PANX3 biological pore protein and application of mutant and single molecule detection thereof

Abstract

The invention belongs to the technical field of characterization of target samples, and provides PANX3 biopore protein and application of a mutant and single molecule detection thereof. The PANX3 biopore 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 biopore protein are mutated into common amino acids except original amino acids, compared with the wild type PANX3 biopore protein, the mutant can be normally expressed and has stable properties, and the current property and the current signal of the mutant are obviously improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of characterizing target samples, and particularly relates to PANX3 biochannel protein, its mutants and applications in single molecule detection. Background Art

[0002] As a single molecule detection technology, biochannel proteins (nanopore proteins) were initially mainly applied to nucleic acid sequencing and epigenetic modification analysis. Nowadays, the application boundaries of this technology are constantly expanding and have extended to broader fields such as protein sensing, chemical reaction monitoring and biophysical property characterization. Its core advantage lies in being able to achieve single molecule-by-molecule analysis without labeling and modification, with advantages such as low cost, high throughput and non-labeling.

[0003] The principle of this technology is derived from the Coulter counting method: when a nano-scale pore forms an ionic current path under electrophoresis drive, the passage of target macromolecules will cause current blockage, and the changes in these current signals can be decoded into detailed information about the molecular properties, concentration and structure. This single molecule-level detection ability is the core advantage that differentiates it from traditional detection methods. In the past two decades, biochannel proteins represented by α-hemolysin (α-HL), Mycobacterium smegmatis porin A (MspA), aerolysin (AeL), etc. have become ideal single molecule detection tools.

[0004] However, in practical applications, the types of biochannel proteins are still not rich enough, and it is necessary to continuously search for more biochannel proteins suitable for nanopore sensing to meet different needs. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides PANX3 biochannel protein, its mutants and applications in single molecule detection, aiming to solve the problems mentioned in the background art.

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

[0007] In the second aspect, the present invention provides a PANX3 biochannel mutant protein, which is obtained by mutating the wild-type PANX3 biochannel protein. The amino acid sequence of the wild-type PANX3 biochannel protein is as shown in SEQ ID NO:1, and the mutation means that one or more amino acids of the wild-type PANX3 biochannel protein are mutated into common amino acids other than the original amino acids; The 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.

[0008] In a third aspect, the present invention provides the application of PANX3 bioporin and its mutants in single-molecule detection, for detecting one or more of polypeptides, proteins, glycosylated peptides, and polymers.

[0009] Further, it is selected from the following A or B: A: One or more of the amino acids I74, R75, and S73 of the wild-type PANX3 bioporin are mutated to common amino acids other than the original amino acids; B: One or more of the amino acids F58 and R20 of the wild-type PANX3 bioporin are mutated to common amino acids other than the original amino acids.

[0010] 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.

[0011] Further, the amino acid R75 of the wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; The amino acid R75 of the wild-type PANX3 bioporin is mutated to R75A or R75S.

[0012] Further, the amino acid S73 of the wild-type PANX3 bioporin is mutated to a common amino acid other than the original amino acid; The amino acid S73 of the wild-type PANX3 bioporin is mutated to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G, or S73M; Preferably, the amino acid S73 of the wild-type PANX3 bioporin is mutated to S73N or S73K.

[0013] Further, in B: the amino acid F58 of the wild-type PANX3 biopore protein is mutated to a common amino acid other than the original amino acid; the amino acid F58 of the wild-type PANX3 biopore protein 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 biopore protein is mutated to F58N.

[0014] Further, the amino acid R20 of the wild-type PANX3 biopore protein is mutated to a common amino acid other than the original amino acid; the amino acid R20 of the wild-type PANX3 biopore protein is mutated to R20N or R20L.

[0015] Fourthly, the present invention provides a method for preparing a PANX3 biopore mutant protein, comprising the following steps: Step S1: Construct a vector of the PANX3 biopore protein or the PANX3 biopore mutant protein; Step S2: Express and purify the PANX3 biopore protein or the PANX3 biopore mutant protein.

[0016] The present invention has the following beneficial effects: (1) The wild-type PANX3 biopore protein is composed of seven identical subunits, which are symmetrically assembled around the center 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 domain, C66-C261 (SS1) and C84-C242 (SS2). Each subunit has an intracellular N-terminal and C-terminal domain, and the overall channel is funnel-shaped. Compared with the existing biopore proteins, its monomer structure has the potential to form nanopores, the expression and purification steps are simple, and it is easy to form stable single pores on the lipid membrane, with a wide range of applications.

