Nanopore protein for molecular detection, cell communication and molecular separation and mutant thereof

Abstract

The invention provides a nanopore protein and a modified nanopore protein, and relates to the technical field of biology. The modified nanopore protein provided by the invention can be used as a wide-aperture nanopore, the length of a hydrophobic region of the provided nanopore protein is as long as 35 angstroms, the length of the hydrophobic region is obviously higher than that of the existing protein, and the integrated assembly effect of the protein on lipid bilayers and artificial membranes is enhanced; the modified mutant provided by the invention is beneficial for biomolecules, compounds and ions to selectively enter a channel under the action of electrophoretic force and electroosmosis force, so that selective transportation of the biomolecules, the compounds and the ions is realized; and the modified nanopore shrinkage region is also beneficial to selective interaction of biomolecules, compounds and ions with the channel, so that selective transportation and recognition of the biomolecules, the compounds and the ions are realized, and the method is suitable for application scenes such as molecular detection, cell communication or molecular separation and the like.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and particularly to nanopore proteins and their mutants that can be used for molecular detection, cell communication, and molecular separation. Background Art

[0002] Nanopore proteins are a class of proteins with nanoscale pores. Nanopore proteins can be used for single-molecule detection and also as selective separation of substances. The basic principle of nanopore protein operation is as follows: Molecules smaller than the nanopore size can pass through the nanopore to generate ion confinement current. Based on the differences in the blocking effects caused by the physicochemical property differences of the substances to be detected, relevant physicochemical information of the substances to be detected can be obtained. This physical process can be used for non-labeled, high-throughput, and low-cost detection of single molecules. Molecules larger than the nanopore size or having the same interaction force as it will be restricted and difficult to pass through the nanopore. This blocking process can be used for selective separation of ions and molecules. Therefore, the application of nanopore proteins has broad prospects.

[0003] Due to their characteristics such as high sensitivity and high repeatability, nanopore proteins have become the main focus in the nanopore field. Current studies have shown that different protein nanopores such as α-hemolysin (α-HL), Mycobacterium smegmatis toxin protein A (MspA), aerolysin, bacteriophage phi29 connector motor protein (phi29 connector), outer membrane protein (OmpG), and the secretion channel structures of bacterial amyloid fibers (β-amyloids) (CsgG, CsgG-CsgF, CsgG-CsgE) can detect nucleic acids, polypeptides, proteins, metal ions, antibiotics, organic pollutants, and analyze changes such as the valence state of metal ions and the configuration and orientation of substances. However, these proteins either easily detach from the membrane or easily generate gating events. In addition, the opening pore diameters of these nanopores are generally less than 10 nM, the capture rate of molecules is also low, and they are prone to depolymerization. Summary of the Invention

[0004] The purpose of this application is to provide nanopore proteins and their mutants that can be used for molecular detection, cell communication, and molecular separation, aiming to solve the problems of easy pore slipping, many gating events, low capture rate, and easy depolymerization of existing nanopore proteins.

[0005] To achieve the above purpose, this application provides a transmembrane channel protein used as a nanopore, and the amino acid sequence of the transmembrane channel protein is as shown in SEQ ID NO.1.

[0006] This application provides a modified nanopore protein, and the modification includes at least one of the following: (1) At least one amino acid in the amino acid sequence shown in SEQ ID NO.1 is replaced; (2) inserting at least one amino acid into the amino acid sequence shown in SEQ ID NO.1; (3) deleting at least one amino acid from the amino acid sequence shown in SEQ ID NO.1; (4) modifying at least one amino acid in the amino acid sequence shown in SEQ ID NO.1.

[0007] In some embodiments, the modification of the nanopore protein includes mutations at one or more positions on L299-V312 of SEQ ID NO.1.

[0008] In some embodiments, the mutations at one or more positions on L299-V312 include at least one of the following: (1) deleting one or more amino acids at positions L302-F309 on L299-V312; (2) adding one or more amino acids between any two amino acids on L299-Q310; Optionally, the added amino acid is at least one of R, H, K, D, E, F, W, Y; (3) replacing one or more amino acids on L299-V312, and the amino acids that can be replaced at each site are as follows: L299: V, M, Y, F, P or W; Q300: N, T, S, M, R, H, K, D, E, F, W or Y; A301: S, T, N, V, L, I, M, R, H, K, D, E, F, W, P or Y; L302-A308: S, T, N, Q, A, I, L, V, M, R, H, K, D, E, F, W or Y; F309: A, V, I, L, M, R, H, K, D, E, W or Y; Q310: S, T, N, I, M, R, H, K, D, E, F, W, Y or P; V312: P, W, F or Y; (4) performing sequence replacement on L299-Q310, and the replacement sequences are selected from any one of the sequences: LQATNSSAQ, LQANNAQ, LQANSNAQ and LQRQFQMQ; Optionally, performing sequence replacement on L299-Q310 with LQRQFQMQ, and mutating 353N on SEQ ID NO.1 to 353Q.

[0009] In some embodiments, the amino acid sequence of the modified nanopore protein is shown in SEQ ID NO.2.

