Application of a panx2 protein and mutants thereof as a biological pore protein

Abstract

This invention belongs to the field of target characterization technology, and provides an application of PANX2 protein and its mutants as bioporins. The bioporins can detect one or more of the following: metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic reagents, adenosine triphosphate (ATP), and monosaccharides. In this invention, one or more amino acids of the wild-type PANX2 protein are mutated to common amino acids other than the original amino acids. Compared to the wild-type PANX2 protein, the PANX2 protein mutants can be expressed normally, are stable, and show a significantly improved current signal during detection, with a longer amplitude and fewer spikes.

Description

Technical Field

[0001] This invention belongs to the field of characterization target analyte technology, and particularly relates to the application of a PANX2 protein and its mutants as a bioporin. Background Technology

[0002] In protein nanopore sensing systems, the core role is played by the nanopore sensor, namely the nanopore protein. Although factors such as temperature, pH, salt type and concentration in the experimental environment can affect the properties of the nanopore and the phenomenon of target molecules blocking ion current, the nanopore protein remains the key to determining the sensing performance. To obtain stable, ungated current records, almost all nanopore proteins require a structurally stable and hydrophilic chemical environment to perform nanopore sensing. Different nanopore proteins, with their unique structures, sizes, lengths, and chemical environments, exhibit irreplaceable advantages in specific studies, such as the precise detection of molecules or subunits with special properties or conformations.

[0003] However, in reality, the methods for precisely modifying natural nanoporous proteins to meet diverse application needs remain relatively limited, primarily relying on protein modification techniques. Therefore, there is a continued need to explore more nanoporous proteins suitable for nanopore sensing to meet different requirements. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of PANX2 protein and its mutants as bioporins, aiming to solve the problems mentioned in the background art.

[0005] In a first aspect, the present invention provides an application of PANX2 protein and its mutants as a bioporin, wherein the bioporin detects one or more of metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic agents, adenosine triphosphate, and monosaccharides.

[0006] Secondly, the present invention provides a PANX2 protein mutant, which is obtained by mutating wild-type PANX2 protein. The amino acid sequence of wild-type PANX2 protein is shown in SEQ ID NO:1. The mutation refers to one or more amino acids of wild-type PANX2 protein being mutated to common amino acids other than the original amino acids.

[0007] The common amino acids mentioned include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0008] Furthermore, the PANX2 protein mutant is selected from either A or B:

[0009] A: One or more of the amino acids R89, D90, and R96 in the wild-type PANX2 protein are mutated to common amino acids other than the original amino acids;

[0010] B: One or more of the amino acids L40 and A36 in the wild-type PANX2 protein are mutated to common amino acids other than the original amino acid.

[0011] Further, in A: the wild-type PANX2 protein has an R89 mutation to a common amino acid other than the original amino acid; the wild-type PANX2 protein has an R89 mutation to R89M, R89P, R89L, R89G, R89I, R89Q, R89E, R89C, R89W, R89H, R89K, R89T, R89A, R89S, R89Y, R89N, R89V, or R89D; preferably, the wild-type PANX2 protein has an R89 mutation to R89T, R89L, or R89N;

[0012] The wild-type PANX2 protein has an amino acid D90 mutation that replaces the original amino acid with a common amino acid; the wild-type PANX2 protein has an amino acid D90 mutation that replaces the original amino acid with D90S.

[0013] The wild-type PANX2 protein has an amino acid R96 mutation to a common amino acid other than the original amino acid; the wild-type PANX2 protein has an amino acid R96 mutation to R96N, R96K, R96H, R96D, R96W, R96S, R96F, R96E, R96P, R96L, R96A, R96V, R96G or R96M; preferably, the wild-type PANX2 protein has an amino acid R96 mutation to R96N or R96K.

[0014] Further, in B: the L40 amino acid of the wild-type PANX2 protein is mutated to a common amino acid other than the original amino acid; the L40 amino acid of the wild-type PANX2 protein is mutated to L40N, L40H, L40Q, L40S, L40F, L40D, L40C, L40I, L40P, L40T, L40G, L40M, L40W or L40R; preferably, the L40 amino acid of the wild-type PANX2 protein is mutated to L40I;

[0015] The wild-type PANX2 protein has an amino acid A36 mutation that is replaced with common amino acids other than the original amino acid; the wild-type PANX2 protein has an amino acid A36 mutation that is replaced with A36N or A36L.

