Application of PANX2 protein and mutant thereof as biopore protein

Abstract

The invention belongs to the technical field of characterization of target analytes, and provides application of PANX2 protein and a mutant thereof as biopore protein. The bioporous protein is used for detecting one or more of metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic agents, adenosine triphosphate and monosaccharides. After one or more amino acids of the wild type PANX2 protein are mutated into common amino acids except original amino acids, compared with the wild type PANX2 protein, the PANX2 protein mutant can be normally expressed and is stable in property, a current signal is obviously improved during detection, the up-down amplitude of the current signal is longer, and burrs are fewer.

Description

Technical Field

[0001] The present invention belongs to the technical field of characterizing target analytes, and particularly relates to the application of PANX2 protein and its mutants as biological pore proteins. Background Art

[0002] In the sensing system of protein nanopores, the core role belongs to the nanopore sensor, that is, the nanopore protein. Although factors such as temperature, pH value, type and concentration of salts in the experimental environment will also affect the characteristics of the nanopore and the phenomenon of target molecules blocking ion current, the nanopore protein is always the key to determining the sensing performance. In order to obtain stable and non-gated current recordings, almost all nanopore proteins need to carry out nanopore sensing work in a chemically stable and hydrophilic environment. Different nanopore proteins show irreplaceable advantages in specific studies due to their unique structures, sizes, lengths and chemical environments, such as accurately detecting molecules and subunits with special properties or conformations.

[0003] However, the reality is that currently, there are still limited means to precisely modify natural nanopore proteins to meet diverse application requirements, mainly relying on protein modification techniques. Therefore, it is necessary to continuously search for more nanopore proteins suitable for nanopore sensing to meet different needs. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides the application of PANX2 protein and its mutants as biological pore proteins, aiming to solve the problems mentioned in the background art.

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

[0006] In the second aspect, the present invention provides a PANX2 protein mutant, which is obtained by mutating the wild-type PANX2 protein, and the amino acid sequence of the wild-type PANX2 protein is as shown in SEQ ID NO: 1. The mutation means that one or more amino acids of the wild-type PANX2 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.

[0007] Further, the PANX2 protein mutant is selected from the following A or B: A: One or more of the amino acids R89, D90, and R96 of the wild-type PANX2 protein are mutated to common amino acids other than the original amino acids; B: One or more of the amino acids L40 and A36 of the wild-type PANX2 protein are mutated to common amino acids other than the original amino acids.

[0008] Further, in A: The amino acid R89 of the wild-type PANX2 protein is mutated to a common amino acid other than the original amino acid; The amino acid R89 of the wild-type PANX2 protein is mutated to R89M, R89P, R89L, R89G, R89I, R89Q, R89E, R89C, R89W, R89H, R89K, R89T, R89A, R89S, R89Y, R89N, R89V, or R89D; Preferably, the amino acid R89 of the wild-type PANX2 protein is mutated to R89T, R89L, or R89N; The amino acid D90 of the wild-type PANX2 protein is mutated to a common amino acid other than the original amino acid; The amino acid D90 of the wild-type PANX2 protein is mutated to D90S; The amino acid R96 of the wild-type PANX2 protein is mutated to a common amino acid other than the original amino acid; The amino acid R96 of the wild-type PANX2 protein is mutated to R96N, R96K, R96H, R96D, R96W, R96S, R96F, R96E, R96P, R96L, R96A, R96V, R96G, or R96M; Preferably, the amino acid R96 of the wild-type PANX2 protein is mutated to R96N or R96K.

[0009] Further, 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; Preferably, the amino acid L40 of the wild-type PANX2 protein is mutated to L40I; 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.

[0010] In a third aspect, the present invention provides a product of a PANX2 protein and its mutant, and the product is a composition, a complex, or a kit.

[0011] Fourthly, the present invention provides a method for preparing a PANX2 protein and its mutants, comprising the following steps: Step S1: Construct a vector of the PANX2 protein or the PANX2 protein mutant; Step S2: Express and purify the PANX2 protein or the PANX2 protein mutant.

