties of the nanopore protein change, making it easier to form oligomers.

[0022] (d) The substitution of the amino acid residue at the position corresponding to valine (V) at position V241 in the amino acid sequence set forth in SEQ ID NO: 1 is with glutamic acid (E), arginine (R), histidine (H), lysine (K), aspartic acid (D), glycine (G), serine (S), threonine (T), asparagine (N) or glutamine (Q), preferably with glutamic acid (E), lysine (K), serine (S) or threonine (T). Valine (V) is a hydrophobic amino acid, and it is believed that by substituting it with a hydrophilic amino acid, the properties of the nanopore protein change, making it easier to form oligomers.

[0023] (e) The substitution of the amino acid residue at the position corresponding to threonine (T) at position 315 in the amino acid sequence set forth in SEQ ID NO: 1 is with phenylalanine (F), tyrosine (Y), tryptophan (W), alanine (A), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M) or proline (P), preferably with valine (V) or isoleucine (I). Threonine (T) is a hydrophilic amino acid, and it is believed that by substituting it with a hydrophobic amino acid, the properties of the nanopore protein are changed, making it easier to form oligomers.

[0024] (f) The substitution of the amino acid residue at the position corresponding to asparagine (N) at position 338 in the amino acid sequence set forth in SEQ ID NO: 1 is with phenylalanine (F), tyrosine (Y), tryptophan (W), alanine (A), cysteine ​​(C), valine (V), leucine (L), isoleucine (I), methionine (M) or proline (P), preferably with phenylalanine (F) or leucine (L). Asparagine (N) is a hydrophilic amino acid, and it is believed that by substituting it with a hydrophobic amino acid, the properties of the nanopore protein are changed, making it easier to form oligomers.

[0025] (g) The substitution of the amino acid residue at the position corresponding to isoleucine (I) at position I339 in the amino acid sequence set forth in SEQ ID NO: 1 is with serine (S), arginine (R), histidine (H), lysine (K), aspartic acid (D), glutamic acid (E), glycine (G), threonine (T), asparagine (N) or glutamine (Q), preferably with lysine (K), serine (S) or threonine (T). Arginine (R) is a hydrophobic amino acid, and it is believed that by substituting it with a hydrophilic amino acid, the properties of the nanopore protein change, making it easier to form oligomers.

[0026] Here, the position corresponding to R302 in the amino acid sequence described in SEQ ID NO: 1 means the position of R302 in the amino acid sequence described in SEQ ID NO: 1 in the case of a mutation in an intermedilysin consisting of the amino acid sequence described in SEQ ID NO: 1. In the case of a mutation in a CDC family protein other than an intermedilysin consisting of the amino acid sequence described in SEQ ID NO: 1, it means the position corresponding to R302 in the amino acid sequence described in SEQ ID NO: 1 when the amino acid sequence of the CDC family protein and the amino acid sequence described in SEQ ID NO: 1 are aligned using a known algorithm. The same applies to amino acid residues at other positions. By aligning the amino acid sequences of the CDC family proteins in this manner, it is possible to determine the corresponding positions in the amino acid sequence of each CDC family protein based on the amino acid sequence of intermedilysin shown in SEQ ID NO: 1, regardless of insertions or deletions in the amino acid sequence. The corresponding positions are considered to be present at the same positions in the three-dimensional structure, and it can be presumed that they have similar effects on the function of the target CDC family protein.

[0027] Specifically, in the present invention, "a position corresponding to position 241 in the amino acid sequence of SEQ ID NO: 1" refers to a position corresponding to position 241 in SEQ ID NO: 1 when the amino acid sequence of a target CDC family protein is compared with the amino acid sequence of SEQ ID NO: 1. This can be identified from Figures 2 and 3 in which the amino acid sequences are aligned by the above-mentioned method. The amino acids at positions corresponding to positions 241, 242, 302, 315, 334, 338, and 339 in SEQ ID NO: 1 in each CDC family protein are as shown in the following table. [Table 2]

[0028] In the nanopore protein of the present invention, it is preferable that the amino acid residue at a position corresponding to R302, H242, T334, V241, T315 or I339 in the amino acid sequence set forth in SEQ ID NO: 1 is mutated by substitution with R302F, H242L, H242I, H242Y, T334V, T334I, V241E, T315V, T315I or I339S, and it is more preferable that the amino acid residue at a position corresponding to R302, H242, V241 or I339 in the amino acid sequence set forth in SEQ ID NO: 1 is mutated by substitution with R302F, H242L, H242F, V241E or I339S.

[0029] The mutation (2) is a mutation in which 5 to 23 consecutive amino acids including Y196-H200 in N178-H200 in the amino acid sequence shown in SEQ ID NO: 1 are deleted, or amino acid residues at positions corresponding to 2 to 13 consecutive amino acids including T517-L518 in W514-V526 are deleted. Here, the 5 to 23 consecutive amino acids including Y196-H200 in N178-H200 in the amino acid sequence shown in SEQ ID NO: 1 refer to consecutive amino acid residues present between N178-H200, and mean 5 to 23 consecutive amino acid residues including Y196-H200, and the length of the amino acids may be, for example, 5 to 20, 5 to 15, or 5 to 10. Of W514-V526 in the amino acid sequence of SEQ ID NO: 1, 2 to 13 consecutive amino acids including T517-L518 refer to consecutive amino acid residues present between T517-L518, and 2 to 13 consecutive amino acid residues including T517-L518, and the length of the amino acids may be, for example, 2 to 10, 2 to 8, or 2 to 5. The amino acid residues at positions corresponding to the amino acid residues in the amino acid sequence of SEQ ID NO: 1 described herein are understood in the same manner as the respective amino acid residues in (1) above.