[0010] In some embodiments, the modified nanopore protein further comprises mutations at one or more positions in the positions of A207 - A221 of SEQ ID NO.1; Optionally, 1 - 30 amino acids are inserted between A207 - A221.

[0011] In some embodiments, at least one proline, at least one negatively charged amino acid, at least two amino acids with polar side chains, and at least one glycine are included in the inserted amino acids; Optionally, the amino acids with polar side chains are selected from S, T, N, Q.

[0012] In some embodiments, the amino acid sequence of the modified nanopore protein is modified, and the modification includes linking at least one of biotin, streptavidin, antibody, maleimide, cysteine, β - cyclodextrin and its derivatives, polypeptide, DNA, phenylboronic acid, nitrilotriacetic acid, azide compound.

[0013] The present application also provides a nanopore protein, which comprises a multimer composed of subunit polypeptides, and the amino acid sequence of the subunit polypeptide is the amino acid sequence of the transmembrane channel protein used as a nanopore as described above, or the amino acid sequence of the modified nanopore protein as described above; Optionally, the multimer comprises a 12 - 15 mer; Optionally, the multimer is a homomultimer or a heteromultimer.

[0014] The present application also provides a nucleic acid encoding the transmembrane channel protein used as a nanopore as described above, or the nucleic acid encoding the modified nanopore protein as described above, or the nucleic acid encoding the nanopore protein as described above.

[0015] The present application also provides the transmembrane channel protein used as a nanopore as described above, or the modified nanopore protein as described above, or the nanopore protein as described above in the application for preparing a biochip or as an ion - selective transport channel; the biochip can detect the electrical and / or optical signals of a substance to be detected; Optionally, the substance to be detected includes at least one of nucleic acid, protein, polysaccharide, neurotransmitter, chiral compound, heavy metal, and toxin; Optionally, the ion - selective transport channel is used for the separation of metal ions or organic compounds; Optionally, the nanopore protein serves as a component of a selective filter membrane; Optionally, the modified nanopore protein serves as a material transport machine between cells, cell - like structures, and vesicles.

[0016] Compared with the prior art, the beneficial effects of the present application include: The hydrophobic region of the nanopore protein provided by the present application is as long as 35 Å, which is significantly longer than that of existing proteins, strengthening the integrated assembly effect of the protein on the lipid bilayer and artificial membrane; the nanopore protein mutants provided by the present application are conducive to the selective entry of biomolecules, compounds and ions into the channel under the action of electrophoresis and electroosmotic forces, realizing the selective transport of biomolecules, compounds and ions; the modified nanopore constriction region is also conducive to the selective interaction of biomolecules, compounds and ions with the channel, realizing the selective transport and recognition of biomolecules, compounds and ions, so as to be applicable to application scenarios such as molecular detection, cell communication or molecular separation.

[0017] The opening of the nanopore protein of the present application is relatively large, and the opening pore diameter is 10-12 nm. The helicase can be directly placed at the pore opening, avoiding the distance between the helicase and the sensing site, which may cause the DNA to fold into a higher-order structure in the nanopore during the sequencing process, such as the sequencing error caused by the formation of the G-4 structure.

[0018] The nanopore protein of the present application can be depolymerized at 100 °C and can also tolerate extreme conditions such as SDS, and can be applied to more extreme conditions. Brief Description of the Drawings

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings required to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be regarded as limiting the scope of the present application.

[0020] Figure 1 It is the molecular sieve diagram and electrophoresis diagram of the nanopore protein; Figure 2 It is the cryo-electron microscopy structure analysis diagram of the nanopore protein; Figure 3 It is the analysis of the hydrophobic region length of the nanopore protein; Figure 4 It is the current signal result diagram of different mutant proteins; Figure 5 It is the electrophysiological characteristic result diagram of different mutant proteins; Figure 6 It is the result diagram of the preparation and testing of the DNA-biotin-streptavidin static chain complex on a single-channel sequencing chip; Figure 7 It is the sequencing performance result diagram of the nanopore protein mutant; Figure 8 It is the sequencing performance result diagram of the nanopore protein mutant; Figure 9 It is the selective transport result diagram of the nanopore protein mutant; Figure 10 Selectivity transmission result graph of the nanopore protein mutant; Figure 11 Polypeptide transmission result graph of the nanopore protein mutant. Detailed implementation manners

[0021] As used herein, the terms: "Prepared from" is synonymous with "comprising". As used herein, the terms "comprising", "including", "having", "containing" or any other variation thereof are intended to cover non-exclusive inclusion. For example, a composition, step, method, article or device containing the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article or device.

[0022] The conjunction "consisting of" excludes any unstated element, step or component. If used in a claim, this phrase will render the claim closed, excluding materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the claim body rather than immediately following the subject, it only limits the elements described in that clause; other elements are not excluded from the claim as a whole.

[0023] When an equivalent, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values and lower preferred values, this should be understood to specifically disclose all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, whether or not the ranges are separately disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range.

[0024] In these examples, unless otherwise specified, the parts and percentages are by mass.

