Panx3 biological pore protein and its mutants and application of single molecule detection

Abstract

The application belongs to the technical field of characterizing target samples, and provides PANX3 biological pore protein, a mutant thereof and application of single molecule detection. The PANX3 biological pore protein is composed of seven identical subunits, the overall channel is funnel-shaped, and the amino acid sequence is shown as SEQ ID NO: 1. After one or more amino acids of the wild type PANX3 biological pore protein are mutated into common amino acids other than the original amino acids, compared with the wild type PANX3 biological pore protein, the mutant can be normally expressed and stable in property, and the current property and current signal of the mutant are obviously improved.

Description

Technical Field

[0001] This invention belongs to the field of target sample characterization technology, and particularly relates to the application of PANX3 bioporin and its mutants in single-molecule detection. Background Technology

[0002] As a single-molecule detection technology, bioporins (nanoporins) were initially mainly used for nucleic acid sequencing and epigenetic modification analysis. Today, the application boundaries of this technology are constantly expanding, extending to broader fields such as protein sensing, chemical reaction monitoring, and biophysical characterization. Its core advantage lies in its ability to achieve single-molecule-level analysis under label-free and modification-free conditions, offering advantages such as low cost, high throughput, and label-free operation.

[0003] The principle behind this technology originates from the Coulter counting method: when nanoscale pores form an ion current under electrophoretic driving, the passage of target macromolecules causes current interruption, and these changes in current signals can be interpreted as detailed information about molecular properties, concentration, and structure. This single-molecule level detection capability is its core advantage over traditional detection methods. Over the past two decades, bioporins, such as α-hemolysin (α-HL), Mycobacterium smegma porin (MspA), and aerolysin (AeL), have become ideal single-molecule detection tools.

[0004] However, in practical applications, the variety of bioporins is still insufficient, and there is a need to continuously search for more bioporins suitable for nanopore sensing to meet different needs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides applications for the detection of PANX3 bioporin and its mutants, with the aim of resolving the problems mentioned in the background art.

[0006] In a first aspect, the present invention provides PANX3 bioporin, which is composed of seven identical subunits and has a funnel-shaped overall channel, and its amino acid sequence is shown in SEQ ID NO:1.

[0007] Secondly, the present invention provides a PANX3 bioporous mutant protein, which is obtained by mutation of wild-type PANX3 bioporous protein. The amino acid sequence of wild-type PANX3 bioporous protein is shown in SEQ ID NO:1. The mutation refers to one or more amino acids of wild-type PANX3 bioporous protein being mutated to common amino acids other than the original amino acids.

[0008] 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.

[0009] Thirdly, this invention provides the application of PANX3 bioporin and its mutants in single-molecule detection, detecting one or more of peptides, proteins, glycosylated peptides, and polymers.

[0010] Furthermore, choose from either A or B:

[0011] A: One or more of the amino acids I74, R75, and S73 in wild-type PANX3 bioporin are mutated to common amino acids other than the original amino acids.

[0012] B: One or more of the amino acids F58 and R20 in wild-type PANX3 bioporins are mutated to common amino acids other than the original amino acids.

[0013] Further, in A: the amino acid I74 of the wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid I74 of the wild-type PANX3 bioporin is mutated to I74M, I74P, I74L, I74G, I74R, I74Q, I74E, I74C, I74W, I74H, I74K, I74T, I74A, I74S, I74Y, I74N, I74V or I74D; preferably, the amino acid I74 of the wild-type PANX3 bioporin is mutated to I74A, I74L or I74N.

[0014] Furthermore, the amino acid R75 of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid R75 of wild-type PANX3 bioporin is mutated to R75A or R75S.

[0015] Furthermore, the S73 amino acid of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the S73 amino acid of wild-type PANX3 bioporin is mutated to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G or S73M; preferably, the S73 amino acid of wild-type PANX3 bioporin is mutated to S73N or S73K.