[0030] As shown in the Example (mut2), when 13 consecutive amino acid residues of L188-H200 in the amino acid sequence described in SEQ ID NO: 1 are deleted, the stability of the mutant nanopore protein is improved. The L188-H200 region of intermedilysin having the amino acid sequence described in SEQ ID NO: 1 is a region that occupies half of the helical structure of the helical region in which it exists, and among them, Y196-H200 is a region that corresponds to one turn of the helix. Therefore, in the mutant CDC family protein of the present invention, even if amino acid residues at positions corresponding to 5 to 23 consecutive amino acids including Y196-H200 in N178-H200 in the amino acid sequence described in SEQ ID NO: 1 are deleted, it is expected that the stability of the mutant nanopore protein can be improved as in mut2. Examples of 5 to 23 consecutive amino acids including Y196-H200 in the amino acid sequence described in SEQ ID NO: 1 include Y196-H200, L188-H200, and N178-H200. In addition, any region containing amino acid residues located at positions corresponding to 5 to 23 consecutive amino acids including Y196-H200 in the amino acid sequence set forth in SEQ ID NO: 1 may be used, and a deletion of consecutive amino acid residues longer than N178-H200 may also be used as long as it does not impair the function of the nanopore protein.

[0031] As shown in the Example (mut18), when 13 consecutive amino acid residues of W514-V526 in the amino acid sequence described in SEQ ID NO: 1 are deleted, the stability of the mutant nanopore protein is improved. According to Non-Patent Document 13, it is known that the LT pair (corresponding to T517-L518 in the amino acid sequence described in SEQ ID NO: 1) in the L1 loop of the D4 region (corresponding to T516-H519 in the amino acid sequence described in SEQ ID NO: 1), which is a motif common to the CDC family, is involved in the recognition and binding of cholesterol in a lipid bilayer membrane. Therefore, in the mutant CDC family protein of the present invention, even if the amino acid residues at the positions corresponding to 2 to 13 consecutive amino acids including T517-L518 in the amino acid sequence described in SEQ ID NO: 1 in W514-V526 are deleted, it is expected that the stability of the mutant nanopore protein can be improved as in mut18. Examples of the 2 to 13 consecutive amino acids including T517-L518 in the amino acid sequence set forth in SEQ ID NO: 1 include T517-L518, T516-H519 (L1 loop), and W514-V526. The deleted region may be any region including amino acid residues at positions corresponding to the 2 to 13 consecutive amino acids including T517-L518 in the amino acid sequence set forth in SEQ ID NO: 1, and may be a deletion of consecutive amino acid residues longer than W514-V526 as long as it does not impair the function of the nanopore protein.

[0032] In the present invention, the "position corresponding to position 178 in the amino acid sequence of SEQ ID NO:1" can be identified by the amino acid sequence alignments in Figures 2 and 3. The amino acids at positions corresponding to positions 178, 188, 196, 200, 514, 516, 517, 518, 519, and 526 in SEQ ID NO:1 in each CDC family protein are as shown in the following table. [Table 3] [Table 4]

[0033] A mutant protein of a CDC family protein containing the mutation (1) or (2) is more likely to form oligomers than a wild-type CDC family protein, and can provide a stable pore protein.

[0034] The mutation (3) is a mutation in which amino acid residues are inserted at a position corresponding to 2 to 25 consecutive amino acid residues including T517-L518 in Q508-D532 in the amino acid sequence of SEQ ID NO: 1 at the C-terminus of the amino acid sequence of SEQ ID NO: 1, or a mutation in which amino acid residues are inserted at a position corresponding to 1 to 24 consecutive amino acid residues including R495 in R480-L503 in the amino acid sequence of SEQ ID NO: 1 between amino acid residues at positions corresponding to L503 and P504 in the amino acid sequence of SEQ ID NO: 1. Here, the 2 to 25 consecutive amino acid residues including T517-L518 in Q508-D532 in the amino acid sequence of SEQ ID NO: 1 refer to consecutive amino acid residues present between Q508-D532, and mean 2 to 25 consecutive amino acid residues including T517-L518, and the length of the amino acids may be, for example, 2 to 20, 2 to 15, 2 to 10, or 2 to 5. The 1 to 24 amino acid residues including R495 among R480-L503 in the amino acid sequence shown in SEQ ID NO: 1 refer to consecutive amino acid residues present between R480-L503, and 1 to 24 consecutive amino acid residues including R495, and the length of the amino acids may be, for example, 1 to 20, 1 to 15, 1 to 10, or 1 to 5. The amino acid residues at positions corresponding to the amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 described here are understood in the same way as the respective amino acid residues in (1) above.