[0025] "Parts by mass" refers to the basic measurement unit representing the mass ratio relationship of multiple components. 1 part can represent any unit mass, such as 1 g or 2.689 g, etc. If we say that the mass part of component A is a parts and the mass part of component B is b parts, it means the mass ratio of component A to component B is a:b. Or, it means the mass of component A is aK and the mass of component B is bK (K is any number representing a multiple factor). It should not be misunderstood that, different from the number of parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0026] "And / or" is used to indicate that either or both of the stated circumstances may occur. For example, A and / or B includes (A and B) and (A or B).

[0027] Throughout all discussions herein, the standard single-letter codes for amino acids are used. These single-letter codes are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Standard substitution notations are also used, i.e., L299F means that L at position 299 is replaced by F.

[0028] This application provides a transmembrane channel protein used as a nanopore, i.e., a nanopore protein, and the amino acid sequence of the transmembrane channel protein is as shown in SEQ ID NO.1.

[0029] Regarding the nanopore protein provided above, this application also provides a modified nanopore protein, and the modification includes at least one of the following: (1) At least one amino acid in the amino acid sequence shown in SEQ ID NO.1 is replaced; (2) At least one amino acid is inserted into the amino acid sequence shown in SEQ ID NO.1; (3) At least one amino acid is deleted from the amino acid sequence shown in SEQ ID NO.1; (4) At least one amino acid in the amino acid sequence shown in SEQ ID NO.1 is modified.

[0030] Among them, for replacement, other amino acids with similar chemical structures, similar chemical properties, or similar side-chain volumes are used to replace amino acids. The introduced amino acid may have polarity, hydrophilicity, hydrophobicity, basicity, acidity, electrical neutrality, or charge similar to the amino acid it replaces. Alternatively, conservative replacement may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the characteristics of the 20 major amino acids.

[0031] For example, C, D, E, K, N, Q, R, S, Y are polar amino acids, A, G, I, L, N are aliphatic amino acids, and F, H, W, Y are aromatic amino acids; A, C, F, I, L, M, P, V, W, Y are hydrophobic amino acids, and D, E, H, K, N, Q, R, S, T are hydrophilic amino acids; A, C, F, I, L, M, N, P, Q, S, T, V, W are electrically neutral amino acids, D, E are negatively charged amino acids, and H, K, R are positively charged amino acids.

[0032] Among them, the modification includes changing the type of amino acid or changing the configuration of the amino acid; changing the type of amino acid includes: changing the electrical property of the amino acid, changing the hydrophobicity of the amino acid, changing the structural rigidity of the amino acid, or changing the size of the side chain group of the amino acid.

[0033] Exemplarily, changing the configuration of an amino acid may include: replacing a D-type amino acid with an L-type amino acid; or, replacing an L-type amino acid with a D-type amino acid.

[0034] Changing the electrical property of an amino acid may include: replacing a negatively charged amino acid with a positively charged or electrically neutral amino acid; or, replacing a positively charged amino acid with a negatively charged or electrically neutral amino acid.

[0035] Changing the hydrophobicity of an amino acid may include: replacing a hydrophobic amino acid with a hydrophilic amino acid; or, replacing a hydrophilic amino acid with a hydrophobic amino acid.

[0036] Changing the structural rigidity of an amino acid includes: replacing an amino acid with a flexible group as the side chain group with an amino acid having a rigid group as the side chain group; or, replacing an amino acid with a rigid group as the side chain group with an amino acid having a flexible group as the side chain group.

[0037] Changing the size of the side chain group of an amino acid includes: replacing an amino acid with a larger side chain group with an amino acid having a smaller side chain group; or, replacing an amino acid with a smaller side chain group with an amino acid having a larger side chain group.

[0038] Amino acids with larger side chain groups can be, for example, tryptophan (W), tyrosine (Y), phenylalanine (F), arginine (R), lysine (K), cysteine (C), etc. Amino acids with smaller side chain groups can be, for example, glycine (G), alanine (A), serine (S), threonine (T), aspartic acid (D), glutamic acid (E), histidine (H), etc.

[0039] In some embodiments, the modification of the nanopore protein includes mutations at one or more positions on L299-V312 of SEQ ID NO.1.

[0040] L299-V312 is the most critical region of the porin, which is the position for changing the minimum size of the porin. It can be regarded as a security checkpoint. Whether a molecule can pass through and be recognized depends on this region. Mutations in this region, when used for single-molecule detection, are to improve the interaction ability between the molecule and the porin, so as to achieve molecular recognition; when used as a cell communication or substance transport channel, it is to restrict which molecules can pass through and which cannot. Modifying the porin is to improve its selectivity or retention ability.

[0041] Among them, the mutations include substitution, insertion, deletion, and modification. The mutations can occur at one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen positions on L299-V312.

[0042] The porin mutants provided in this application are beneficial to improving the interaction between biomolecules, compounds, and ions and the nanopore, strengthening the integrated assembly effect of the pore protein, and also beneficial to promoting the effective capture, translocation, and blockage of biomolecules, compounds, and ions by the nanopore, realizing the selective transport of biomolecules, compounds, and ions, so as to be applicable to application scenarios such as molecular detection, cell communication, or molecular separation.