[0012] Fifthly, the present invention provides a membrane layer embedded with the PANX2 protein and its mutants, and the membrane layer is a lipid bilayer.

[0013] The present invention has the following beneficial effects: (1) The wild-type PANX2 protein is composed of seven identical subunits, which are symmetrically assembled around the center axis forming a transmembrane channel. Each subunit contains four transmembrane domains TM1-TM4. The N-terminus and C-terminus of TM1 and TM4 are respectively anchored intracellularly. TM1 and TM2 are helices within the pore, and TM3 and TM4 are arranged on the periphery of the channel. The overall channel is funnel-shaped. Compared with the existing nanopore proteins, its cloning expression and purification processes are simple, easy for protein assembly of biological nanopores, with a wide application range, applicable to rapid detection of pathogens and gene expression.

[0014] (2) The 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 agents, adenosine triphosphate, and monosaccharides. And through mutation experiments, it is found that after one or more amino acids of the wild-type PANX2 protein are mutated to common amino acids other than the original amino acids, it can be normally expressed. Compared with the wild-type PANX2 protein, the PANX2 protein mutant has stable properties, and the current signal is significantly improved during detection, with a longer up and down amplitude of the current signal and fewer burrs. Description of the Drawings

[0015] 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 PANX2 protein channel of the present invention.

[0016] Figure 2 It is a side view of the surface structure model of the wild-type PANX2 protein channel of the present invention.

[0017] Figure 3 It is a top view of the ribbon structure model of the wild-type PANX2 protein channel of the present invention.

[0018] Figure 4 It is a side view of the ribbon structure model of the wild-type PANX2 protein channel of the present invention.

[0019] Figure 5Purification electrophoresis diagrams of the wild-type PANX2 protein and the PANX2 protein mutant with amino acid L40 mutated to L40I in Example 1 and Example 4 of the present invention; in the figure, lane 1: protein Marker; lane 2: elution sample of wild-type PANX2; lane 3: elution sample of PANX2 L40I mutant; lane 4: elution sample of wild-type PANX2; lane 5: elution sample of PANX2 L40I mutant; lane 6: elution sample of PANX2 L40I mutant.

[0020] Figure 6 This is the current detection result diagram when adenosine triphosphate passes through the pore of the wild-type PANX2 protein in Example 3 of the present invention.

[0021] Figure 7 This is the nanopore opening current detection result diagram of the PANX2 protein mutant with amino acid L40 mutated to L40I in Example 5 of the present invention.

[0022] Figure 8 This is the current detection result diagram when adenosine triphosphate passes through the pore of the PANX2 protein mutant with amino acid L40 mutated to L40I in Example 6 of the present invention.

[0023] Figure 9 This is the statistical chart of the pore passing time and the number of events when adenosine triphosphate passes through the pore of the PANX2 protein mutant with amino acid L40 mutated to L40I in Example 6 of the present invention. Detailed implementation manners

[0024] 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.

[0025] 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.

[0026] The embodiments of the present invention provide an application of a PANX2 protein and its mutant as a biological pore protein, and the biological pore protein detects one or more of metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic agents, adenosine triphosphate, and monosaccharides.

[0027] In some embodiments, the PANX2 protein mutant is obtained by mutating the wild-type PANX2 protein, and the amino acid sequence of the wild-type PANX2 protein is shown in SEQ ID NO:1. The mutation means that one or more amino acids of the wild-type PANX2 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.

[0028] In some embodiments, the PANX2 protein mutant is selected from the following A or B: A: One or more of the amino acids R89, D90, and R96 of the wild-type PANX2 protein are mutated into common amino acids other than the original amino acids. B: One or more of the amino acids L40 and A36 of the wild-type PANX2 protein are mutated into common amino acids other than the original amino acids.

[0029] In some embodiments, in A: The amino acid R89 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid R89 of the wild-type PANX2 protein is mutated into R89M, R89P, R89L, R89G, R89I, R89Q, R89E, R89C, R89W, R89H, R89K, R89T, R89A, R89S, R89Y, R89N, R89V, or R89D.

[0030] In some embodiments, the amino acid R89 of the wild-type PANX2 protein is mutated into R89T, R89L, or R89N.