[0035] As shown in the Example (mut29), when 25 consecutive amino acid residues of Q508-C-terminus (D532) in the amino acid sequence described in SEQ ID NO: 1 are inserted into the C-terminus of the amino acid sequence described in SEQ ID NO: 1, the resulting mutant nanopore protein can open a pore of 10 nm or more with a higher probability than the wild type. As described above, according to Non-Patent Document 13, it is known that the LT pair (corresponding to T517-L518 in the amino acid sequence described in SEQ ID NO: 1) in the L1 loop of the D4 region (corresponding to T516-H519 in the amino acid sequence described in SEQ ID NO: 1), which is a motif common to the CDC family, is involved in the recognition and binding of cholesterol in a lipid bilayer membrane. Therefore, in the mutant CDC family protein of the present invention, it is expected that the stability of the mutant nanopore protein can be improved similarly to mut29 even if amino acid residues at positions corresponding to 2 to 25 consecutive amino acid residues including T517-L518 in Q508-D532 in the amino acid sequence described in SEQ ID NO: 1 are inserted. Examples of the 2 to 25 consecutive amino acid residues including T517-L518 in Q508-D532 in the amino acid sequence shown in SEQ ID NO: 1 include T517-L518, T516-H519 (L1 loop), and Q508-D532. The region to be inserted may be any region including amino acid residues at positions corresponding to the 2 to 25 consecutive amino acid residues including T517-L518 in Q508-D532 in the amino acid sequence shown in SEQ ID NO: 1, and may be an insertion of consecutive amino acid residues longer than Q508-D532 as long as it does not impair the function of the nanopore protein.

[0036] As shown in the Example (mut32), when 24 consecutive amino acid residues of R480-L503 in the amino acid sequence of SEQ ID NO: 1 are inserted into the C-terminus of the amino acid sequence of SEQ ID NO: 1, the resulting mutant nanopore protein can open a pore of 10 nm or more with a higher probability than the wild type. According to Non-Patent Document 14, it is known that the undecapeptideloop (corresponding to G485-495R in the amino acid sequence of SEQ ID NO: 1) in the D4 region and R in the loop (corresponding to R495 in the amino acid sequence of SEQ ID NO: 1) affect the binding to the membrane and the membrane penetrating activity. Therefore, in the mutant CDC family protein of the present invention, it is expected that the stability of the mutant nanopore protein can be improved similarly to mut32 even if amino acid residues are inserted at positions corresponding to 1 to 24 consecutive amino acid residues including R495 in R480-L503 in the amino acid sequence of SEQ ID NO: 1. Examples of 1 to 24 consecutive amino acid residues including R495 among R480-L503 in the amino acid sequence set forth in SEQ ID NO: 1 include 495R (R in the loop), 485G-495R (membrane contact loop motif called undecapeptide), and R480-L503. Note that the region to be inserted may be any region including amino acid residues at positions corresponding to 1 to 24 consecutive amino acid residues including R495 among R480-L503, and insertion of consecutive amino acid residues longer than R480-L503 may be possible as long as it does not impair the function of the nanopore protein.

[0037] In the present invention, the "position corresponding to position 480 in the amino acid sequence of SEQ ID NO:1" can be identified from the amino acid sequence alignments in Figures 2 and 3. The amino acids at positions corresponding to positions 480, 485, 495, 503, 504, 508, and the C-terminus (position 532) of SEQ ID NO:1 in each CDC family protein are as shown in the following table. [Table 5]

[0038] A mutant protein of a CDC family protein containing the mutation (3) can provide a pore protein having a higher probability of opening a large-sized pore than a wild-type CDC family protein. Here, a large-sized pore means a pore having a pore size (diameter) of 10 nm or more, and may be, for example, 10 nm to 50 nm, 10 nm to 40 nm, 10 nm to 30 nm, 10 nm to 20 nm, or 10 nm to 15 nm.

[0039] The nanopore protein of the present invention can be obtained by transforming a nucleic acid (e.g., DNA) encoding the nanopore protein into an appropriate host cell and expressing it. For example, in the case of intermedilysin, the base sequence of the nucleic acid encoding Streptococcusintermedius-derived intermedilysin (SEQ ID NO: 1) is shown in SEQ ID NO: 2. A nucleic acid encoding an intermedilysin having a predetermined mutation can be prepared by a method known to those skilled in the art. For example, as described in the Examples, a nucleic acid having the base sequence shown in SEQ ID NO: 2 can be used as a template to prepare a nucleic acid encoding a mutant protein having a desired mutation using a primer designed to have a mutation at a predetermined position, and a nucleic acid encoding a mutant protein can also be prepared by artificial synthesis.

[0040] The nucleic acid can be inserted into an appropriate expression vector and transformed into a host cell by a method known to those skilled in the art. Examples of vectors include, but are not limited to, pET22b+ (Sigma-Aldrich), pBluescriptII KS(+), pPCR-Script Amp SK(+) (Agilent Technologies), pT7Blue (Merck Millipore), pCRII (Thermo Fisher Scientific), and pCR-TRAP (Gene Hunter). The nucleic acid can also be directly inserted into the chromosome of the host organism, for example, by using homologous recombination. The host cell is not particularly limited, and examples thereof include Escherichia coli, Corynebacterium grutamicum, Brevibacillus choshinensis, Streptomyces violaceoruber, yeasts of the genus Zygosaccharomyces, Schizosaccharomyces, Saccharomyces, yeasts of the genus Pichia, yeasts of the genus Candida, filamentous fungi of the genus Aspergillus oryzae, and filamentous fungi of the genus Trichoderma.