[0043] In some embodiments, the mutation at one or more positions on L299-V312 can be deleting one or more amino acids at positions L302-F309 on L299-V312, that is, deleting one, two, three, four, five, six, seven, or eight amino acids at L302-F309. For example, it can be deleting L302-A304, or deleting N306-A307, or deleting F309. The research in this application found that after deleting the amino acids at positions L302-F309, the change of current with voltage becomes larger compared with the wild type, which indicates that deleting amino acids increases the pore size.

[0044] In some embodiments, the mutation at one or more positions on L299-V312 can be adding one or more amino acids between any two amino acids on L299-Q310. For example, it can be adding one, two, three, four, five, six, seven, or eight amino acids between any two amino acids.

[0045] Optionally, the added amino acid is at least one of R, H, K, D, E, F, W, Y.

[0046] In some embodiments, the mutation at one or more positions on L299-V312 may be the replacement of one or more amino acids on L299-V312. For example, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen amino acids on L299-V312 may be replaced. The amino acids that can be replaced at each site are as follows: L299: V, M, Y, F, P, or W; Q300: N, T, S, M, R, H, K, D, E, F, W, or Y; A301: S, T, N, V, L, I, M, R, H, K, D, E, F, W, P, or Y; L302 - A308: S, T, N, Q, A, I, L, V, M, R, H, K, D, E, F, W, or Y; F309: A, V, I, L, M, R, H, K, D, E, W, or Y; Q310: S, T, N, I, M, R, H, K, D, E, F, W, Y, or P; V312: P, W, F, or Y.

[0047] The replacement of the amino acid on L299 with the large - side - chain amino acid W improves the electrophysiological baseline stability of the nanopore protein channel. Considering that large - side - chain amino acids such as V, M, Y, F, P have the same properties as W, the replacement of these amino acids is considered an equivalent replacement.

[0048] After mutating A301 to L and N, the electrophysiological baseline of the nanopore protein channel becomes more stable, indicating that the increase in the side - chain size of this amino acid is also beneficial to the stability of the channel. In addition to L and N, amino acids with the same characteristics such as T, S, V, I, M, R, H, K, D, E, F, W, P, and Y belong to equivalent replacements.

[0049] After mutating V312 to P, the stability of the electrophysiological baseline of the nanopore protein channel is also improved. Therefore, in some embodiments, mutating V312 to amino acids with larger side - chains such as W, F, Y is also equivalent.

[0050] After A303 mutates to F, G305 mutates to W, A308 mutates to F, and Q310 mutates to Y, the ion transport of the obtained nanopore protein mutants is reduced, indicating that replacing the amino acids between L299-Q310 with large-side-chain hydrophobic amino acids can reduce ion transportability. This is because replacing with amino acids with larger side chains can narrow the pore diameter of the nanopore protein, which is beneficial for hindering the passage of molecules. This hindrance can be used for selective substance transport, such as water filtration. Similarly, it can also be known that replacing the amino acids between L299-Q310 with hydrophilic amino acids with smaller side chain groups can improve the ion transport performance. This is because replacing with amino acids with smaller side chains can increase the pore diameter of the nanopore protein, which is beneficial for promoting the passage of molecules.

[0051] In some embodiments, replacing one or more amino acids between L299-V312 with large-side-chain amino acids, or changing the side chain length of one or more amino acids, is an equivalent method.

[0052] In some embodiments, the mutation at one or more positions on L299-V312 can be a sequence replacement of L299-Q310, and the replaced sequence is selected from any one of the sequences: LQATNSSAQ, LQANNAQ, LQANSNAQ. The present application research finds that the obtained nanopore protein mutants after these replacements can be integrated on a chip on a large scale to form stable and uniform pores, have high-efficiency sequencing performance, can transform the nanopore protein into a sequencing nanopore, and can also be used as a selective transport channel for ions.

[0053] In some embodiments, L299-Q310 can be replaced with the LQRQFQMQ sequence, and 353N on SEQ ID NO.1 can be mutated to 353Q. The research finds that the obtained nanopore protein mutant can interact with polypeptides, and this replacement enhances the channel's ability to transport polypeptides and can be used for protein sequencing.

[0054] In some embodiments, L299-Q310 is subjected to sequence replacement, and the replaced sequence is LQATNSSAQ. The amino acid sequence of the modified nanopore protein obtained is shown in SEQ ID NO.2.

[0055] In some embodiments, the modified nanopore protein further includes mutations at one or more positions on A207-A221 of SEQ ID NO.1; Optionally, 1-30 amino acids are inserted between A207-A221.

[0056] These mutations narrow the size of the cap gate, construct two constriction regions inside the nanopore protein, and achieve the pore size regulation of the modified nanopore protein.

[0057] In some embodiments, the inserted amino acids contain at least one proline, at least one negatively charged amino acid, at least two amino acids with polar side chains, and at least one glycine; Optionally, the amino acids with polar side chains are selected from S, T, N, and Q.

[0058] In some embodiments, the amino acid sequence of the modified nanopore protein is modified, and the modification includes connecting biotin, streptavidin, antibody, maleimide, cysteine, β-cyclodextrin and its derivatives, polypeptide, DNA, phenylboronic acid, nitrilotriacetic acid, azide, etc., and other molecules connected to these molecules. These connected molecules can change the size, electrical property, and affinity of the nanopore.