[0031] In some embodiments, the amino acid D90 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid D90 of the wild-type PANX2 protein is mutated into D90S.

[0032] In some embodiments, the amino acid R96 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid R96 of the wild-type PANX2 protein is mutated into R96N, R96K, R96H, R96D, R96W, R96S, R96F, R96E, R96P, R96L, R96A, R96V, R96G, or R96M.

[0033] In some embodiments, the amino acid R96 of the wild-type PANX2 protein is mutated into R96N or R96K.

[0034] 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.

[0035] In some embodiments, the amino acid L40 of the wild-type PANX2 protein is mutated to L40I.

[0036] 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.

[0037] In some embodiments, the present invention provides a product of a PANX2 protein and its mutants, and the product is a composition, a complex or a kit.

[0038] In some embodiments, the present invention provides a method for preparing a PANX2 protein and its mutants, comprising the following steps: Step S1: Construct a vector of the PANX2 protein or a mutant of the PANX2 protein; Step S2: Express and purify the PANX2 protein or a mutant of the PANX2 protein.

[0039] In some embodiments, the present invention provides a membrane layer incorporating the PANX2 protein and its mutants, and the membrane layer is a lipid bilayer.

[0040] Example 1: Expression and purification of wild-type PANX2 protein 1. Gene construction and vector design (1) Synthesis of the target gene and cloning of the initial vector: Synthesize the PANX2 (SEQ ID NO:2) DNA fragment. Clone the fragment into the modified yeast expression vector pPICZ-B, and the vector contains: C-terminal fusion tag: PreScission protease cleavage site Tag (for subsequent purification and detection).

[0041] (2) Transformation: Use the Pichia pastoris strain SMD1163H and transfer the constructed expression vector into it by a standard method.

[0042] (3) Induced expression: Culture the transformed yeast cells to the logarithmic growth phase and induce the expression of PANX2EM by methanol.

[0043] (4)Cell disruption: The induced yeast cells were collected and disrupted by grinding with a grinder. The disrupted cells were resuspended in buffer A (50 mM Tris-HCl, pH 8.0, 150 mM NaCl), and protease inhibitor cocktail was added: leupeptin (2.5 μg / ml), pepstatin A (1 μg / ml), AEBSF (100 μg / ml), aprotinin (3 μg / ml), benzamidine (1 mM), PMSF (200 μM). DNase I (GoldBio, D-300-1) was added to digest nucleic acids.

[0044] (5)Membrane protein extraction: 1% (w / v) detergent LMNG (lauryl maltose neopentyl glycol) was added, and the mixture was stirred at 4°C for 2 hours. After centrifugation at 30000×g for 1 hour, the supernatant containing the target protein was collected.

[0045] 2. Affinity chromatography purification (1)Cobalt ion resin binding: The supernatant was incubated with 3 ml of cobalt ion resin at 4°C for 3 hours to bind the protein with the tag. Tag-bound protein.

[0046] (2)Washing of miscellaneous proteins: 10 column volumes of washing buffer (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 20 mM imidazole, 85 μM GDN) were used to wash away non-specifically bound proteins. The detergent was replaced with GDN (glycyrrhizin) to maintain the stability of the membrane protein.

[0047] (3)Elution of target protein: The bound protein was eluted with elution buffer (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 200 mM imidazole, 85 μM GDN), and the eluate was collected.

[0048] 3. Tag removal and size exclusion chromatography (1)Tag cleavage: PreScission protease was added to the eluate and incubated at 4°C overnight to cleave the C-terminal tag. Tag.

[0049] (2)Gel filtration chromatography: The digested protein was purified by a size exclusion chromatography column using a buffer of 20 mM Tris-HCl (pH = 8.0), 150 mM NaCl, and 40 μM GDN. The single symmetric peak of the target protein was collected (monitored by UV absorption at 280 nm).