[0016] Thirdly, the present invention provides a product of PANX2 protein and its mutants, which is a composition, complex or kit.

[0017] Fourthly, the present invention provides a method for preparing PANX2 protein and its mutants, comprising the following steps:

[0018] Step S1: Construct the PANX2 protein or a PANX2 protein mutant vector;

[0019] Step S2: Expression and purification of PANX2 protein or PANX2 protein mutant.

[0020] Fifthly, the present invention provides a membrane layer incorporating PANX2 protein and its mutants, wherein the membrane layer is a lipid bilayer.

[0021] The present invention has the following beneficial effects:

[0022] (1) The wild-type PANX2 protein consists of seven identical subunits that are symmetrically assembled around a central axis forming a transmembrane channel. Each subunit contains four transmembrane domains TM1-TM4. The N-terminus and C-terminus of TM1 and TM4 are anchored intracellularly, respectively. TM1 and TM2 are spirally arranged inside the pore, while TM3 and TM4 are arranged around the periphery of the channel, resulting in a funnel-shaped overall channel. Compared to existing nanoporous proteins, its cloning, expression, and purification processes are simple, it is easy to assemble proteins in biological nanopores, and it has a wide range of applications, making it suitable for rapid pathogen detection and gene expression.

[0023] (2) PANX2 protein and its mutants can detect small molecule compounds such as metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic reagents, adenosine triphosphate, and monosaccharides. Mutation experiments have shown that when one or more amino acids of wild-type PANX2 protein are mutated to common amino acids other than the original amino acids, it can be expressed normally. Compared with wild-type PANX2 protein, the PANX2 protein mutant is more stable, and the current signal is significantly improved during detection, with a longer amplitude and fewer spikes. Attached Figure Description

[0024] Exemplary embodiments of the present invention can be more fully understood by referring to the following figures:

[0025] Figure 1 This is a top view of the surface structure model of the wild-type PANX2 protein channel of this invention.

[0026] Figure 2 This is a side view of the surface structure model of the wild-type PANX2 protein channel of the present invention.

[0027] Figure 3This is a top view of the wild-type PANX2 protein channel ribbon structure model of the present invention.

[0028] Figure 4 This is a side view of the wild-type PANX2 protein channel ribbon structure model of the present invention.

[0029] Figure 5 This is an electrophoresis diagram of the purification of wild-type PANX2 protein and PANX2 protein mutant after amino acid L40 is mutated to L40I in Examples 1 and 4 of the present invention; in the figure, lane 1: protein marker; lane 2: wild-type PANX2 eluted sample; lane 3: PANX2 L40I mutant eluted sample; lane 4: wild-type PANX2 eluted sample; lane 5: PANX2 L40I mutant eluted sample; lane 6: PANX2 L40I mutant eluted sample.

[0030] Figure 6 This is a graph showing the current detection results of wild-type PANX2 protein with adenosine triphosphate passing through a pore in Example 3 of the present invention.

[0031] Figure 7 The image shows the nanopore opening current detection results for the PANX2 protein mutant after amino acid L40 was mutated to L40I in Example 5 of this invention.

[0032] Figure 8 The image shows the current detection results when adenosine triphosphate (ATP) is passed through a pore, representing the PANX2 protein mutant after amino acid L40 is mutated to L40I in Example 6 of this invention.

[0033] Figure 9 This is a statistical graph showing the time and number of events during the permeation of the PANX2 protein mutant after amino acid L40 is mutated to L40I in Example 6 of the present invention. Detailed Implementation

[0034] To make the technical problems to be solved, the technical solutions, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0036] This invention provides an application of PANX2 protein and its mutants as bioporins, which can be used to detect one or more of the following: metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic reagents, adenosine triphosphate, and monosaccharides.

[0037] In some embodiments, the PANX2 protein mutant is obtained by mutating the wild-type PANX2 protein. The amino acid sequence of the wild-type PANX2 protein is shown in SEQ ID NO:1. Mutation refers to one or more amino acids of the wild-type PANX2 protein being mutated to common amino acids other than the original amino acid.

[0038] Common amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0039] In some embodiments, the PANX2 protein mutant is selected from either A or B:

[0040] A: One or more of the amino acids R89, D90, and R96 in the wild-type PANX2 protein are mutated to common amino acids other than the original amino acids;

[0041] B: One or more of the amino acids L40 and A36 in the wild-type PANX2 protein are mutated to common amino acids other than the original amino acid.