[0059] The present application also provides a nanopore protein, which includes a multimer composed of subunit polypeptides, and the amino acid sequence of the subunit polypeptide is the same as the amino acid sequence of the transmembrane channel protein used as a nanopore as described above, or the amino acid sequence of the modified nanopore protein as described above; Optionally, the multimer includes a 12 - 15 mer, for example, it can be a 12 mer, 13 mer, 14 mer, or 15 mer, which can realize the regulation of the pore diameter of the nanopore protein.

[0060] It should be noted that in some feasible embodiments, the nanopore protein can include any number of subunit polypeptides to enable the nanopore protein to form a large enough cavity structure, thereby allowing target analytes such as polynucleotides to pass through.

[0061] Optionally, the multimer is a homomultimer or a heteromultimer.

[0062] It should be noted that a homomultimer refers to: the amino acid sequences of all subunit polypeptides in the nanopore protein are the same; a heteromultimer refers to: the amino acid sequence of at least one subunit polypeptide in the nanopore protein is different from the amino acid sequences of other subunit polypeptides.

[0063] The present application also provides a nucleic acid, which encodes the transmembrane channel protein used as a nanopore as described above, or the nucleic acid encodes the modified nanopore protein as described above, or the nucleic acid encodes the nanopore protein as described above.

[0064] The present application also provides the use of the transmembrane channel protein used as a nanopore as described above, or the modified nanopore protein as described above, or the nanopore protein as described above in the preparation of a biochip or as an ion-selective transport channel; the nanopore protein and the modified nanopore protein can be integrated into the chip alone or in a high-throughput manner, and can also be used in a system that can form a phospholipid bilayer. In this use, it is mainly to detect the electrical signals and optical signals at both ends of the nanopore; Optionally, the substance to be detected includes at least one of nucleic acid, protein, polysaccharide, neurotransmitter, chiral compound, heavy metal, and toxin.

[0065] Optionally, the ion-selective transport channel is used for the separation of metal ions or organic compounds; Optionally, the nanopore protein serves as a component of the selective filter membrane. The nanopore protein can be filled into ultrafiltration and nanofiltration membranes. In this process, the nanopore protein can act as a retention component, blocking some molecules from passing through and allowing some other molecules to pass through. The above application also includes serving as an ion-selective transport channel for the separation of metal ions, organic compounds, etc.; Optionally, the modified nanopore protein serves as a material transport machine between cells, cell-like structures, and vesicles. The modified nanopore protein can be assembled into engineered cells or vesicles, serving as a component of the protein molecular machine in the cell or alone as a cell molecular machine to achieve the material transport between two cells or vesicles.

[0066] The embodiments of the present application will be described in detail below in conjunction with specific examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. For those conditions not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not specified by the manufacturer can be obtained as conventional products through commercial purchase.

[0067] Example 1 Method for Protein Expression and Purification Protein expression: Synthesize the gene sequence encoding the nanopore protein (the gene sequence corresponding to the amino acid sequence shown in SEQ ID NO.1), add a strep II-tag sequence (sequence: WSHPQFEK) at the C-terminus of the gene sequence, and transform it into the E.coli C43 expression strain. Screen for single colonies on an agar plate containing 100 μg / mL antibiotic. Pick a single colony and culture it at 37 °C with a rotation speed of 200 rpm until the OD600 reaches 1.2 - 1.6. Then, perform an enlarged culture at a ratio of 1:200 (seed solution / culture medium). After the OD600 reaches 0.6, add IPTG and lower the temperature to 20 °C and continue culturing for 14 - 20 hours. Centrifuge to collect the bacterial cells (centrifugation condition: 4000g), and wash them once with phosphate buffer with a pH of 8.0.

[0068] Purification of the protein: The cells after the above washing were added to buffer A, and the mass concentration of the cells in buffer A was 0.1 g / mL; then the cells were lysed by ultrasonic disruption, and cell debris was removed by centrifugation (centrifugation conditions: 4000 g), and then centrifuged at 200000 g (Beckman, 70Ti rotor) for 1.5 h. Buffer B was added to the centrifuged system to fully dissolve the membrane, and the mixture was mixed at 4°C for 1 h, and the supernatant was obtained by filtration through a 0.22 μm filter. Among them, buffer A contains NaCl and Tris-HCl, the concentration of NaCl in buffer A is 300 mM, the concentration of Tris-HCl in buffer A is 50 mM, and the pH of buffer A is 8.0. Buffer B contains NaCl, Tris-HCl, LDAO (lauryldimethylamine oxide) and DDM (dodecyl-β-D-maltoside), the concentration of NaCl in buffer B is 300 mM, the concentration of Tris-HCl in buffer B is 50 mM, the concentration of LDAO in buffer B is 0.01 g / mL, the concentration of DDM in buffer B is 0.01 g / mL, and the pH of buffer A is 8.0.