[0050] 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 Top view of the ribbon structure model of wild-type PANX2 protein channel, where parts with the same color represent one protein monomer. Figure 4 Side view of the ribbon structure model of wild-type PANX2 protein channel, where parts with the same color represent one protein monomer. The results show that wild-type PANX2 protein consists of seven identical subunits, which are symmetrically assembled around the axis of the transmembrane channel. Each subunit contains 4 transmembrane domains TM1 - TM4. The N-terminus and C-terminus of TM1 and TM4 are anchored intracellularly respectively. TM1 and TM2 are in the pore helix, and TM3 and TM4 are arranged on the periphery of the channel. The overall channel is funnel-shaped.

[0051] Example 2: Characterization of wild-type PANX2 protein The 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 as shown in SEQ ID NO:2.

[0052] (1) Preparation of lipid bilayer: Dissolve 2 mg of DPhPC (dipalmitoyl phosphatidylcholine) in chloroform, and drop it on both sides of a Teflon film (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 both the cis chamber and the trans chamber.

[0053] (2) Protein insertion: Add the purified wild-type PANX2 protein (8 ng / ml) to the cis chamber. The Axopatch 200B integrated patch clamp amplifier applies a voltage of 180 mV (positive on the opposite side) between the two chambers and measures the ionic current passing through the pore. Add the wild-type PANX2 protein to the grounded cis chamber at a concentration of 8.0 ng / ml. Once a pore is inserted, the compartment is rinsed with the experimental buffer to avoid further insertion. 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 by Origin. The results show that the open pore current signal of wild-type PANX2 protein has a large noise.

[0055] Example 3: Detection of adenosine triphosphate using wild-type PANX2 protein (1) Preparation of lipid bilayer: Dissolve 2 mg of DPhPC in chloroform, and drop it on both sides of a Teflon film (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 both the cis chamber and the trans chamber.

[0056] (2)Protein insertion: The purified wild-type PANX2 protein (8 ng / ml) was added to the cis chamber. The Axopatch 200B integrated patch clamp amplifier applied a voltage of 180 mV (positive on the contralateral side) between the two chambers and measured the ionic current passing through the pore. The wild-type PANX2 protein was added to the grounded cis chamber at a concentration of 8.0 ng / ml. Once a pore was inserted, the compartment was rinsed with the experimental buffer to avoid further insertion. 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 and digitized at 500 kHz. Data acquisition was controlled by Origin.

[0057] (3)ATP samples were added to the cis chamber, a voltage of 160 mV was applied, and the current was recorded using an Axopatch 200B amplifier (bandwidth 10 kHz). The data was digitized at a sampling rate of 50 kHz, and real-time filtering was performed using Origin software (4-pole Bessel filter, cut-off frequency 2 kHz).

[0058] For the wild-type PANX2 protein, the current detection results when ATP passed through the pore are as Figure 6 shown. The results show that the current signal has a large amount of noise and many spikes.

[0059] Example 4: Expression and purification of the PANX2 protein mutant after the amino acid L40 was mutated to L40I 1. Gene construction and vector design (1)Transformation: Using the Pichia pastoris strain SMD1163H, the expression vector of the PANX2 protein mutant with the amino acid L40 mutated to L40I was transferred by standard methods.

[0060] (2)Induced expression: The transformed yeast cells were cultured to the logarithmic growth phase, and the expression of PANX2EM was induced by methanol.

[0061] (3)Cell disruption: The induced yeast cells were collected and disrupted by grinding with a grinder. The disrupted cells were resuspended in a buffer (50 mM Tris-HCl, pH = 8.0, 150 mM NaCl), and protease inhibitor cocktail was added: leupeptin (2.5 μg / ml), pepstatin A (1 μg / ml), AEBSF (100 μg / ml), aprotinin (3 μg / ml), benzamidine (1 mM), PMSF (200 μM). DNase I (GoldBio, D-300-1) was added to digest nucleic acids.

[0062] (4)Membrane protein extraction: 1% (w / v) detergent LMNG (lauryl maltose neopentyl glycol) was added, and the mixture was stirred and extracted at 4 °C for 2 hours. Centrifugation was performed at 30,000 × g for 1 hour, and the supernatant containing the target protein was collected.