[0042] In some embodiments, in A: the wild-type PANX2 protein has an amino acid R89 mutation to a common amino acid other than the original amino acid; the wild-type PANX2 protein has an amino acid R89 mutation to R89M, R89P, R89L, R89G, R89I, R89Q, R89E, R89C, R89W, R89H, R89K, R89T, R89A, R89S, R89Y, R89N, R89V, or R89D.

[0043] In some embodiments, the wild-type PANX2 protein is mutated to amino acid R89 as R89T, R89L, or R89N.

[0044] In some embodiments, the D90 amino acid of the wild-type PANX2 protein is mutated to a common amino acid other than the original amino acid; the D90 amino acid of the wild-type PANX2 protein is mutated to D90S.

[0045] In some embodiments, the wild-type PANX2 protein has an amino acid R96 mutation to a common amino acid other than the original amino acid; the wild-type PANX2 protein has an amino acid R96 mutation to R96N, R96K, R96H, R96D, R96W, R96S, R96F, R96E, R96P, R96L, R96A, R96V, R96G, or R96M.

[0046] In some embodiments, the wild-type PANX2 protein has an amino acid mutation at R96, which is either R96N or R96K.

[0047] In some embodiments, in B: the amino acid L40 of the wild-type PANX2 protein is mutated to a common amino acid other than the original amino acid; the amino acid L40 of the wild-type PANX2 protein is mutated to L40N, L40H, L40Q, L40S, L40F, L40D, L40C, L40I, L40P, L40T, L40G, L40M, L40W, or L40R.

[0048] In some embodiments, the amino acid L40 of the wild-type PANX2 protein is mutated to L40I.

[0049] In some embodiments, the amino acid A36 of the wild-type PANX2 protein is mutated to a common amino acid other than the original amino acid; the amino acid A36 of the wild-type PANX2 protein is mutated to A36N or A36L.

[0050] In some embodiments, the present invention provides a product of PANX2 protein and its mutants, wherein the product is a composition, complex or kit.

[0051] In some embodiments, the present invention provides a method for preparing PANX2 protein and its mutants, comprising the following steps:

[0052] Step S1: Construct the PANX2 protein or a PANX2 protein mutant vector;

[0053] Step S2: Expression and purification of PANX2 protein or PANX2 protein mutant.

[0054] In some embodiments, the present invention provides a membrane layer incorporating PANX2 protein and its mutants, the membrane layer being a lipid bilayer.

[0055] Example 1: Expression and purification of wild-type PANX2 protein

[0056] 1. Gene construction and vector design

[0057] (1) Target gene synthesis and initial vector cloning: The PANX2 (SEQ ID NO:2) DNA fragment was synthesized. The fragment was cloned into the modified yeast expression vector pPICZ-B, which contained: a C-terminal fusion tag and a PreScission protease cleavage site. Labels (used for subsequent purification and detection).

[0058] (2) Transformation: Pichia pastoris strain SMD1163H was used to transform into the constructed expression vector using standard methods.

[0059] (3) Induction of expression: Transformed yeast cells were cultured to the logarithmic growth phase and PANX2EM expression was induced by methanol.

[0060] (4) Cell disruption: Collect induced yeast cells and disrupt them using a grinder. Resuspend the disrupted cells in buffer A (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) and add protease inhibitor cocktails: leupropeptidase (2.5 μg / ml), pepsin inhibitor A (1 μg / ml), AEBSF (100 μg / ml), aprotinin (3 μg / ml), benzoamidine (1 mM), and PMSF (200 μM). Add DNase I (GoldBio, D-300-1) to digest nucleic acids.

[0061] (5) Membrane protein extraction: Add 1% (w / v) detergent LMNG (lauryl maltose neopentyl glycol), stir and extract at 4°C for 2 hours. Centrifuge at 30000×g for 1 hour and collect the supernatant containing the target protein.

[0062] 2. Affinity chromatography purification

[0063] (1) Cobalt ion resin binding: The supernatant was incubated with 3 ml of cobalt ion resin at 4°C for 3 hours, and the mixture was then subjected to cobalt ion resin binding. Tag-binding protein.

[0064] (2) Washing away contaminating proteins: Non-specifically bound proteins were washed away with 10 column volumes of washing buffer (20 mM Tris-HCl, pH=8.0, 150 mM NaCl, 20 mM imidazole, 85 μM GDN). The detergent was replaced with GDN (glycosyldiosgenin) to maintain membrane protein stability.