[0041] A surfactant may be used for eluting and recovering the pore protein expressed from the host cell. The surfactant may be a nonionic surfactant or an amphoteric surfactant. As surfactants, BIGCHAP(N,N-Bis(3-D-gluconamidopropyl)cholamide), deoxy-BIGCHAP(N,N-Bis(3-D-gluconamidopropyl)cholamide) l)deoxycholamide), N-Octyl-β-D-glucopyranoside, N-Octyl-β-D-maltopyranoside, N-Decyl-β-D-maltopyranoside , N-Dodecyl-β-D-maltopyranoside, 3-Oxatridecyl-α-D-mannopyranoside, n-Heptyl-β-D-thioglucopyranoside, n-Octyl-β-D-thioglucopyranoside, n-Nonyl-β-D-thiomaltopyranoside, α-D-Glucopyranosyl-α-D-glucopyranoside Examples of such surfactants include nonionic surfactants such as monododecanoate, α-D-Glucopyranosyl-α-D-glucopyranoside monomyristate, n-Octanoyl-N-methyl-D-glucamine, n-Nonanoyl-N-methyl-D-glucamine, and n-Decanoyl-N-methyl-D-glucamine, and zwitterionic surfactants such as CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonate), CHAPSO (3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxypropanesulfonate), Sulphobetaine-8, Sulphobetaine-10, Sulphobetaine-12, Sulphobetaine-14, and Sulphobetaine-16.

[0042] The present inventors have found that the presence of a surfactant in a lysate obtained by adding a surfactant to a host cell causes oligomerization of the nanopore protein. Therefore, the lysate may be directly applied to a nanopore sensing system, or the nanopore protein may be purified from the lysate and then an effective amount of a surfactant capable of stabilizing the nanopore protein may be added to the resulting protein suspension.

[0043] Nanopore proteins can be purified using commercially available protein purification kits, purification columns, extraction, precipitation, ultracentrifugation, and the like.

[0044] The nanopore protein of the present invention can be used as a nanopore sensor, as described below, and therefore can be used in a nanopore sensing system.

[0045] (Nanopore sensor) The nanopore sensor of the present invention includes the nanopore protein of the present invention. The nanopore sensor of the present invention may include only the nanopore protein, or may include a lipid bilayer membrane that mimics a cell membrane or a polymer in addition to the nanopore protein, and may further include a medium for holding the nanopore protein and the lipid bilayer membrane, and an additive for stabilizing the nanopore protein and the lipid bilayer membrane.

[0046] The nanopore sensor of the present invention can detect minute objects, such as metal ions, small molecules, nucleic acids (DNA or RNA), proteins, bacteria, and viruses. The size of the nanopore protein used varies depending on the object to be detected, but the nanopore sensor of the present invention has a pore size of 1 nm to 50 nm, and therefore can be suitably used to detect a wide range of biomolecules. The nanopore sensor of the present invention can be suitably used to detect biomolecules, such as nucleic acids and proteins, particularly at the single molecule level.

[0047] It is preferable that the nanopore sensor embeds a nanopore protein corresponding to the sensor portion (detection portion) in the lipid bilayer membrane. The mechanism of detecting a minute object is, for example, as described in Non-Patent Document 8, when a voltage is applied to both sides of the lipid bilayer membrane, a current flows. When the test substance is caught in the nanopore (opening) of the nanopore protein, the current value in the nanopore temporarily decreases (blocking), and when the test substance passes through or is kicked out of the nanopore, the current value is restored, thereby enabling the presence of the target molecule to be detected. A small detection target has a small change in the current value when passing through, and a large detection target has a large change in the current value when passing through. Therefore, not only can the presence of the detection target be detected by the presence or absence of a change in the current value, but when the test substance is a mixture of multiple detection targets of different sizes, the presence of small or large minute objects can be distinguished. In addition, by selecting a pore of about the same diameter as the detection target, the time of residence in the pore can be increased, and the detection sensitivity can be increased. The identity of the test substance is determined by the time and degree of current block, as well as the change in the current level.

[0048] The method of embedding a nanopore protein in a lipid bilayer membrane is not particularly limited, and for example, a nanopore protein can be added to an artificially synthesized lipid bilayer membrane, which causes the nanopore protein to spontaneously associate and insert into the membrane. The method of forming a lipid bilayer membrane is not particularly limited, and for example, a lipid bilayer membrane formation method using a droplet contact method includes a method of forming a lipid bilayer membrane by inserting a silver-silver chloride microelectrode with a cavity filled with an aqueous solution at the tip into the interface of a solution in which a lipid solution and an aqueous solution are layers (Non-Patent Document 9), and other methods using microdevices (Non-Patent Documents 5 and 10).

[0049] One embodiment provides a kit for nanopore sensing, comprising a nanopore protein of the invention and a lipid bilayer membrane or a component thereof.

[0050] (Nanopore sensing method) The nanopore sensing method is a nanopore sensing method using the nanopore protein of the present invention or the nanopore sensor of the present invention.

[0051] The nanopore sensing method is a highly sensitive molecular analysis method for detecting and measuring minute objects. The nanopore sensing method is a sensing technology for electrically measuring minute objects passing through the nanopore of a nanopore protein, and is specifically described in, for example, Non-Patent Documents 6, 8, and 11.