[0017] (2) After one or more amino acids of the wild-type PANX3 biopore protein are mutated to common amino acids other than the original amino acids, compared with the wild-type PANX3 biopore protein, the mutant can be normally expressed and has stable properties, and its current properties and current signals are significantly improved. Description of the Drawings

[0018] By referring to the following drawings, the exemplary embodiments of the present invention can be more fully understood: Figure 1 It is a top view of the surface structure model of the wild-type PANX3 biopore protein channel of the present invention.

[0019] Figure 2This is a side view of the surface structure model of the wild-type PANX3 biopore protein of the present invention.

[0020] Figure 3 This is a top view of the ribbon structure model of the wild-type PANX3 biopore protein channel of the present invention.

[0021] Figure 4 This is a side view of the ribbon structure model of the wild-type PANX3 biopore protein channel of the present invention.

[0022] Figure 5 This is the purification electrophoresis diagram of the wild-type PANX3 biopore protein and the PANX3 biopore mutant protein with amino acid S73 mutated to S73N in Examples 1 and 3 of the present invention; in the figure, lane 1: protein Marker; lane 2: elution sample of wild-type PANX3; lane 3: elution sample of mutant PANX3 S73N; lane 4: elution sample of wild-type PANX3; lane 5: elution sample of mutant PANX3 S73N; lane 6: elution sample of mutant PANX3 S73N.

[0023] Figure 6 This is the detection result diagram of the nanopore opening current of the PANX3 biopore mutant protein with amino acid S73 mutated to S73N in Example 6 of the present invention.

[0024] Figure 7 This is the detection result diagram of the current when the polypeptide chain passes through the nanopore of the PANX3 biopore mutant protein with amino acid S73 mutated to S73N in Example 7 of the present invention.

[0025] Figure 8 This is the detection result diagram of the current when the glycosylated peptide passes through the nanopore of the PANX3 biopore mutant protein with amino acid F58 mutated to F58N in Example 8 of the present invention. Detailed implementation manners

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by 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 only used to explain the present invention and are not used to limit the present invention.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and do not limit the present invention.

[0028] The embodiments of the present invention provide a PANX3 biopore protein. The PANX3 biopore protein is composed of seven identical subunits, and the overall channel is funnel-shaped. The amino acid sequence is as shown in SEQ ID NO: 1.

[0029] In some embodiments, the present invention provides a PANX3 biological pore mutant protein, which is obtained by mutating the wild-type PANX3 biological pore protein. The amino acid sequence of the wild-type PANX3 biological pore protein is shown in SEQ ID NO:1. The mutation means that 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. 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.

[0030] In some embodiments, the present invention provides the application of the PANX3 biological pore protein and its mutants in single-molecule detection, for detecting one or more of polypeptides, proteins, glycosylated peptides, and polymers.

[0031] In some embodiments, it is selected from the following A or B: A: One or more of the amino acids I74, R75, and S73 of the wild-type PANX3 biological pore protein are mutated into common amino acids other than the original amino acids; B: One or more of the amino acids F58 and R20 of the wild-type PANX3 biological pore protein are mutated into common amino acids other than the original amino acids.

[0032] In some embodiments, in A: the amino acid I74 of the wild-type PANX3 biological pore protein is mutated into a common amino acid other than the original amino acid; the amino acid I74 of the wild-type PANX3 biological pore protein is mutated into I74M, I74P, I74L, I74G, I74R, I74Q, I74E, I74C, I74W, I74H, I74K, I74T, I74A, I74S, I74Y, I74N, I74V, or I74D.

[0033] In some embodiments, the amino acid I74 of the wild-type PANX3 biological pore protein is mutated into I74A, I74L, or I74N.

[0034] In some embodiments, the amino acid R75 of the wild-type PANX3 biological pore protein is mutated into a common amino acid other than the original amino acid; the amino acid R75 of the wild-type PANX3 biological pore protein is mutated into R75A or R75S.

[0035] In some embodiments, the amino acid S73 of the wild-type PANX3 biopore protein is mutated to a common amino acid other than the original amino acid; the amino acid S73 of the wild-type PANX3 biopore protein is mutated to S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G or S73M.

[0036] In some embodiments, the amino acid S73 of the wild-type PANX3 biopore protein is mutated to S73N or S73K.