[0063] 2. Affinity Chromatography Purification (1)Cobalt Ion Resin Binding: The supernatant was incubated with 3 ml of cobalt ion resin at 4 °C for 3 hours to bind the tagged protein.

[0064] (2)Washing of Non-specific 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 (glycogenin) to maintain the stability of membrane proteins.

[0065] (3)Elution of Target Protein: The bound protein was eluted with elution buffer (20 mM Tris-HCl, pH = 8.0, 150 mM NaCl, 200 mM imidazole, 85 μM GDN), and the eluate was collected.

[0066] 3. Tag Removal and Size Exclusion Chromatography (1)Tag Cleavage: PreScission protease was added to the eluate and incubated overnight at 4 °C to cleave the C-terminal tag.

[0067] (2)Gel Filtration Chromatography: The enzymatically digested protein was purified by size exclusion chromatography column using a buffer of 20 mM Tris-HCl (pH = 8.0), 150 mM NaCl, and 40 μM GDN. The single symmetric peak of the target protein was collected (monitored by ultraviolet absorption at 280 nm).

[0068] The purification electrophoresis results of the wild-type PANX2 protein in Example 1 and Example 4 and the PANX2 protein mutant with amino acid L40 mutated to L40I are as Figure 5 shown.

[0069] Example 5: Characterization of the PANX2 Protein Mutant with Amino Acid L40 Mutated to L40I (1)Lipid Bilayer Preparation: 2 mg of DPhPC was dissolved in chloroform and dropped on both sides of a Teflon film (pore size 20 μm), and 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 both the cis chamber and the trans chamber.

[0070] (2)Protein insertion: The purified PANX2 protein mutant with the amino acid L40 mutated to L40I (8 ng / ml) was added to the cis chamber. The Axopatch 200B integrated patch clamp amplifier (Axon Instruments) applied a voltage of 180 mV across the two chambers (positive on the opposite side) and measured the ionic current passing through the pore. Once a pore was inserted, the compartment was rinsed with the experimental buffer to avoid further insertion. All experiments were completed at room temperature (23 ± 1 °C).

[0071] 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 PANX2 protein mutant with the amino acid L40 mutated to L40I, the detection results of the nanopore opening current are as Figure 7 shown. The results indicate that compared with the wild-type PANX2 protein, the current properties of the PANX2 protein mutant were significantly improved and the current noise decreased.

[0072] Example 6: Detection of adenosine triphosphate using the PANX2 protein mutant with the amino acid L40 mutated to L40I (1)Lipid bilayer preparation: 2 mg of DPhPC was dissolved in chloroform and dropped onto both sides of a Teflon film (pore size 20 μm), and 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 both the cis chamber and the trans chamber.

[0073] (2)Protein insertion: The purified PANX2 protein mutant with the amino acid L40 mutated to L40I (8 ng / ml) was added to the cis chamber. The Axopatch 200B integrated patch clamp amplifier applied a voltage of 180 mV across the two chambers (positive on the opposite side) and measured the ionic current passing through the pore. Once a pore was inserted, the compartment was rinsed with the experimental buffer to avoid further insertion. 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 and digitized at 500 kHz. Data acquisition was controlled by Origin.

[0074] (3)The adenosine triphosphate sample was added to the cis chamber, a voltage of 160 mV was applied, and the current was recorded using the Axopatch 200B amplifier (bandwidth 10 kHz). The data was digitized at a sampling rate of 50 kHz, and real-time filtering was performed using Origin software (4-pole Bessel filter, cut-off frequency 2 kHz).

[0075] For the PANX2 protein mutant with the amino acid L40 mutated to L40I, the detection results of the current when adenosine triphosphate passed through the pore are as Figure 8As shown; for the PANX2 protein mutant after the amino acid L40 is mutated to L40I, the statistical results of the pore passing time and the number of events when adenosine triphosphate passes through the pore are as Figure 9 shown. The results show that: compared with the wild-type PANX2 protein, the current properties of the PANX2 protein mutant have significantly improved current signals, with longer up and down amplitudes of the current signal and fewer spikes, and the adenosine triphosphate pore passing time has better consistency.