[0016] Further, in B: the amino acid F58 of the wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid F58 of the wild-type PANX3 bioporin is mutated to F58N, F58H, F58Q, F58S, F58L, F58D, F58C, F58I, F58P, F58T, F58G, F58M, F58W or F58R; preferably, the amino acid F58 of the wild-type PANX3 bioporin is mutated to F58N.

[0017] Furthermore, the amino acid R20 of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid R20 of wild-type PANX3 bioporin is mutated to R20N or R20L.

[0018] Fourthly, the present invention provides a method for preparing PANX3 bioporous mutant protein, comprising the following steps:

[0019] Step S1: Construct a PANX3 bioporin or PANX3 biopor mutant protein vector;

[0020] Step S2: Construct, express, and purify PANX3 bioporin or PANX3 biopor mutant protein.

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

[0022] (1) Wild-type PANX3 bioporin consists of seven identical subunits that are symmetrically assembled around a central axis forming a transmembrane channel. Each subunit consists of four transmembrane domains (TMDs) and two extracellular loops (ELs). Two disulfide bonds (SS1 and SS2) stabilize the extracellular domains, C66-C261 (SS1) and C84-C242 (SS2). Each subunit has intracellular N-terminal and C-terminal domains, and the overall channel is funnel-shaped. Compared with existing bioporins, its monomeric structure has the potential to form nanopores, the expression and purification steps are simple, and it is easy to form stable monopores on lipid membranes, making it widely applicable.

[0023] (2) When one or more amino acids of wild-type PANX3 bioporin are mutated to common amino acids other than the original amino acids, the mutant can be expressed normally and has stable properties compared with wild-type PANX3 bioporin, and its current properties and current signal are significantly improved. 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 PANX3 bioporin channel of the present invention.

[0026] Figure 2 This is a side view of the surface structure model of wild-type PANX3 bioporin of the present invention.

[0027] Figure 3 This is a top view of the wild-type PANX3 bioporin channel ribbon structure model of the present invention.

[0028] Figure 4 This is a side view of the wild-type PANX3 bioporin channel ribbon structure model of the present invention.

[0029] Figure 5 This is an electrophoresis diagram of the purification of wild-type PANX3 bioporin and PANX3 bioporous mutant protein after amino acid S73 was mutated to S73N in Examples 1 and 3 of the present invention; in the figure, lane 1: protein marker; lane 2: wild-type PANX3 eluted sample; lane 3: mutant PANX3 S73N eluted sample; lane 4: wild-type PANX3 eluted sample; lane 5: mutant PANX3 S73N eluted sample; lane 6: mutant PANX3 S73N eluted sample.

[0030] Figure 6 The image shows the nanopore opening current detection results of the PANX3 biopore mutant protein after amino acid S73 was mutated to S73N in Example 6 of this invention.

[0031] Figure 7 The image shows the current detection results when the polypeptide chain passes through a nanopore, representing the PANX3 biopore mutant protein obtained by mutating amino acid S73 to S73N in Example 7 of this invention.

[0032] Figure 8 The image shows the current detection results when the glycosylated peptide passes through a nanopore, representing the PANX3 biopore mutant protein after amino acid F58 is mutated to F58N in Example 8 of this invention. Detailed Implementation

[0033] 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.

[0034] 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.

[0035] This invention provides PANX3 bioporin, which is composed of seven identical subunits and has a funnel-shaped overall channel. The amino acid sequence is shown in SEQ ID NO:1.

[0036] In some embodiments, the present invention provides a PANX3 bioporous mutant protein, which is obtained by mutation of wild-type PANX3 bioporous protein. The amino acid sequence of wild-type PANX3 bioporous protein is shown in SEQ ID NO:1. Mutation refers to one or more amino acids of wild-type PANX3 bioporous protein being mutated to common amino acids other than the original amino acids.

[0037] 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.

[0038] In some embodiments, the present invention provides the application of PANX3 bioporin and its mutants in single-molecule detection to detect one or more of peptides, proteins, glycosylated peptides, and polymers.