[0037] In some embodiments, in B: the amino acid F58 of the wild-type PANX3 biopore protein is mutated to a common amino acid other than the original amino acid; the amino acid F58 of the wild-type PANX3 biopore protein is mutated to F58N, F58H, F58Q, F58S, F58L, F58D, F58C, F58I, F58P, F58T, F58G, F58M, F58W or F58R.

[0038] In some embodiments, the amino acid F58 of the wild-type PANX3 biopore protein is mutated to F58N.

[0039] In some embodiments, the amino acid R20 of the wild-type PANX3 biopore protein is mutated to a common amino acid other than the original amino acid; the amino acid R20 of the wild-type PANX3 biopore protein is mutated to R20N or R20L.

[0040] In some embodiments, the present invention provides a method for preparing a PANX3 biopore mutant protein, comprising the following steps: Step S1: Construct a vector for the PANX3 biopore protein or the PANX3 biopore mutant protein; Step S2: Express and purify the PANX3 biopore protein or the PANX3 biopore mutant protein.

[0041] Example 1: Expression and purification of the wild-type PANX3 biopore protein 1. Gene construction and vector design (1) Synthesis of the target gene and cloning of the initial vector: Synthesize the PANX3 (SEQ ID NO:2) DNA fragment. Clone the fragment into the pcDNA3.4 vector and add an mEGFP-6xHis tag at the C-terminus.

[0042] (2)Transfection of COS-7 cells: Culture COS-7 cells in low-glucose DMEM medium containing 1x penicillin / streptomycin and 10% fetal bovine serum. For each 35 mm culture dish, prepare 1 μg of DNA containing hPANX3-mEGFP-6xHis and 4 μg of PEI-MAX for transfection. Seed the transfected cells into a glass-bottom culture dish. Fix the cells with phosphate buffer containing 3.7% formaldehyde. Stain the cells with wheat germ agglutinin-Fluor 647. Capture and analyze the fluorescence images using a confocal laser scanning microscope.

[0043] (3)Transfection of Expi293F cells: Use Expi293F cells and culture them in HE400AZ medium. When the cell density reaches cells / ml, use 0.8 μg of DNA containing hPANX3-6xHis and 3.2 μg of PEI-MAX for transfection per 1 ml of medium. Harvest the cells overexpressing hPANX3-6xHis 48 hours after transfection.

[0044] (4)Cell lysis: Sonicate the harvested cells in TBS buffer (50 mM Tris-HCl, pH = 8.0, 150 mM NaCl) containing protease inhibitors.

[0045] (5)Membrane fraction collection: Collect the membrane fraction by ultracentrifugation. Suspend the membrane fraction in TBS buffer supplemented with 2% n-dodecyl-β-D-maltoside, 0.4% cholesterol hemisuccinate, and protease inhibitors.

[0046] 2. Purification by affinity chromatography (1)Gently mix and incubate at 4°C for 2 hours to solubilize the membrane proteins. Load the solubilized proteins onto TALON resin.

[0047] (2)Wash the contaminating proteins: Wash with SEC buffer containing 15 mM imidazole (TBS buffer containing 0.003% glyco-diosgenin) at 25 column volumes (each column volume is 10 ml).

[0048] (3)Elute the target protein: Elute with SEC buffer containing 250 mM imidazole at 5 column volumes.

[0049] (4)Size exclusion chromatography: Further purify through a size exclusion chromatography column and collect the single symmetric peak of the target protein (monitored by UV absorption at 280 nm).

[0050] The top view of the surface structure model of wild-type PANX3 biochannel protein is as shown in Figure 1As shown; the side view of the surface structure model of the wild-type PANX3 bioporin channel is as Figure 2 shown; the top view of the ribbon structure model of the wild-type PANX3 bioporin channel is as Figure 3 shown; the side view of the ribbon structure model of the wild-type PANX3 bioporin channel is as Figure 4 shown. The results show that: the wild-type PANX3 bioporin is composed of seven identical subunits, which are symmetrically assembled around the center axis forming a transmembrane channel, and the overall channel is funnel-shaped.

[0051] Example 2: Characterization of wild-type PANX3 bioporin The 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 as shown in SEQ ID NO:2.