[0052] The nanopore sensing method may include a step of preparing a nanopore sensing system including a nanopore sensor and an electrical detection device (preparation step), a step of adding a test substance to the nanopore sensing system (addition step), a step of measuring the current value of the nanopore sensing system by the electrical detection device (measurement step), and a step of determining that the test substance contains the micro object to be measured when the current value after the addition of the test substance is substantially different from that before the addition (determination step). The nanopore sensor is as described above.

[0053] Examples of electrical detection devices include PICO2 (manufactured by Tesla Corporation) and Orbit (manufactured by Nanion Technologies, Inc.) and the like. An appropriate device can be selected depending on the size of the nanopore sensing system and the minute object to be measured.

[0054] (Nanopore Protein Suspension) The nanopore protein suspension of the present invention comprises a nanopore protein, which may be a membrane protein or a transmembrane toxin, and a surfactant in an amount sufficient to stabilize the nanopore protein, which may be a wild-type or mutant nanopore protein. Examples of nanopore proteins include Aerоlysin, MspA, ОmpG, VDAC, SP1, Escherichia coli-derived cytolysin (Colicin E1, Ia, A, N), phi29, FhuA, CsgG, Fragaceatoxin C, TatA, GspD2, Perforin-1, Equinatoxin II, Enterolobin, CDC family (Streptolysin O, Pneumolysin, Perfringolysin O, Intermedilysin, Listeriolysin O, Vaginolysin, alveolysin, Ivanolysin, Lectinolysin, Seeligeriolysin, Suilysin), and mutant proteins thereof.

[0055] The surfactant is as described above. The effective amount of the surfactant capable of stabilizing the nanopore protein can be appropriately selected by those skilled in the art depending on the type of surfactant and the content of the nanopore protein. For example, sulfobetaine-14 or dodecylmaltoside is 0.1 mM to 10 mM, and SDS is 0.1 mM to 80 mM. The presence of the surfactant allows the nanopore protein to be stably present in the suspension. EXAMPLES

[0056] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0057] 1. Design of Intermedilysin (ILY) Mutants ILY exhibits three conformations as shown in Figure 1. We hypothesized that the introduction of amino acid mutations that stabilize the prepore and pore structures of ILY, or the removal of motifs that act as steric hindrances to the interaction between monomers, might affect the interaction between monomers and, therefore, the stability of the pore.

[0058] To verify the hypothesis, we designed a mutant as shown in Figure 4A. First, since R302 of Streptococcusintermedius-derived intermedilysin (ILY) (SEQ ID NO: 1) is located in a hydrophobic field inside the monomer, this was mutated to hydrophobic amino acid residues (R302F, R302M, R302L, R302I) based on the hydrophobicity scale (Non-Patent Document 12) to stabilize the prepore structure and pore structure by amplifying the hydrophobic interaction (Figure 4A(a)). Next, since it is thought that amino acid residues that are thought to be in contact with the membrane are more stable if they are hydrophobic, the hydrophilic amino acid residues located in the corresponding parts were mutated to hydrophobic amino acid residues based on the hydrophobicity scale (Non-Patent Document 12). (S240L, H242L, H242I, H242F, H242Y, T314V, T315I, T334V, T334I, N338L, N338F; Fig. 4A(b)). In addition, since it is believed that the amino acid residues that are likely to be in contact with the aqueous solution portion are more stable if they are hydrophilic, the hydrophobic amino acid residues located in the corresponding portions were mutated to hydrophilic amino acid residues based on the hydrophobicity scale (Non-Patent Document 12) (V241T, V241S, V241K, V241E, I339T, I339S, I339K, I339E; Fig. 4A(b)). Finally, we designed mutants in which a part of the helix that could sterically hinder the interaction between monomers was deleted (ΔL188-H200) and mutants in which the C-terminus was deleted (ΔW514-V526, ΔQ508-V526) (Figure 4A(c)).

[0059] On the other hand, although the CDC family including ILY is known to form a relatively large pore of several tens of nm (Non-Patent Document 1), large pores are not always formed, and pores of various sizes from several nm to several tens of nm may be formed. Therefore, the present inventors aimed at a protein nanopore sensing system using ILY, and designed an ILY mutant with increased motifs present in the membrane contact region in the prepore structure and the membrane-spanning region in the pore structure to examine whether the probability of opening a large pore can be increased by modifying ILY (Figure 4B). According to Non-Patent Document 13, it is known that the LT pair (corresponding to T517-L518 in ILY) in the L1 loop (corresponding to T516-H519 in ILY) of the D4 region, which is a motif common to the CDC family, is involved in the recognition and binding of cholesterol in a lipid bilayer membrane. Therefore, it was decided to insert amino acid residues at a position corresponding to Q508-D532 including the LT pair and the L1 loop. In addition, according to Non-Patent Document 14, it is known that the undecapeptide loop of the D4 region (corresponding to G485-495R in ILY) and R in the loop (corresponding to R495 in ILY) affect membrane binding and membrane penetrating activity. Therefore, we decided to insert amino acid residues at a position corresponding to R480-L503, including R495 and the undecapeptide loop.