[0069] Then the supernatant was injected into a Strep-Tactin XT Resin chromatography column, washed with solution C, and the protein was obtained by adding the eluent and collecting. The collected protein was further separated into polymers and monomers by gel chromatography molecular sieve to obtain the nanopore protein AcGspD shown in SEQ ID NO.1. Among them, solution C contains NaCl, Tris-HCl and LDAO, and the concentrations of NaCl and Tris-HCl in solution C are 300 mM and 50 mM respectively, and the concentration of LDAO in solution C is 0.001 g / mL; the eluent contains NaCl, Tris-HCl, LDAO and desthiobiotin, and the concentrations of NaCl, Tris-HCl and desthiobiotin in the eluent are 300 mM, 50 mM and 5 mM respectively, and the concentration of LDAO in the eluent is 0.001 g / mL.

[0070] Figure 1 It is the molecular sieve diagram and electrophoresis diagram of the AcGspD polymer protein (14-mer) obtained after the above purification. Figure 1 Part A in it is the molecular sieve diagram of the purified nanopore protein AcGspD, where the abscissa represents the elution volume and the ordinate represents the elution intensity. Figure 1 Part B in it is the electrophoresis diagram of the purified nanopore protein AcGspD, where the left side is the marker and the 4 bands on the right side are the polymer proteins after molecular sieve.

[0071] From Figure 1It can be seen that the method for expressing and purifying the protein in Example 1 can obtain multimeric proteins. The multimeric structure and size of the purified nanopore protein AcGspD were analyzed, and the analysis results are as Figure 2 and Figure 3 shown. Figure 2 In part A of Figure 2 , it is a typical classified average map of the top-view particles of the nanopore protein, part B is a side-view display of the three-dimensional reconstruction of the nanopore protein, and part C is an overlay map of the model of the symmetric nanopore protein (represented by pink cartoons) and the cryo-EM density (white). These results illustrate that the purified protein has structural homogeneity.

[0072] Figure 3 For the analysis of the hydrophobic region length of the nanopore protein, where Ac is the nanopore protein AcGspD of this application, T2S is the type II secretin protein GspD, T3S is the type III secretin InvG, and T4S is the type IV secretin PilQ. From Figure 3 it can be seen that the hydrophobic region of the nanopore protein of this application is up to 35 Å, which is significantly higher than the length of existing proteins.

[0073] Example 2 Electrophysiological Function Test of Protein Channels The electrophysiological function of the protein channel was evaluated by planar lipid membrane (PLM) experiments. The experimental device includes a Teflon film with a central hole of 100 μm in diameter, which divides the chamber into two 1 ml compartments. The pore diameter was pretreated by dropping 1 μl of hexadecane (1% v / v, dissolved in n-hexane) and allowing it to stand for 20 minutes. Before the experiment, the pore diameter was conditioned with 1 μl of hexadecane solution (1% v / v, dissolved in n-hexane) for 20 minutes. After drying, 700 μl of buffer (1 M KCl, 50 mM Tris-HCl, pH 8.0) was added to each compartment. Subsequently, Ag / AgCl electrodes were inserted into the two chambers for measuring ion current.

[0074] To form the lipid membrane, 2 μl of a 10 mg / ml DPhPC solution (dissolved in decane) was added to each compartment. The lipid monolayer was formed at the air-water interface by adjusting the solution level, and then carefully adjusted to form a lipid bilayer to complete the construction of the membrane. The integrity of the membrane was evaluated by detecting its tolerance at a voltage of 380 mV and its stability at 200 mV. Subsequently, the protein (in the form of liposomes) with a concentration of 1 - 5 nM was added to the cis chamber (the chamber where the voltage is applied). At a fixed voltage of +180 mV, the current was recorded using Axopatch 200B (Molecular Devices).

[0075] In this process, we tested the current signals of nanopore proteins and different nanopore protein mutants, and the results are as Figure 4 shown. The baseline of the wild-type protein (AcGspD) is not very stable, indicating relatively high noise. However, after selective mutation between L299-V312 and increasing the side chains of amino acids, it was found that the current baseline became more stable. Especially for L299W, A301L / N, V312P, the baselines became more stable compared to the WT. This shows that increasing the size of the amino acid side chains between L299-Q310 can improve the stability of the baseline. These amino acids can also be Y, F, and M, etc.

[0076] Example 3 Permeability of the Channel Through the methods of the above examples, we evaluated the changes in channel characteristics after deleting amino acids between L299-V312. After deleting L302-A304, Del N306-A307, and Del F309, the mutant proteins were purified by the method of Example 1, and the electrophysiological characteristics of the channels were studied by Example 2, and the current values at different voltages were recorded. The results are as Figure 5 shown. It can be seen that after deleting amino acids, the change of current with voltage becomes larger compared to the wild type, which indicates that deleting amino acids increases the pore size. Therefore, it can be concluded that deleting any amino acids between L302-F309 may change the pore size.