[0052] A single wild-type PANX3 bioporin pore was established in the lipid bilayer. First, the detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM , 0.1 mM EDTA) was injected into both chambers, with a volume of 1.2 ml / side to ensure the liquid levels on both sides of the membrane were balanced. 0.5 μl of 1,2-diphytanyl-n-glycerol-3-phosphocholine (Avanti Polar Lipids) lipid solution was aspirated with a pipette tip and blown on a Teflon horizontal pore with a diameter of about 20 μm to form an artificial lipid bilayer.

[0053] The wild-type PANX3 bioporin solution (8 ng / ml) was added to the cis chamber, and the trans chamber was maintained as a buffer blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and the current change was monitored in real time. After the protein was inserted, the cis chamber was rinsed 3 times with buffer (50 μl each time) to remove unbound protein and avoid pore interference. All experiments were completed at room temperature (23 ± 1 °C).

[0054] The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz, digitized at 500 kHz, and data acquisition was controlled with Origin. The results show that: the open pore current signal of the wild-type PANX3 bioporin has a large noise and many current spikes.

[0055] Example 3: Expression and purification of the PANX3 bioporin mutant protein with amino acid S73 mutated to S73N 1. Gene construction and vector design (1)Synthesis of the target gene and cloning into the initial vector: Synthesize the DNA fragment of the PANX3 biological pore protein with the amino acid S73 mutated to S73N. Clone the fragment into the pcDNA3.4 vector and add the mEGFP-6xHis tag at the C-terminus.

[0056] (2)Transfection of COS-7 cells: Culture COS-7 cells in low-glucose DMEM medium containing 1x penicillin / streptomycin and 10% fetal bovine serum. For each 35 mm culture dish, prepare 1 μg of DNA containing hPANX3-mEGFP-6xHis and 4 μg of PEI-MAX for transfection. Seed the transfected cells into a glass-bottom culture dish. Fix the cells with phosphate-buffered saline containing 3.7% formaldehyde. Stain the cells with wheat germ agglutinin-Fluor 647. Capture and analyze the fluorescence images using a confocal laser scanning microscope.

[0057] (3)Transfection of Expi293F cells: Use Expi293F cells and culture them in HE400AZ medium. When the cell density reaches cells / ml, use 0.8 μg of DNA containing hPANX3-6xHis and 3.2 μg of PEI-MAX for transfection per 1 ml of medium. Harvest the cells overexpressing hPANX3-6xHis 48 hours after transfection.

[0058] (4)Cell lysis: Ultrasonically lyse the harvested cells in TBS buffer (50 mM Tris-HCl, pH = 8.0, 150 mM NaCl) containing protease inhibitors.

[0059] (5)Collection of membrane fraction: Collect the membrane fraction by ultracentrifugation. Suspend the membrane fraction in TBS buffer supplemented with 2% n-dodecyl-β-D-maltoside, 0.4% cholesterol hemisuccinate, and protease inhibitors.

[0060] 2. Purification by affinity chromatography (1)Gently mix and incubate at 4°C for 2 hours to solubilize the membrane proteins. Load the solubilized proteins onto the TALON resin.

[0061] (2)Wash away the contaminating proteins: Wash with SEC buffer containing 15 mM imidazole (TBS buffer containing 0.003% glyco-diosgenin) at 25 column volumes (each column volume is 10 ml).

[0062] (3)Elute the target protein: Elute with SEC buffer containing 250 mM imidazole at 5 column volumes.

[0063] (4)Size exclusion chromatography: Further purification was carried out through a size exclusion chromatography column, and a single symmetric target protein peak was collected (monitored by ultraviolet absorption at 280 nm).

[0064] The purification electrophoresis results of the wild-type PANX3 biopore protein in Example 1 and the PANX3 biopore mutant protein with amino acid S73 mutated to S73N in Example 3 are as Figure 5 shown.

[0065] Example 4: Detection of polypeptide chains using wild-type PANX3 biopore protein A single wild-type PANX3 biopore protein pore was established in the lipid bilayer. First, the detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM , 0.1 mM EDTA) was injected into both chambers, with a volume of 1.2 ml / side, ensuring that the liquid levels on both sides of the membrane were balanced. 0.5 μl of 1,2-diphytanyl-n glycerol-3-phosphocholine (Avanti Polar Lipids) lipid solution was aspirated with a pipette tip and blown out on a Teflon horizontal pore diameter of about 20 μm to form an artificial lipid bilayer.