[0065] (3) Target protein elution: Elute the binding protein with elution buffer (20 mM Tris-HCl, pH=8.0, 150 mM NaCl, 200 mM imidazole, 85 μM GDN) and collect the eluent.

[0066] 3. Label removal and size exclusion chromatography

[0067] (1) Tag cutting: Add PreScission protease to the elution buffer, incubate overnight at 4°C, and remove the C-terminus. Label.

[0068] (2) Gel filtration chromatography: The enzyme-digested protein was purified by size exclusion chromatography using a buffer of 20 mM Tris-HCl (pH=8.0), 150 mM NaCl and 40 μM GDN. A single symmetrical target protein peak was collected (monitored by UV absorption at 280 nm).

[0069] Figure 1 Top view of the surface structure model of wild-type PANX2 protein channel. Figure 2 Side view of the surface structure model of wild-type PANX2 protein channel. Figure 3 This is a top view of the channel ribbon structure model of the wild-type PANX2 protein, where the parts of the same color represent a single protein monomer. Figure 4 This is a side view of the channel ribbon structure model of the wild-type PANX2 protein, where the parts of the same color represent a single protein monomer. The results indicate that the wild-type PANX2 protein consists of seven identical subunits, which are symmetrically assembled around a central axis forming the transmembrane channel. Each subunit contains four transmembrane domains TM1-TM4. The N-terminus and C-terminus of TM1 and TM4 are anchored intracellularly, respectively. TM1 and TM2 are spirally arranged within the pores, while TM3 and TM4 are arranged on the periphery of the channel, resulting in a funnel-shaped overall channel.

[0070] Example 2: Characterization of wild-type PANX2 protein

[0071] Wild-type PANX2 protein with amino acids as shown in SEQ ID NO:1 was obtained, and the DNA sequence of its encoding gene is shown in SEQ ID NO:2.

[0072] (1) Preparation of lipid bilayer: 2 mg of DPhPC (dispalmitoylphosphatidylcholine) was dissolved in chloroform and dropped onto both sides of a Teflon membrane (pore size 20 μm). The solvent was evaporated to form a stable bilayer. 1 ml of detection buffer (0.5 M KCl, 10 mM HEPES, pH=8.0) was added to each of the cis and trans chambers.

[0073] (2) Protein insertion: Purified wild-type PANX2 protein (8 ng / ml) was added to the cis chamber. An Axopatch 200B integrated patch-clamp amplifier was used to apply a voltage of 180 mV between the two chambers (positive on the reverse side) and the ion current through the well was measured. Wild-type PANX2 protein was added to the grounded cis chamber at a concentration of 8.0 ng / ml. Once a well was inserted, the chamber was flushed with experimental buffer to prevent further insertion. All experiments were performed at room temperature (23 ± 1 °C).

[0074] The analog signal was low-pass filtered at 100 kHz using a 4-pole Bessel filter and digitized at 500 kHz. Data acquisition was controlled by Origin. The results showed that the pore current signal of wild-type PANX2 protein had large noise.

[0075] Example 3: Detection of adenosine triphosphate (ATP) using wild-type PANX2 protein

[0076] (1) Preparation of lipid bilayer: Dissolve 2 mg of DPhPC in chloroform and drop it onto both sides of a Teflon membrane (pore size 20 μm). Evaporate the solvent to form a stable bilayer. Add 1 ml of detection buffer (0.5 M KCl, 10 mM HEPES, pH=8.0) to each of the cis and trans chambers.

[0077] (2) Protein insertion: Purified wild-type PANX2 protein (8 ng / ml) was added to the cis chamber. An Axopatch 200B integrated patch-clamp amplifier was used to apply a voltage of 180 mV between the two chambers (positive on the reverse side) and the ion current through the well was measured. Wild-type PANX2 protein was added to the grounded cis chamber at a concentration of 8.0 ng / ml. Once a well was inserted, the chamber was flushed with experimental buffer to prevent further insertion. All experiments were performed at room temperature (23 ± 1 °C). Analog signals were low-pass filtered at 100 kHz using a 4-pole Bessel filter and digitized at 500 kHz. Data acquisition was controlled using Origin.