[0077] Example 4 Analysis of the Channel Protein's Resolution of Different Nucleotides To evaluate whether the channel can distinguish bases, we constructed a sequence-substituted mutant SEQ ID NO.2 (AcGspD_Mut). The DNA-biotin-streptavidin static chain complex was prepared and tested on a single-channel sequencing chip. This experiment was carried out on MePore, and the polyA and PolyT DNA strands were provided by Beijing Tsingke Biotechnology Co., Ltd. The 3' ends of these DNA strands were biotinylated. The DNA strands were incubated with monomeric streptavidin at a 1:1 ratio (final concentration of 9.9 μM) for 20 minutes at room temperature to generate the DNA-biotin-streptavidin static chain complex for each polynucleotide chain. Subsequently, the complex was diluted to 2 μM with primer buffer. In the sequencing chip with 30 μL buffer (10 mM HEPES, 150 mM potassium ferricyanide, 200 mM NaCl, 1 mM MgCl2, pH 8.0), the nanopore protein mutant AcGspD_Mut prepared as described above was added to the chip at a 10,000-fold dilution. When the experimental script was run, it was set at 140 mV and 180 mV, and a reverse potential pulse (0 mV for 0.1 second, -140 mV for 2 seconds, and then 0 mV for 2 seconds) was applied every 5 seconds. 2 μl of each DNA-biotin-streptavidin complex was added in sequence, and data was recorded for 15 minutes for each complex.

[0078] The results are as Figure 6 shown, Figure 6 where part a shows the schematic diagram of the detection, part b shows the molecular formulas of the two nucleotides detected, parts c and e show the temporal signal changes of the polynucleotide at 140 mV and 180 mV voltages, and parts d and f show the changes in the current blockage ratio of the two polynucleotides at 140 mV and 180 mV voltages. Obviously, the nanopore protein mutant AcGspD_Mut can distinguish A and T, and this discrimination degree does not change at different voltages.

[0079] Example 5 Evaluation of the Sequencing Performance of the Nanopore Protein Mutant In this test example, the nanopore protein mutant AcGspD_Mut for sequencing obtained in Example 4 was used, and the test schematic diagram is as Figure 7As shown in part a of the figure. The nanopore protein mutant was diluted 1:1000 using a buffer containing 500mM KCl and added to a single-channel sequencing chip (provided by Shenzhen Meili Nanopore). The wells were inserted at a gradient voltage of 0-300mV. After the wells were inserted, the excess wells were washed with the above buffer to remove the excess wells. The chip was rinsed twice with a sequencing mixture at pH 8.0. The prepared DNA sample with characteristic sequence was sequenced, 1μg of DNA analyte was mixed and connected with 40nM adapter mixture (provided by Shenzhen Meili Nanopore), and the sequencing sample was obtained by magnetic bead purification. The purified sequencing sample was added to the sequencing chip and sequenced at a voltage of 180mV. The results are shown in Figure 7 shown.

[0080] Depend on Figure 7 The results in part b show that the opening current of the sequencing nanopore protein mutant AcGspD_Mut is 220pA, indicating that the pore formed by the nanopore protein mutant provided by the present application is stable. Figure 7 From the results in part b, we can see that the blocking current ratio generated by sequencing is 0.75 when the 1100 kbp sequence is used for library construction test. Figure 7 The results in part b show that the current change caused by the base is about 50 pA. Based on the above results, it can be seen that the sequencing performance of the nanopore protein mutant provided in the present application is consistent with the sequencing performance of MspA and CsgG reported in the prior art. That is, the nanopore protein mutant provided in the present application can be integrated on a large scale on the chip to form a stable and uniform pore; at the same time, it has efficient sequencing performance.

[0081] Depend on Figure 7 The results in part c show that the current signal generated by sequencing can be segmented by neural network training, indicating that the nanopore protein mutant has the practicality of sequencing. Figure 7 As shown in part d, the nanopore sequencing signal provided by the present invention can accurately match the DNA sequence, wherein the dotted line in the figure represents the model of the MspA nanopore protein, and the solid line represents the model of the nanopore protein provided by the present invention. This also shows that the nanopore protein mutant provided by the present application is completely inconsistent with MspA in signal generation and is a new type of nanopore protein.

[0082] In addition, the sequencing capabilities of the other two mutants were further evaluated, and it was found that DNA sequencing could also be achieved by replacing L299-Q310 with LQANNAQ and LQANSNAQ, e.g. Figure 8 This indicates that random substitution of the amino acid sequence on L299-Q310 can achieve the same function.

[0083] Example 6 Selective transport of nanopore proteins This experiment was carried out in a 6-well plate. First, a 1:1 mixture of hexadecane and silicone oil containing 5 mM DPhPC was prepared, and 2 mL of the above mixture was pipetted into the 6-well plate. Then, buffer A (1 M NaCl, 20 mM HEPES, 500 mM CaCl2, pH 8.0) was prepared, and 500 nL was pipetted into the above phospholipid mixture to form the first vesicle ( Figure 9 the left vesicle in

[0084] Fluo-8 was excited by a 473 nm laser beam through total internal reflection (Vortran Laser Technology, CA, USA; power of 3–4 mW in the back focal plane of the objective lens). The laser was transmitted through a 60×, 1.49 NA TIRF oil immersion objective lens (Nikon). The emitted light (λem. max. of Fluo-8 = 514 nm) passed through a 525 / 39 nm emission filter (Brightline Basic; Semrock, NY, USA), and the images were acquired by an electron multiplying CCD (iXon3 897; Andor, UK) at a frame rate of 120.34 Hz. The device was mounted on a temperature-controlled stage (PE94; Linkam, UK), and the objective lens was heated by a custom-made resistive heater. The temperature was monitored by a thermocouple placed in an adjacent well of the imaging droplet.