[0066] The wild-type PANX3 biopore protein solution (8 ng / ml) was added to the cis chamber, and the trans chamber was maintained as a buffer blank. A transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) was applied, and the current change was monitored in real time. After the protein was inserted, the cis chamber was rinsed 3 times with buffer (50 μl each time) to remove unbound proteins and avoid pore interference.

[0067] The polypeptide chain sample (SEQ ID NO:3: RRRRRR, hexameric arginine, +6 charge, unmodified 10 nM) was added, a voltage of 160 mV was applied (optimized voltage to reduce the influence of chain folding), the sampling rate was 50 kHz, and the low-pass filter was 2 kHz. All experiments were completed at room temperature (23 ± 1 °C). The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz, digitized at 500 kHz, and the data acquisition was controlled by Origin. The results showed that when the wild-type PANX3 biopore protein detected the polypeptide chain, the current signal had a large noise, many current spikes, and a large fluctuation amplitude of the current baseline.

[0068] Example 5: Detection of glycosylated peptides using wild-type PANX3 biopore protein A single wild-type PANX3 biopore protein pore was established in the lipid bilayer. First, the detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM , 0.1 mM EDTA), with a volume of 1.2 ml / side, ensuring the liquid levels on both sides of the membrane are balanced. Pipette 0.5 μl of 1,2-diphytanyl-nglycerol-3-phosphocholine (Avanti Polar Lipids) lipid solution and blow it out on a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0069] Add the wild-type PANX3 bioporin solution (8 ng / ml) to the cis chamber and maintain the trans chamber as a buffer blank. Apply a transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV) and monitor the current changes in real time. After protein insertion, rinse the cis chamber 3 times with buffer (50 μl each time) to remove unbound proteins and avoid pore interference.

[0070] Add the glycosylated peptide sample (GalNAc-Ser-YYY, the glycosylation site is serine-linked N-acetylgalactosamine, and it is negatively charged overall due to the carboxyl group on the sugar; 10 nM), apply a voltage of 160 mV, a sampling rate of 50 kHz, and a low-pass filter of 2 kHz. All experiments were completed at room temperature (23 ± 1 °C). The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz, digitized at 500 kHz, and data acquisition was controlled with Origin. The results showed that when the wild-type PANX3 bioporin detected the glycosylated peptide, the current signal had a large noise, many current spikes, a large fluctuation amplitude of the current baseline, and there were some pore-blocking phenomena.

[0071] Example 6: Characterization of the PANX3 bioporin mutant protein after amino acid S73 was mutated to S73N After amino acid S73 was mutated to S73N, a single PANX3 bioporin mutant protein pore was established in the lipid bilayer. First, inject the detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM , 0.1 mM EDTA) into both chambers, with a volume of 1.2 ml / side, ensuring the liquid levels on both sides of the membrane are balanced. Pipette 0.5 μl of 1,2-diphytanyl-n glycerol-3-phosphocholine (Avanti Polar Lipids) lipid solution and blow it out on a Teflon horizontal pore with a diameter of approximately 20 μm to form an artificial lipid bilayer.

[0072] Add the PANX3 biochannel mutant protein solution (8 ng / ml) with the amino acid S73 mutated to S73N to the cis chamber, and keep the buffer blank in the trans chamber. Apply a transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV), and monitor the current change in real time. After the protein is inserted, rinse the cis chamber 3 times with buffer (50 μl each time) to remove unbound protein and avoid pore interference. All experiments were completed at room temperature (23 ± 1 °C).

[0073] The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz, digitized at 500 kHz, and data acquisition was controlled by Origin. For the PANX3 biochannel mutant protein with the amino acid S73 mutated to S73N, the results of nanopore open pore current detection are as Figure 6 shown. The results show that compared with the wild-type PANX3 biochannel protein, the current properties generated by the PANX3 biochannel mutant protein detection are stable, the current signal width is narrower, and there are fewer spikes.

[0074] Example 7: Detection of polypeptide chains using the PANX3 biochannel mutant protein with the amino acid S73 mutated to S73N After the amino acid S73 was mutated to S73N, a single PANX3 biochannel mutant protein pore was established in the lipid bilayer. First, inject the detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM , 0.1 mM EDTA) into both chambers, with a volume of 1.2 ml / side, to ensure that the liquid levels on both sides of the membrane are balanced. Pipette 0.5 μl of 1,2-diphytanyl-n-glycerol-3-phosphocholine (Avanti Polar Lipids) lipid solution and blow it out on a Teflon horizontal pore with a diameter of about 20 μm to form an artificial lipid bilayer.