[0039] In some embodiments, the selection is made from either A or B:

[0040] A: One or more of the amino acids I74, R75, and S73 in wild-type PANX3 bioporin are mutated to common amino acids other than the original amino acids.

[0041] B: One or more of the amino acids F58 and R20 in wild-type PANX3 bioporins are mutated to common amino acids other than the original amino acids.

[0042] In some embodiments, in A: the amino acid I74 of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid I74 of wild-type PANX3 bioporin is mutated to I74M, I74P, I74L, I74G, I74R, I74Q, I74E, I74C, I74W, I74H, I74K, I74T, I74A, I74S, I74Y, I74N, I74V or I74D.

[0043] In some embodiments, the amino acid I74 of wild-type PANX3 bioporin is mutated to I74A, I74L, or I74N.

[0044] In some embodiments, the amino acid R75 of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid R75 of wild-type PANX3 bioporin is mutated to R75A or R75S.

[0045] In some embodiments, the S73 amino acid of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the S73 amino acid of wild-type PANX3 bioporin is mutated to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G or S73M.

[0046] In some embodiments, the amino acid S73 of wild-type PANX3 bioporin is mutated to S73N or S73K.

[0047] In some embodiments, in B: the amino acid F58 of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid F58 of wild-type PANX3 bioporin is mutated to F58N, F58H, F58Q, F58S, F58L, F58D, F58C, F58I, F58P, F58T, F58G, F58M, F58W or F58R.

[0048] In some embodiments, the amino acid F58 of wild-type PANX3 bioporin is mutated to F58N.

[0049] In some embodiments, the amino acid R20 of wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; the amino acid R20 of wild-type PANX3 bioporin is mutated to R20N or R20L.

[0050] In some embodiments, the present invention provides a method for preparing PANX3 biopore mutant protein, comprising the following steps:

[0051] Step S1: Construct a PANX3 bioporin or PANX3 biopor mutant protein vector;

[0052] Step S2: Construct, express, and purify PANX3 bioporin or PANX3 biopor mutant protein.

[0053] Example 1: Expression and purification of wild-type PANX3 bioporin

[0054] 1. Gene construction and vector design

[0055] (1) Synthesis of target gene and initial vector cloning: The PANX3 (SEQ ID NO:2) DNA fragment was synthesized. The fragment was cloned into the pcDNA3.4 vector, and an mEGFP-6xHis tag was added to the C-terminus.

[0056] (2) COS-7 cell transfection: COS-7 cells were cultured in low-glucose DMEM medium containing 1x penicillin / streptomycin and 10% fetal bovine serum. For each 35 mm culture dish, 1 μg of DNA containing hPANX3-mEGFP-6xHis and 4 μg of PEI-MAX were prepared for transfection. The transfected cells were seeded into glass-bottom culture dishes. Cells were fixed with phosphate buffer containing 3.7% formaldehyde. Cells were stained with wheat germ lectin-Fluor 647. Fluorescence images were captured and analyzed using a confocal laser scanning microscope.

[0057] (3) Expi293F cell transfection: Expi293F cells were cultured in HE400AZ medium. When the cell density reached... At a cell / ml ratio, 0.8 μg of hPANX3-6xHis DNA and 3.2 μg of PEI-MAX were used per 1 ml of culture medium for transfection. Cells overexpressing hPANX3-6xHis were harvested 48 hours after transfection.

[0058] (4) Cell disruption: The harvested cells were disrupted by sonication in TBS buffer containing protease inhibitors (50 mM Tris-HCl, pH=8.0, 150 mM NaCl).

[0059] (5) Membrane fraction collection: Membrane fractions were collected by ultracentrifugation. The membrane fractions were then suspended in TBS buffer containing 2% n-dodecyl-β-D-maltodextrin, 0.4% cholesterol hemisuccinate, and a protease inhibitor.