[0060] 2. Construction of Plasmids for Expression of ILY Mutants A gene (SEQ ID NO: 2) encoding Streptococcus intermedius-derived intermedilysin (ILY) (SEQ ID NO: 1) was cloned into a plasmid vector pET22b+ (Sigma-Aldrich) to obtain a plasmid for expressing wild-type ILY (designated "pET22b+-ILY"). The expression plasmids for ILY mutants were prepared by PCR using a predetermined primer set and pET22b+-ILY as a template. The names of each mutant, the mutation sites, and the primer sets of the forward primer (primer Fw) and reverse primer (primer Rv) used to prepare each mutant are shown in Table 6. A gene sequence encoding a His tag sequence for protein purification was placed at the 3' end of the ILY mutant gene. [Table 6]

[0061] KOD One PCR Master Mix (Toyobo) was used for PCR, and PCR conditions were determined according to the three-step cycle in the attached manual (denaturation was performed at 98°C for 10 seconds, annealing was performed at Tm-5°C for each primer for 5 seconds, and extension was performed at 68°C for 40 seconds). The number of PCR cycles was 6.

[0062] 1.0 μL of DpnI (New England Biolab) was added to the solution after PCR, and the mixture was treated at 37° C. for 1 hour. 2.5 μL of Ligation high Ver.2 (Toyobo Co., Ltd.), 0.5 μL of T4 polynucleotide Kinase (5 U / μL) (Toyobo Co., Ltd.), and 3.5 μL of MilliQ water were added to 1.0 μL of the reaction solution treated with DpnI, and the mixture was treated at 16° C. for 1 hour. E. coli JM109 strain was transformed with the obtained plasmid and seeded on LB agar medium containing ampicillin. The grown colony was inoculated into LB liquid medium (LB-amp medium) containing 50 μg / mL of ampicillin, and cultured with shaking, and the plasmid was isolated using FastGene Plasmid Mini Kit (Nihon Genetics Co., Ltd.). The base sequence of the region encoding the ILY mutant in the plasmid was analyzed by entrusting it to FASMAC Co., Ltd., and it was confirmed that the plasmid for expressing the target mutant was correctly constructed.

[0063] 3. Recombinant Expression of ILY Mutants The ILY mutant recombinant production strain, prepared by transforming E. coli BL21 with the ILY mutant expression plasmid, was inoculated into 5.0 mL of medium (50 μg / mL ampicillin, 1% tripeptone, 0.5% yeast extract, 25 mM (NH4)2SO4, 50 mM KH2PO4, 50 mM Na2HPO4, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 1 mM magnesium sulfate) and cultured in a test tube with shaking at 25°C for 24 hours.

[0064] 4. Interaction Analysis between Monomers of ILY Mutants by Polyacrylamide Gel Electrophoresis and Western Blotting After the culture, the cells were collected by centrifugation at 8,000×g for 10 minutes. The pellet was mixed with 0.5 ml of BugBuster (Merck) containing Benzonaze ​​nuclease (Millipore), Lysozyme (Millipore), and protease inhibitor cocktail (Roche) at the recommended concentrations, and incubated at room temperature for 20 minutes to lyse the cells. After centrifugation at 8,000×g for 10 minutes, the supernatant was used for analysis. 3 μl of the supernatant, 5 μl of 2×buffer (280 mM KCl, 20 mM DTT, 20 mM MOPS, pH 7), and 2 μl of MilliQ were mixed, and a portion was incubated at 37°C for 17 hours. Then, 2.5 μl of SDS sample buffer was added, and the mixture was applied to a 6% polyacrylamide gel and electrophoresed at 4°C and 30 mA for 46 minutes. The gel was blotted at 70 mA at 4°C for 80 minutes, and then monomeric mutants were detected using a primary antibody (Abcam, Biotin Anti-6xHis tag antibody), a secondary antibody (Vector Laboratories, streptavidin-HRP), and a detection reagent (Cytiva).

[0065] 5. Purification of ILY Mutants E. coli BL21(DE3) transformed with a plasmid carrying the gene for the monomer mutant was inoculated into 100 ml of medium (50 μg / mL ampicillin, 1% tripeptone, 0.5% yeast extract, 25 mM (NH4)2SO4, 50 mM KH2PO4, 50 mM Na2HPO4, 0.5% glycerol, 0.05% glucose, 0.2% α-lactose, 1 mM magnesium sulfate) placed in a shake flask and cultured for 24 hours with shaking at 25 °C. The mixture was then centrifuged at 8,000 × g for 10 minutes to collect the bacteria. The pellet of 50 ml of culture was mixed with 5 ml of BugBuster (Merck) containing Benzonaze, Lysozyme, and a protease inhibitor cocktail at the recommended concentrations, and incubated at room temperature for 20 minutes to lyse the bacteria. After ultracentrifugation at 45,000×rpm for 30 minutes, the supernatant was removed, mixed with 6 ml of solubilization buffer containing protease cocktail, and disrupted using a Dounce-type glass homogenizer. After mixing by inversion at 4°C for 1 hour, ultracentrifugation was performed at 45,000×rpm for 30 minutes. 400 μl NTA-Agarose (QIAGEN) was added to the supernatant, and mixed by inversion at 4°C for 1 hour. The mixture was loaded onto a Micro Bio-spin column, and the liquid was removed by gravity. After washing with Wash buffer, the target protein was eluted with Elution buffer containing 300 mM imidazole.