[0075] Add the PANX3 biochannel mutant protein solution (8 ng / ml) with the amino acid S73 mutated to S73N to the cis chamber, and keep the buffer blank in the trans chamber. Apply a transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV), and monitor the current change in real time. After the protein is inserted, rinse the cis chamber 3 times with buffer (50 μl each time) to remove unbound protein and avoid pore interference.

[0076] Add a polypeptide chain sample (SEQ ID NO:3: RRRRRR, hexameric arginine, +6 charge, unmodified, 10 nM), apply a voltage of 160 mV (optimized voltage to reduce the influence of chain folding), sampling rate of 50 kHz, and low-pass filter (2 kHz). All experiments were completed at room temperature (23 ± 1 °C).

[0077] The analog signal was low-pass filtered with a 4-pole Bessel filter at 100 kHz, digitized at 500 kHz, and data acquisition was controlled by Origin. For the PANX3 biological pore mutant protein after the amino acid S73 was mutated to S73N, the current detection results when the polypeptide chain passed through the nanopore are as Figure 7 shown. The results show that: compared with the wild-type PANX3 biological pore protein, the PANX3 biological pore mutant protein has a stable current property when detecting the polypeptide chain, with fewer current signal spikes and a longer up-and-down amplitude.

[0078] Example 8: Detection of glycosylated peptides using the PANX3 biological pore mutant protein after the amino acid F58 was mutated to F58N After the amino acid F58 was mutated to F58N, a single PANX3 biological pore mutant protein pore was established in the lipid bilayer. First, inject the detection buffer (0.5 M KCl, 10 mM HEPES-KOH, pH = 8.0, 2 mM of , 0.1 mM EDTA) into both chambers, with a volume of 1.2 ml / side, to ensure the liquid levels on both sides of the membrane are balanced. Use a pipette tip to aspirate 0.5 μl of 1,2-diphytanyl-n glycerol-3-phosphocholine (Avanti Polar Lipids) lipid solution and blow it out on a Teflon horizontal pore with a diameter of about 20 μm to form an artificial lipid bilayer.

[0079] Add the PANX3 biological pore mutant protein solution (8 ng / ml) after the amino acid F58 was mutated to F58N to the cis chamber, and maintain the buffer blank in the trans chamber. Apply a transmembrane voltage of 160 mV (Axopatch 200B amplifier, voltage accuracy ±0.1 mV), and monitor the current change in real time. After the protein was inserted, rinse the cis chamber 3 times with the buffer (50 μl each time) to remove the unbound protein and avoid pore interference.

[0080] Add a glycosylated peptide sample (GalNAc-Ser-YYY, the glycosylation site is serine linked to N-acetylgalactosamine, and it is negatively charged overall due to the carboxyl group on the sugar; 10 nM), apply a voltage of 160 mV, sampling rate of 50 kHz, and low-pass filter at 2 kHz. All experiments were completed at room temperature (23 ± 1 °C).

[0081] The analog signal is low-pass filtered with a 4-pole Bessel filter at 100 kHz, digitized at 500 kHz, and data acquisition is controlled by Origin. For the PANX3 biological pore mutant protein after the amino acid F58 is mutated to F58N, the current detection results when the glycosylated peptide passes through the nanopore are as Figure 8 shown. The results show that: compared with the wild-type PANX3 biological pore protein, the current properties generated by the PANX3 biological pore mutant protein for detecting glycosylated peptides are stable, with fewer current signal spikes and a narrower width.

[0082] The narrowest pore diameter of the wild-type PANX3 protein is 1.32 nm. Therefore, the PANX3 protein and its mutants are more suitable for detecting large molecular compounds such as polypeptides, proteins, glycosylated peptides, and polymers.

[0083] In summary, the present invention discovers that the wild-type PANX3 biological pore protein is composed of seven identical subunits, which are symmetrically assembled around the center 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 domain, C66-C261 (SS1) and C84-C242 (SS2). Each subunit has an intracellular N-terminal and C-terminal domain. The overall channel is funnel-shaped. Compared with existing biological pore proteins, its monomer structure has the potential to form nanopores, the expression and purification steps are simple, and it is easy to form a stable single pore on the lipid membrane, with a wide range of applications.

[0084] After one or more amino acids of the wild-type PANX3 biological pore protein are mutated to 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 has stable properties, and its current properties and current signals are significantly improved.