[0076] The narrowest pore diameter of the wild-type PANX2 protein is 0.9 nm. The PANX2 protein and its mutants are more suitable for detecting small molecule compounds such as metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs (the diameter of the drug is smaller than the protein pore diameter), small molecule diagnostic agents (the diameter of the diagnostic agent is smaller than the protein pore diameter), adenosine triphosphate, and monosaccharides.

[0077] Amino acid sequence: SEQ ID NO:1: MHHLLEQSADMATALLAGEKLRELILPGAQDDKAGALAALLLQLKLELPFDRVVTIGTVLVPILLVTLVFTKNFAEEPIYCYTPHNFTRDQALYARGYCWTELRDALPGVDASLWPSLFEHKFLPYALLAFAAIMYVPALGWEFLASTRLTSELNFLLQEIDNCYHRAAEGRAPKIEKQIQSKGPGITEREKREIIENAEKEKSPEQNLFEKYLERRGRSNFLAKLYLARHVLILLLSAVPISYLCTYYATQKQNEFTCALGASPDGAAGAGPAVRVSCKLPSVQLQRIIAGVDIVLLCVMNLIILVNLIHLFIFRKSNFIFDKLHKVGIKTRRQWRRSQFCDINILAMFCNENRDHIKSLNRLDFIT

[0078] DNA sequence SEQ ID NO:2:

[0079] 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. Application of PANX2 protein and its mutants as biological porins, characterized in that: The biological porin detects one or more of metal ions, inorganic salts, amino acids, nucleotides, small molecule drugs, small molecule diagnostic agents, adenosine triphosphate, and monosaccharides.

2. A PANX2 protein mutant, 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 as shown in SEQ ID NO:

1. The mutation means that one or more amino acids of the wild-type PANX2 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. The PANX2 protein mutant according to claim 2, wherein: The PANX2 protein mutant is selected from the following A or B: A: One or more of the amino acids R89, D90, and R96 of the wild-type PANX2 protein are mutated into common amino acids other than the original amino acids. B: One or more of the amino acids L40 and A36 of the wild-type PANX2 protein are mutated into common amino acids other than the original amino acids.

4. The PANX2 protein mutant according to claim 3, wherein: In A: The amino acid R89 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid R89 of the wild-type PANX2 protein is mutated into R89M, R89P, R89L, R89G, R89I, R89Q, R89E, R89C, R89W, R89H, R89K, R89T, R89A, R89S, R89Y, R89N, R89V, or R89D. The amino acid D90 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid D90 of the wild-type PANX2 protein is mutated into D90S. The amino acid R96 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid R96 of the wild-type PANX2 protein is mutated into R96N, R96K, R96H, R96D, R96W, R96S, R96F, R96E, R96P, R96L, R96A, R96V, R96G, or R96M.

5. The PANX2 protein mutant according to claim 3, wherein: In A: The amino acid R89 of the wild-type PANX2 protein is mutated into R89T, R89L, or R89N; the amino acid R96 of the wild-type PANX2 protein is mutated into R96N or R96K.

6. The PANX2 protein mutant according to claim 4, characterized in that: In B: The amino acid L40 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid L40 of the wild-type PANX2 protein is mutated into L40N, L40H, L40Q, L40S, L40F, L40D, L40C, L40I, L40P, L40T, L40G, L40M, L40W, or L40R. The amino acid A36 of the wild-type PANX2 protein is mutated into a common amino acid other than the original amino acid; the amino acid A36 of the wild-type PANX2 protein is mutated into A36N or A36L.

7. The PANX2 protein mutant according to claim 4, wherein: In B: The amino acid L40 of the wild-type PANX2 protein is mutated into L40I.

8. A product of PANX2 protein and its mutants, characterized in that: The product is a composition, a complex, or a kit.

9. A method for preparing a PANX2 protein and its mutants, characterized in that: It includes the following steps: Step S1: Construct a PANX2 protein or PANX2 protein mutant vector; Step S2: Express and purify the PANX2 protein or PANX2 protein mutant.

10. A membrane layer embedded with PANX2 protein and its mutants, characterized in that: The membrane layer is a lipid bilayer.

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