[0078] (3) Add the adenosine triphosphate sample to the cis chamber, apply a voltage of 160mV, record the current using an Axopatch 200B amplifier (bandwidth 10kHz), digitize the data at a sampling rate of 50kHz, and filter it in real time using Origin software (4-pole Bessel filter, cutoff frequency 2kHz).

[0079] The current detection results of wild-type PANX2 protein and adenosine triphosphate during pore passage are as follows: Figure 6 As shown, the results indicate that the current signal has significant noise and numerous spikes.

[0080] Example 4: Expression and purification of the PANX2 protein mutant after amino acid L40 is mutated to L40I

[0081] 1. Gene construction and vector design

[0082] (1) Transformation: Pichia pastoris strain SMD1163H was used to transform the PANX2 protein mutant expression vector with amino acid L40 mutated to L40I using standard methods.

[0083] (2) Induction of expression: Transformed yeast cells were cultured to the logarithmic growth phase and PANX2EM expression was induced by methanol.

[0084] (3) Cell disruption: Collect induced yeast cells and disrupt them using a grinder. Resuspend the disrupted cells in buffer (50 mM Tris-HCl, pH=8.0, 150 mM NaCl), and add protease inhibitor cocktails: leupropeptidase (2.5 μg / ml), pepsin inhibitor A (1 μg / ml), AEBSF (100 μg / ml), aprotinin (3 μg / ml), benzalkonium chloride (1 mM), and PMSF (200 μM). Add DNase I (GoldBio, D-300-1) to digest nucleic acids.

[0085] (4) Membrane protein extraction: Add 1% (w / v) detergent LMNG (lauryl maltose neopentyl glycol), stir and extract at 4°C for 2 hours. Centrifuge at 30000×g for 1 hour and collect the supernatant containing the target protein.

[0086] 2. Affinity chromatography purification

[0087] (1) Cobalt ion resin binding: The supernatant was incubated with 3 ml of cobalt ion resin at 4°C for 3 hours, and the mixture was then subjected to cobalt ion resin binding. Tag-binding protein.

[0088] (2) Washing away contaminating proteins: Non-specifically bound proteins were washed away with 10 column volumes of washing buffer (20 mM Tris-HCl, pH=8.0, 150 mM NaCl, 20 mM imidazole, 85 μM GDN). The detergent was replaced with GDN (glycosyldiosgenin) to maintain membrane protein stability.

[0089] (3) Target protein elution: Elute the binding protein with elution buffer (20mM Tris-HCl, pH=8.0, 150mM NaCl, 200mM imidazole, 85μM GDN) and collect the eluent.

[0090] 3. Label removal and size exclusion chromatography

[0091] (1) Tag cutting: Add PreScission protease to the elution buffer, incubate overnight at 4°C, and remove the C-terminus. Label.

[0092] (2) Gel filtration chromatography: The enzyme-digested protein was purified by size exclusion chromatography using a buffer of 20 mM Tris-HCl (pH=8.0), 150 mM NaCl and 40 μM GDN. A single symmetrical target protein peak was collected (monitored by UV absorption at 280 nm).

[0093] The purification electrophoresis results of wild-type PANX2 protein and the PANX2 protein mutant with amino acid L40 mutated to L40I in Examples 1 and 4 are as follows: Figure 5 As shown.

[0094] Example 5: Characterization of the PANX2 protein mutant after amino acid L40 is mutated to L40I

[0095] (1) Preparation of lipid bilayer: Dissolve 2 mg of DPhPC in chloroform and drop it onto both sides of a Teflon membrane (pore size 20 μm). Evaporate the solvent to form a stable bilayer. Add 1 ml of detection buffer (0.5 M KCl, 10 mM HEPES, pH=8.0) to each of the cis and trans chambers.

[0096] (2) Protein insertion: The purified PANX2 protein mutant (8 ng / ml) with amino acid L40 mutated to L40I was added to the cis compartment. An Axopatch 200B integrated patch-clamp amplifier (Axon Instruments) was used to apply a voltage of 180 mV between the two compartments (positive on the reverse side) and the ion current through the well was measured. Once a well was inserted, the compartment was flushed with experimental buffer to prevent further insertion. All experiments were performed at room temperature (23 ± 1 °C).

[0097] The analog signal was low-pass filtered at 100 kHz using a 4-pole Bessel filter and digitized at 500 kHz, with data acquisition controlled by Origin. The nanopore opening current detection results for the PANX2 protein mutant, resulting from the L40-L40I mutation, are as follows: Figure 7 As shown, the results indicate that the current properties of the PANX2 protein mutant are significantly improved and the current noise is reduced compared to the wild-type PANX2 protein.