[0060] 2. Affinity chromatography purification

[0061] (1) Gently mix and incubate at 4°C for 2 hours to dissolve the membrane protein. Load the dissolved protein onto TALON resin.

[0062] (2) Washing off contaminants: Wash with SEC buffer containing 15 mM imidazole (TBS buffer containing 0.003% diosgenin) in 25 column volumes (10 ml per column).

[0063] (3) Target protein elution: Elution was performed with SEC buffer containing 250 mM imidazole in 5 column volumes.

[0064] (4) Size exclusion chromatography: Further purification was performed using a size exclusion chromatography column to collect a single symmetrical target protein peak (monitored by UV absorption at 280 nm).

[0065] Top view of the surface structure model of wild-type PANX3 bioporin channel as shown in the figure. Figure 1As shown; a side view of the surface structure model of wild-type PANX3 bioporin channels. Figure 2 As shown; Top view of the wild-type PANX3 bioporin channel ribbon structure model. Figure 3 As shown; a side view of the wild-type PANX3 bioporin channel ribbon structure model is shown. Figure 4 As shown. The results indicate that wild-type PANX3 bioporin consists of seven identical subunits that symmetrically assemble around a central axis forming a transmembrane channel, resulting in a funnel-shaped overall channel.

[0066] Example 2: Characterization of wild-type PANX3 bioporins

[0067] Wild-type PANX3 bioporin 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.

[0068] A single wild-type PANX3 biopore protein pore was constructed in a lipid bilayer. Detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH 8.0, 2 mM HCl) was first injected into both chambers. 0.1 mM EDTA was added, with a volume of 1.2 ml / side, ensuring liquid levels were balanced on both sides of the membrane. 0.5 μL of 1,2-bis(phytanyl-n-glycerol-3-phosphocholine) (Avanti Polar Lipids) lipid solution was pipetted through a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0069] Wild-type PANX3 bioporin solution (8 ng / ml) was added to the cis-compartment, while the trans-compartment was kept buffer-blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and current changes were monitored in real time. After protein insertion, the cis-compartment was rinsed three times with buffer (50 μl each time) to remove unbound protein and avoid pore interference. All experiments were performed at room temperature (23 ± 1 °C).

[0070] 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 results showed that the pore-opening current signal of wild-type PANX3 bioporin had significant noise and numerous current spikes.

[0071] Example 3: Expression and purification of PANX3 biopore mutant protein after amino acid S73 is mutated to S73N

[0072] 1. Gene construction and vector design

[0073] (1) Synthesis of target gene and initial vector cloning: A PANX3 bioporin DNA fragment with amino acid S73 mutated to S73N was synthesized. The fragment was cloned into the pcDNA3.4 vector, and an mEGFP-6xHis tag was added to the C-terminus.

[0074] (2) COS-7 cell transfection: COS-7 cells were cultured in low-glucose DMEM medium containing 1x penicillin / streptomycin and 10% fetal bovine serum. For each 35 mm culture dish, 1 μg of DNA containing hPANX3-mEGFP-6xHis and 4 μg of PEI-MAX were prepared for transfection. The transfected cells were seeded into glass-bottom culture dishes. Cells were fixed with phosphate buffer containing 3.7% formaldehyde. Cells were stained with wheat germ lectin-Fluor 647. Fluorescence images were captured and analyzed using a confocal laser scanning microscope.

[0075] (3) Expi293F cell transfection: Expi293F cells were cultured in HE400AZ medium. When the cell density reached... At a cell / ml ratio, 0.8 μg of hPANX3-6xHis DNA and 3.2 μg of PEI-MAX were used per 1 ml of culture medium for transfection. Cells overexpressing hPANX3-6xHis were harvested 48 hours after transfection.

[0076] (4) Cell disruption: The harvested cells were disrupted by sonication in TBS buffer containing protease inhibitors (50 mM Tris-HCl, pH=8.0, 150 mM NaCl).