[0066] 6. Monomer-monomer interaction analysis of purified ILY mutants 2 μl of purified ILY mutant (final concentration 500 nM), 5 μl of 2× buffer (280 mM KCl, 20 mM DTT, 20 mM MOPS (pH 7)), and 3 μl of MilliQ were mixed and incubated at 37°C for 17 hours. Then, 2.5 μl of SDS sample buffer was added, and the mixture was applied to a 6% polyacrylamide gel and electrophoresed at 4°C and 30 mA for 46 minutes. The gel was blotted at 70 mA for 80 minutes at 4°C, and monomer mutants were detected using a primary antibody (Abcam, Biotin Anti-6xHis tag antibody), a secondary antibody (Vector Laboratories, streptavidin-HRP), and a detection reagent (Cytiva).

[0067] 7. Electrochemical Evaluation of Purified ILY Mutants 4.7 μl of a buffer suitable for each protein was added to the hole on the positive (+) electrode side of the microdevice (Non-Patent Document 5). 4.7 μl of a buffer containing a membrane protein (final concentration 1 μM) was added to the negative (-) electrode side. Then, 0.5 ul of a decane solution containing DphPC:Chresterol=1:1 (mol / mol) was added to each hole, and the hole was set in PICO2 (Tesla). Under a voltage of 100 mV, a lipid bilayer membrane (membrane capacitance 65-70 pF) suitable for the insertion of the membrane protein was formed using a tracing rod, and a stepwise increase in the current value due to pore insertion was observed (Non-Patent Document 6). The experiment was performed under buffer conditions of 140 mM KCl, 10 mM DTT, and 10 mM MOPS (pH 7). The ABF file was converted and analyzed using Clampex (free software).

[0068] 8. Analysis of interactions between monomers of ILY mutants by polyacrylamide gel electrophoresis and Western blotting Each of the designed mutants was expressed in E. coli, and the E. coli was lysed with BugBuster (Merck). The interaction of the monomers contained in the lysate with other monomers was analyzed by SDS-PAGE and Western blotting. The results of analyzing the lysate containing each mutant after incubating it at 37°C for 17 hours are shown in Figure 5. As shown in Figure 5, the mutants with higher bands that are thought to be dimerized and trimerized monomers compared to the wild type (WT) were mut2, mut4, mut9, mut10, mut20, and mut26.

[0069] The results of analysis of mut2, mut4, mut9, mut10, mut20, and mut26 without incubation after purification with His-tag-NiNTA are shown in Figure 6. As a result, no bands that would suggest multimerization were observed in WT, whereas clear dimer and trimer bands were confirmed in mut4, mut9, and mut10.

[0070] 9. Electrochemical Evaluation of Purified ILY Mutants The pore stability of mut9, one of the mutants selected by SDS-PAGE and Western blotting, was evaluated electrochemically. It is known that the stability of a pore can be measured by the shape of the electric signal observed when a pore is formed on a lipid bilayer membrane (Non-Patent Document 7). Specifically, when a protein forms a stable pore and penetrates the membrane, a step-like signal called a Step signal is observed (Non-Patent Document 7). On the other hand, when lipids are mixed between proteins and an unstable pore is formed, a Multi signal is observed, and when a protein enters the lipid membrane and the pore collapses on the membrane over time, an Erratic signal is observed (Non-Patent Document 7). In order to compare the pore stability of WT and mut9, the signals observed when each formed a pore on the membrane were classified and it was examined which signal was observed most frequently in terms of probability. Step signals were counted as stable and other signals were counted as unstable, and the occurrence probability was graphed in Figure 7. The probability of forming a stable pore was 46% for WT, while it was improved to 76% for mut9.

[0071] 10. Promotion of oligomerization by surfactants While examining the oligomerization of mutants in the lysate, the present inventors found that the addition of BugBuster (Merck) used for lysis to purified wild-type ILY promoted oligomerization (Figure 8, lane 1: purified ILY, lane 2: purified ILY with BugBuster (Merck). Each was incubated at 37°C for 17 hours before electrophoresis). To examine which surfactant in BugBuster (Merck) promoted oligomerization, a nonionic surfactant (lane 3: CHAPS, lane 4: CHAPSO) and an amphoteric surfactant (lane 5: sulfobetaine-12, lane 6: sulfobetaine-16, lane 7: sulfobetaine-10, lane 8: sulfobetaine-14) were added to purified ILY, and the mixture was incubated at 37°C for 17 hours for analysis. As a result, all surfactants promoted oligomerization compared to when no surfactants were added, but the effect of zwitterionic surfactants tended to be stronger than that of nonionic surfactants (Figure 8). Among them, sulfobetaine-14 promoted oligomerization the most (Figure 8). This result suggests that surfactants such as sulfobetaine-14 may contribute to pore stabilization.

[0072] 11. Electrochemical Evaluation of ILY Mutants Directed to Form Large-diameter Pores Next, we expressed and purified mutants mut29, mut30, mut31, and mut32, designed to form large pores with a high probability, and subjected them to electrochemical evaluation. The stability of these four mutants was higher than that of the WT (Figure 9). In addition, when these mutants formed pores, we examined the probability of opening pores of 10 nm or more or less than 10 nm, and found that mut29 and mut32 opened pores of 10 nm or more with a higher probability than the WT (Figure 10A).

[0073] A graph comparing the pore diameters of the mutant mut32, which had the highest probability of forming pores of 10 nm or more, and the WT is shown in Figure 10B. WT has peaks at diameters of 1 to 2.5 nm and 2.5 to 3.5 nm, whereas mut32 has a peak at 10 to 13.5 nm. This result also shows that mut32 is more likely to form pores with diameters larger than those of the WT, and is more likely to form pores of 10 to 13.5 nm.