[0098] Example 6: Detection of adenosine triphosphate (ATP) using the PANX2 protein mutant with amino acid L40 mutated to L40I.

[0099] (1) Preparation of lipid bilayer: Dissolve 2 mg of DPhPC in chloroform and drop it onto both sides of a Teflon membrane (pore size 20 μm). Evaporate the solvent to form a stable bilayer. Add 1 ml of detection buffer (0.5 M KCl, 10 mM HEPES, pH=8.0) to each of the cis and trans chambers.

[0100] (2) Protein insertion: The purified PANX2 protein mutant (8 ng / ml) with amino acid L40 mutated to L40I was added to the cis chamber. An Axopatch 200B integrated patch-clamp amplifier was used to apply a voltage of 180 mV between the two chambers (positive on the reverse side) and the ion current through the well was measured. Once a well was inserted, the chamber was flushed with experimental buffer to prevent further insertion. All experiments were performed at room temperature (23 ± 1 °C). Analog signals were low-pass filtered at 100 kHz using a 4-pole Bessel filter and digitized at 500 kHz. Data acquisition was controlled using Origin.

[0101] (3) Add the adenosine triphosphate sample to the cis chamber, apply a voltage of 160mV, record the current using an Axopatch 200B amplifier (bandwidth 10kHz), digitize the data at a sampling rate of 50kHz, and filter it in real time using Origin software (4-pole Bessel filter, cutoff frequency 2kHz).

[0102] The PANX2 protein mutant, resulting from the L40 mutation of amino acid L40 to L40I, showed the following current detection results during adenosine triphosphate (ATP) permeation: Figure 8 As shown; the statistical results of the perforation time and number of events during ATP perforation of the PANX2 protein mutant after amino acid L40 mutation to L40I are as follows. Figure 9 As shown in the figure. The results indicate that, compared with the wild-type PANX2 protein, the PANX2 protein mutant exhibits significantly improved current properties, with longer current amplitudes, fewer spikes, and better consistency in adenosine triphosphate (ATP) pore passage time.

[0103] The narrowest pore size of the wild-type PANX2 protein is 0.9 nm. PANX2 protein and its mutants are more suitable for detecting metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs (the diameter of the drug is smaller than the protein pore size), small molecule diagnostic reagents (the diameter of the diagnostic reagent is smaller than the protein pore size), adenosine triphosphate, monosaccharides and other small molecule compounds.

[0104] amino acid sequence:

[0105] SEQ ID NO:1:

[0106] MHHLLEQSADMATALLAGEKLRELILPGAQDDKAGALAALLLQLKLELPFDRVVTIGTVLVPILLVTLVFTKNFAEEPIYCYTPHNFTRDQALYARGYCWTELRDALPGVDASLWPSLFEHKFLPYALLAFAAIMYVPALGWEFLASTRLTSELNFLLQEIDNCYHRAAEGRAPKIEKQIQSKGPGITEREKREIIENAEKEKSPEQNLFEKYLERRGRSNFLAKLYLARHVLILLLSAVPISYLCTYYATQKQNEFTCALGASPDGAAGAGPAVRVSCKLPSVQLQRIIAGVDIVLLCVMNLIILVNLIHLFIFRKSNFIFDKLHKVGIKTRRQWRRSQFCDINILAMFCNENRDHIKSLNRLDFIT

[0107] DNA sequence

[0108] SEQ ID NO:2:

[0109]

[0110] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of a PANX2 protein mutant as a bioporin in single-molecule detection, characterized in that: The PANX2 protein mutant is obtained by mutating the wild-type PANX2 protein. The amino acid sequence of the wild-type PANX2 protein is shown in SEQ ID NO:

1. The mutation refers to the mutation of amino acid L40 of the wild-type PANX2 protein to L40I.

2. The application as described in claim 1, characterized in that: The bioporin is used to detect one or more of the following: metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic reagents, adenosine triphosphate, and monosaccharides.

3. The application as described in claim 2, characterized in that: The method for preparing PANX2 protein mutants includes the following steps: Step S1: Construct the PANX2 protein mutant vector; Step S2: Expression and purification of the PANX2 protein mutant.

4. The application as described in claim 3, characterized in that: A lipid bilayer embeds a PANX2 protein mutant.

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