[0077] (5) Membrane fraction collection: Membrane fractions were collected by ultracentrifugation. The membrane fractions were then suspended in TBS buffer containing 2% n-dodecyl-β-D-maltodextrin, 0.4% cholesterol hemisuccinate, and a protease inhibitor.

[0078] 2. Affinity chromatography purification

[0079] (1) Gently mix and incubate at 4°C for 2 hours to dissolve the membrane protein. Load the dissolved protein onto TALON resin.

[0080] (2) Washing off contaminants: Wash with SEC buffer containing 15 mM imidazole (TBS buffer containing 0.003% diosgenin) in 25 column volumes (10 ml per column).

[0081] (3) Target protein elution: Elution was performed with SEC buffer containing 250 mM imidazole in 5 column volumes.

[0082] (4) Size exclusion chromatography: Further purification was performed using a size exclusion chromatography column to collect a single symmetrical target protein peak (monitored by UV absorption at 280 nm).

[0083] The purification electrophoresis results of wild-type PANX3 bioporin and PANX3 biopor mutant protein with amino acid S73 mutated to S73N in Examples 1 and 3 are as follows: Figure 5 As shown.

[0084] Example 4: Detection of polypeptide chains using wild-type PANX3 bioporin

[0085] A single wild-type PANX3 biopore protein pore was constructed in a lipid bilayer. Detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH 8.0, 2 mM HCl) was first injected into both chambers. 0.1 mM EDTA was added, with a volume of 1.2 ml / side, ensuring liquid levels were balanced on both sides of the membrane. 0.5 μL of 1,2-bis(phytanyl-n-glycerol-3-phosphocholine) lipid solution was pipetted through a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0086] Wild-type PANX3 bioporin solution (8 ng / ml) was added to the cis-compartment, while the trans-compartment was kept buffer-blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and current changes were monitored in real time. After protein insertion, the cis-compartment was rinsed three times with buffer (50 μl each time) to remove unbound protein and avoid pore interference.

[0087] A polypeptide chain sample (SEQ ID NO:3: RRRRRR, hexamethylenetetramine, +6 charge, unmodified 10 nM) was added, and a voltage of 160 mV was applied (voltage optimized to reduce the influence of chain folding). The sampling rate was 50 kHz, and a low-pass filter was applied at 2 kHz. All experiments were performed at room temperature (23 ± 1 °C). 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 using Origin. The results showed that the current signal had significant noise, numerous current spikes, and large fluctuations in the current baseline when detecting polypeptide chains using wild-type PANX3 bioporin.

[0088] Example 5: Detection of glycosylated peptides using wild-type PANX3 bioporin

[0089] A single wild-type PANX3 biopore protein pore was constructed in a lipid bilayer. Detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH 8.0, 2 mM HCl) was first injected into both chambers. 0.1 mM EDTA was added, with a volume of 1.2 ml / side, ensuring liquid levels were balanced on both sides of the membrane. 0.5 μL of 1,2-bis(phytanyl-nglycerol-3-phosphocholine) lipid solution was pipetted through a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0090] Wild-type PANX3 bioporin solution (8 ng / ml) was added to the cis-compartment, while the trans-compartment was kept buffer-blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and current changes were monitored in real time. After protein insertion, the cis-compartment was rinsed three times with buffer (50 μl each time) to remove unbound protein and avoid pore interference.

[0091] A glycosylated peptide sample (GalNAc-Ser-YYY, glycosylation site is serine linked to N-acetylgalactosamine, negatively charged overall due to the presence of carboxyl groups on the glycosyl group; 10 nM) was added, a voltage of 160 mV was applied, the sampling rate was 50 kHz, and a low-pass filter was applied at 2 kHz. All experiments were performed at room temperature (23 ± 1 °C). 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 results showed that the current signal had significant noise, numerous current spikes, and large fluctuations in the current baseline when detecting glycosylated peptides using wild-type PANX3 bioporin, indicating some pore blockage.