[0074] 12.Detection of proteins using mut32 Using mut32, which had the highest probability of forming pores of 10 nm or more, we attempted to detect protein / nucleic acid complexes. As a protein complex, we used a ternary complex of dCas9, which is a deactivated version of the DNA cleavage activity of Cas9 known as the CRISPR-Cas9 system, cesA, a plasmid derived from Bacillus cereus, and a guide strand RNA with a sequence complementary to cesA.

[0075] 0.15μg / μl dCas9 NLS (manufactured by Nippon Gene Co., Ltd.) 6μl, 2.3μM sgRNA (targeting sequence number 46, sequence number 47 of the cesA gene, prepared with EnGen sgRNA Synthesis Kit manufactured by NEB Co., Ltd.) 3μl were placed at room temperature for 5 minutes. 5μl of 89ng / μl cesA plasmid prepared from a cereulide-containing strain, 2μl of 10×H buffer, and 4μl of MilliQ (trademark) water were added to the mixed solution, and incubated at 37°C for 60 minutes. 5μl of the solution was taken, and 7.5μl of buffer (280mM KCl, 20mM DTT, 20mM MOPS (pH7)), 1.8μl of MilliQ (trademark) water, and 0.7μl of 22μM mut32 (final concentration 1000nM) were added and used for evaluation with nanopore.

[0076] Figure 11 shows the electrical signals observed when dCas9 / sgRNA / cesA was not added and when it was added. When dCas9 / sgRNA / cesA was not added, a constant current value (open signal) was observed due to pore formation, while when dCas9 / sgRNA / cesA was added, a blocking signal was observed in which the current value dropped rapidly from the open signal. It is known that the blocking signal is caused by the ion current inside the pore being inhibited by the entry of a molecule into the pore, suggesting that protein sensing using mut32 is possible.

Claims

1. A nanopore protein which is a mutant protein of a cholesterol-dependent pore-forming cytolytic toxin (CDC) family protein, comprising at least one of the following mutations (1) to (3): (1) Any one of the following substitutions (a) to (g) in the amino acid sequence set forth in SEQ ID NO:1: (a) substitution of the amino acid residue at the position corresponding to arginine at position 302 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline; (b) substitution of an amino acid residue at a position corresponding to histidine at position 242 in a CDC family protein other than the proteins having the amino acid sequences set forth in SEQ ID NOs: 48, 51, and 55 with leucine, phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, isoleucine, methionine, or proline; (c) substitution of an amino acid residue at a position corresponding to threonine at position 334 of a CDC family protein other than the proteins having the amino acid sequences set forth in SEQ ID NOs: 49, 53, and 15 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline; (d) substitution of the amino acid residue at the position corresponding to valine at position 241 with glutamic acid, arginine, histidine, lysine, aspartic acid, glycine, serine, threonine, asparagine, or glutamine. (e) substitution of the amino acid residue at the position corresponding to threonine at position 315 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (f) substitution of the amino acid residue at the position corresponding to asparagine 338 with phenylalanine, tyrosine, tryptophan, alanine, cysteine, valine, leucine, isoleucine, methionine, or proline. (g) substitution of the amino acid residue at the position corresponding to isoleucine at position 339 with serine, arginine, histidine, lysine, aspartic acid, glutamic acid, glycine, threonine, asparagine, or glutamine (2) A mutation in which 5 to 23 consecutive amino acids including Y196-H200 in N178-H200 in the amino acid sequence set forth in SEQ ID NO:1 are deleted, or amino acid residues at positions corresponding to 2 to 13 consecutive amino acids including T517-L518 in W514-V526 are deleted. (3) A mutation in which an amino acid residue is inserted at a position corresponding to 2 to 25 consecutive amino acid residues, including T517-L518, of Q508-D532 in the amino acid sequence of SEQ ID NO: 1, at the C-terminus of the amino acid sequence of SEQ ID NO: 1, or a mutation in which an amino acid residue is inserted at a position corresponding to 1 to 24 consecutive amino acid residues, including R495, of R480-L503 in the amino acid sequence of SEQ ID NO: 1, between amino acid residues corresponding to L503 and P504 in the amino acid sequence of SEQ ID NO: 1

2. 2. The nanopore protein of claim 1, wherein the amino acid residues at positions corresponding to R302, H242, T334, V241, T315, N338 or I339 in the amino acid sequence set forth in SEQ ID NO:1 are mutated by substitution with R302F, R302M, R302L, R302I, H242L, H242I, H242Y, T334V, T334I, V241T, V241S, V241K, V241E, T315V, T315I, N338L, N338F, I339T, I339S or I339K.

3. 3. The nanopore protein of claim 2, wherein the amino acid residue at a position corresponding to R302, H242, T334, V241, T315 or I339 in the amino acid sequence set forth in SEQ ID NO:1 is mutated by substitution with R302F, H242L, H242I, H242Y, T334V, T334I, V241E, T315V, T315I or I339S.

4. A nanopore sensor comprising the nanopore protein according to any one of claims 1 to 3.

5. A nanopore sensing method using the nanopore sensor according to claim 4.

6. A nanopore protein suspension comprising a nanopore protein and a surfactant in an amount sufficient to stabilize the nanopore protein.

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