[0085] Amino acid sequence: SEQ ID NO:1: MSLAHTAAEYMLSDALLPDRRGPRLKGLRLELPLDRIVKFVAVGSPLLLMSLAFAQEFSSGSPISCFSPSNFSIRQAAYVDSSCWDSLLHHKQDGPGQDKMKSLWPHKALPYSLLALALLMYLPVLLWQYAAVPALSSDLLFIISELDKSYNRSIRLVQHMLKIRQKSSDPYVFWNELEKARKERYFEFPLLERYLACKQRSHSLVATYLLRNSLLLIFTSATYLYLGHFHLDVFFQEEFSCSIKTGLLSDETHVPNLITCRLTSLSIFQIVSLSSVAIYTILVPVIIYNLTRLCRWDKRLLSVYEMLPAFDLLSRKMLGCPINDLNVILLFLRANISELISFSWLSVLCVLKDTTTQKHNIDTVVDFMTLLAGLEPSKPKHLTNSACDEHP

[0086] DNA sequence: SEQ ID NO:2:

[0087] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. PANX3 biochannel protein, characterized in that: The PANX3 biochannel protein is composed of seven identical subunits, and the overall channel is funnel-shaped. The amino acid sequence is shown in SEQ ID NO:

1.

2. The PANX3 biological pore mutant protein is characterized in that: It is obtained by mutating the wild-type PANX3 biochannel protein. The amino acid sequence of the wild-type PANX3 biochannel protein is shown in SEQ ID NO:

1. The mutation means that one or more amino acids of the wild-type PANX3 biochannel protein are mutated into common amino acids other than the original amino acids. The 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.

3. Application of PANX3 pannexin and its mutants in single molecule detection, characterized in that: Detect one or more of polypeptides, proteins, glycosylated peptides, and polymers.

4. The PANX3 biological pore mutant protein according to claim 2, characterized in that: Selected from the following A or B: A: One or more of the amino acids I74, R75, and S73 of the wild-type PANX3 biochannel protein are mutated into common amino acids other than the original amino acids. B: One or more of the amino acids F58 and R20 of the wild-type PANX3 biochannel protein are mutated into common amino acids other than the original amino acids.

5. The PANX3 biological pore mutant protein according to claim 4, characterized in that: In A: The amino acid I74 of the wild-type PANX3 biochannel protein is mutated into a common amino acid other than the original amino acid; the amino acid I74 of the wild-type PANX3 biochannel protein is mutated into I74M, I74P, I74L, I74G, I74R, I74Q, I74E, I74C, I74W, I74H, I74K, I74T, I74A, I74S, I74Y, I74N, I74V, or I74D.

6. The PANX3 biological pore mutant protein according to claim 5, characterized in that: The amino acid R75 of the wild-type PANX3 biochannel protein is mutated into a common amino acid other than the original amino acid; the amino acid R75 of the wild-type PANX3 biochannel protein is mutated into R75A or R75S.

7. The PANX3 biological pore mutant protein according to claim 6, characterized in that: The amino acid S73 of the wild-type PANX3 biochannel protein is mutated into a common amino acid other than the original amino acid; the amino acid S73 of the wild-type PANX3 biochannel protein is mutated into S73N, S73K, S73H, S73D, S73W, S73R, S73F, S73E, S73P, S73L, S73A, S73V, S73G, or S73M.

8. The PANX3 biological pore mutant protein according to claim 4, characterized in that: In B: The amino acid F58 of the wild-type PANX3 biochannel protein is mutated into a common amino acid other than the original amino acid; the amino acid F58 of the wild-type PANX3 biochannel protein is mutated into F58N, F58H, F58Q, F58S, F58L, F58D, F58C, F58I, F58P, F58T, F58G, F58M, F58W, or F58R.

9. A PANX3 biological pore mutant protein according to claim 8, characterized in that: The amino acid R20 of the wild-type PANX3 biochannel protein is mutated into a common amino acid other than the original amino acid; the amino acid R20 of the wild-type PANX3 biochannel protein is mutated into R20N or R20L.

10. A method for preparing PANX3 biochannel protein and its mutants, characterized in that: Includes the following steps: Step S1: Construct a PANX3 biochannel protein or PANX3 biochannel mutant protein vector. Step S2: Construct the expression and purification of PANX3 biochannel protein or PANX3 biochannel mutant protein.

Images