[0092] Example 6: Characterization of PANX3 biopore mutant protein after amino acid S73 is mutated to S73N

[0093] After the amino acid S73 was mutated to S73N, a single PANX3 biopore mutant protein pore was constructed in a lipid bilayer. Detection buffer (0.5M KCl, 10mM HEPES-KOH, pH=8.0, 2mM...) was first injected into both chambers. 0.1 mM EDTA was added, with a volume of 1.2 ml / side, ensuring liquid levels were balanced on both sides of the membrane. 0.5 μL of 1,2-bis(phytanyl-n-glycerol-3-phosphocholine) lipid solution was pipetted through a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0094] A 8 ng / ml solution of the PANX3 biopore mutant protein (S73-S73N mutant) was added to the cis-compartment, while the trans-compartment was kept buffer-blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and current changes were monitored in real time. After protein insertion, the cis-compartment was rinsed three times with buffer (50 μl each time) to remove unbound protein and avoid pore interference. All experiments were performed at room temperature (23 ± 1 °C).

[0095] 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 PANX3 biopore mutant protein, resulting from the S73 mutation of amino acid S73 to S73N, are as follows: Figure 6 As shown, the results indicate that, compared to wild-type PANX3 bioporin, the PANX3 bioporin mutant protein produces a more stable current signal with a narrower current signal width and fewer spikes.

[0096] Example 7: Detection of polypeptide chains using PANX3 biopore mutant protein with amino acid S73 mutated to S73N.

[0097] After the amino acid S73 was mutated to S73N, a single PANX3 biopore mutant protein pore was constructed in a lipid bilayer. Detection buffer (0.5M KCl, 10mM HEPES-KOH, pH=8.0, 2mM...) was first injected into both chambers. 0.1 mM EDTA was added, with a volume of 1.2 ml / side, ensuring liquid levels were balanced on both sides of the membrane. 0.5 μL of 1,2-bis(phytanyl-n-glycerol-3-phosphocholine) lipid solution was pipetted through a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0098] A 8 ng / ml solution of the PANX3 biopore mutant protein (S73-S73N mutant) was added to the cis-compartment, while the trans-compartment was kept buffer-blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and current changes were monitored in real time. After protein insertion, the cis-compartment was rinsed three times with buffer (50 μl each time) to remove unbound protein and avoid pore interference.

[0099] The polypeptide chain sample (SEQ ID NO:3: RRRRRR, hexamethylenetetramine, +6 charge, unmodified 10 nM) was added, and a voltage of 160 mV was applied (voltage optimized to reduce the influence of chain folding), with a sampling rate of 50 kHz and low-pass filtering (2 kHz). All experiments were performed at room temperature (23 ± 1 °C).

[0100] 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 current detection results of the PANX3 biopore mutant protein (S73 mutated to S73N) when the polypeptide chain passed through the nanopore are shown below. Figure 7 As shown, the results indicate that, compared to wild-type PANX3 bioporin, the PANX3 bioporin mutant protein has stable current properties in detecting the current generated by the polypeptide chain, fewer current signal spikes, and a longer amplitude.

[0101] Example 8: Detection of glycosylated peptides using PANX3 biopore mutant protein with amino acid F58 mutated to F58N.

[0102] After the amino acid F58 was mutated to F58N, a single PANX3 biowell mutant protein well was constructed in a lipid bilayer. Detection buffer (0.5M KCl, 10mM HEPES-KOH, pH=8.0, 2mM) was first injected into both chambers. 0.1 mM EDTA was added, with a volume of 1.2 ml / side, ensuring liquid levels were balanced on both sides of the membrane. 0.5 μL of 1,2-bis(phytanyl-n-glycerol-3-phosphocholine) lipid solution was pipetted through a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0103] A 8 ng / ml solution of the PANX3 biopore mutant protein (F58 mutated to F58N) was added to the cis-compartment, while the trans-compartment was kept buffer-blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and current changes were monitored in real time. After protein insertion, the cis-compartment was rinsed three times with buffer (50 μl each time) to remove unbound protein and avoid pore interference.

[0104] A glycosylated peptide sample (GalNAc-Ser-YYY, glycosylation site is serine linked to N-acetylgalactosamine, the whole is negatively charged due to the presence of carboxyl groups on the glycosyl group; 10 nM) was added, a voltage of 160 mV was applied, the sampling rate was 50 kHz, and a low-pass filter of 2 kHz was used. All experiments were performed at room temperature (23 ± 1 °C).

[0105] 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 current detection results of the PANX3 biopore mutant protein (F58 mutated to F58N) glycosylated peptide passing through nanopores are shown below. Figure 8 As shown, the results indicate that, compared to wild-type PANX3 bioporin, the PANX3 bioporin mutant protein exhibits stable current characteristics in detecting glycosylated peptides, with fewer current signal spikes and a narrower signal width.

[0106] The narrowest pore size of the wild-type PANX3 protein is 1.32 nm, making PANX3 protein and its mutants more suitable for detecting large molecular compounds such as peptides, proteins, glycosylated peptides, and polymers.

[0107] In summary, this invention reveals that the wild-type PANX3 bioporin consists of seven identical subunits that are symmetrically assembled around a central axis forming a transmembrane channel. Each subunit comprises four transmembrane domains (TMDs) and two extracellular loops (ELs). Two disulfide bonds (SS1 and SS2) stabilize the extracellular domains, C66-C261 (SS1) and C84-C242 (SS2). Each subunit also possesses intracellular N-terminal and C-terminal domains. The overall channel is funnel-shaped. Compared to existing bioporins, its monomeric structure possesses the potential to form nanopores, its expression and purification steps are simple, and it easily forms stable monopores on lipid membranes, making it widely applicable.

[0108] When one or more amino acids of wild-type PANX3 bioporin are mutated to common amino acids other than the original amino acids, the mutant can be expressed normally and has stable properties compared to wild-type PANX3 bioporin, and its current properties and current signal are significantly improved.

[0109] amino acid sequence:

[0110] SEQ ID NO:1:

[0111] MSLAHTAAEYMLSDALLPDRRGPRLKGLRLELPLDRIVKFVAVGSPLLLMSLAFAQEFSSGSPISCFSPSNFSIRQAAYVDSSCWDSLLHHKQDGPGQDKMKSLWPHKALPYSLLALALLMYLPVLLWQYAAVPALSSDLLFIISELDKSYNRSIRLVQHMLKIRQKSSDPYVFWNELEKARKERYFEFPLLERYLACKQRSHSLVATYLLRNSLLLIFTSATYLYLGHFHLDVFFQEEFSCSIKTGLLSDETHVPNLITCRLTSLSIFQIVSLSSVAIYTILVPVIIYNLTRLCRWDKRLLSVYEMLPAFDLLSRKMLGCPINDLNVILLFLRANISELISFSWLSVLCVLKDTTTQKHNIDTVVDFMTLLAGLEPSKPKHLTNSACDEHP

[0112] DNA sequence:

[0113] SEQ ID NO:2:

[0114]

[0115] The above description is merely 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 PANX3 bioporous mutant protein in single-molecule detection, characterized by: The PANX3 biopore mutant protein is selected from either A or B: A: The amino acid S73 of wild-type PANX3 bioporin is mutated to S73N; B: The amino acid F58 of wild-type PANX3 bioporin is mutated to F58N; The amino acid sequence of wild-type PANX3 bioporin is shown in SEQ ID NO:

1.

2. The application as described in claim 1, characterized in that: Detects one or more of peptides, proteins, glycosylated peptides, and polymers.

3. The application as described in claim 2, characterized in that: Detects peptides or glycosylated peptides.

4. The application as described in claim 3, characterized in that: The method for preparing PANX3 bioporous mutant protein includes the following steps: Step S1: Construct the PANX3 biopore mutant protein vector; Step S2: Expression and purification of PANX3 biopore mutant protein.

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