[0085] As Figure 9 shown in part a of Figure 9 , in the experimental group with the addition of wild-type nanopore protein, it was found that the volume of the vesicle decreased. However, the fluorescence intensity inside the vesicle did not change. This indicates that through osmosis, water molecules in the right vesicle can enter the left through the vesicle, while calcium ions do not enter the right vesicle through this nanopore. In comparison, Figure 9 part b of 2+ shows that water molecules can enter the left vesicle through the nanopore protein mutant AcGspD_Mut (i.e., 2+ Ac-Mut in 2+Transport channels and other channels with similar transport functions are regarded as tools for intercellular communication.

[0086] In addition to the above substitution mutations, selective point mutations were also performed on amino acid sites. As shown in Table 1, after the mutations, the same tests were carried out, and the results are as Figure 10 shown. After 90 minutes, the vesicles became smaller, indicating that water molecule transport could occur, but the fluorescence did not change, indicating a decrease in ion transport. These results show that replacing the large side-chain hydrophobic amino acids between L299-Q310 can reduce ion transportability. Similarly, it can also be known that replacing them with hydrophilic amino acids can improve ion transport performance.

[0087] Table 1 Mutation sites of nanopore proteins

[0088] Example 7 Polypeptide transport ability of protein mutants After replacing L299-Q310 with LQRQFQMQ, 353N was further mutated to 353Q. Under the conditions of 0.5M KCl, 20mM HEPES, pH 8.0, the transport of polypeptides by this mutant protein was explored, and the results are as Figure 11 shown. At a voltage of 180 mV, a signal of blocked current could be observed, indicating that the altered mutant could interact with polypeptides. This shows that replacing the sequence between L299-Q310 is used for protein sequencing.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0090] In addition, those skilled in the art can understand that although some of the embodiments herein include certain features included in other embodiments rather than other features, the combination of the features of different embodiments means that it is within the scope of the present application and forms different embodiments. For example, in the above claims, any one of the claimed embodiments can be used in any combination. The information disclosed in this background art section is only intended to deepen the understanding of the overall background art of the present application, and should not be regarded as an admission or any form of suggestion that this information constitutes the prior art known to those skilled in the art.

Claims

1. A transmembrane channel protein used as a nanopore, characterized in that, The amino acid sequence of the transmembrane channel protein is as shown in SEQ ID NO.

1.

2. A modified nanopore protein, characterized in that, The modification includes at least one of the following: (1) At least one amino acid in the amino acid sequence shown in SEQ ID NO.1 is replaced; (2) At least one amino acid is inserted into the amino acid sequence shown in SEQ ID NO.1; (3) At least one amino acid is deleted from the amino acid sequence shown in SEQ ID NO.1; (4) At least one amino acid in the amino acid sequence shown in SEQ ID NO.1 is modified.

3. The modified nanopore protein according to claim 2, wherein, The modification of the nanopore protein includes mutations at one or more positions on L299-V312 of SEQ ID NO.

1.

4. The modified nanopore protein according to claim 3, wherein, The mutations at one or more positions on L299-V312 include at least one of the following: (1) Deleting one or more amino acids at positions L302-F309 on L299-V312; (2) Adding one or more amino acids between any two amino acids on L299-Q310; Optionally, the added amino acid is at least one of R, H, K, D, E, F, W, Y; (3) Replacing one or more amino acids on L299-V312, and the amino acids that can be replaced at each site are as follows: L299: V, M, Y, F, P or W; Q300: N, T, S, M, R, H, K, D, E, F, W or Y; A301: S, T, N, V, L, I, M, R, H, K, D, E, F, W, P or Y; L302-A308: S, T, N, Q, A, I, L, V, M, R, H, K, D, E, F, W or Y; F309: A, V, I, L, M, R, H, K, D, E, W or Y; Q310: S, T, N, I, M, R, H, K, D, E, F, W, Y or P; V312: P, W, F or Y; (4) Performing sequence replacement on L299-Q310, and the replacement sequence is selected from any one of the sequences: LQATNSSAQ, LQANNAQ, LQANSNAQ, and LQRQFQMQ; Optionally, the amino acid sequence of the modified nanopore protein is as shown in SEQ ID NO.2; Optionally, L299-Q310 is replaced with the sequence LQRQFQMQ, and 353N on SEQ ID NO.1 is mutated to 353Q.

5. The modified nanopore protein according to claim 3, wherein, The modified nanopore protein further includes mutations at one or more positions on A207-A221 of SEQ ID NO.1; Optionally, 1-30 amino acids are inserted between A207-A221.

6. The modified nanopore protein according to claim 5, wherein Among the inserted amino acids, there is at least one proline, at least one negatively charged amino acid, at least two amino acids with polar side chains, and at least one glycine; Optionally, the amino acids with polar side chains are selected from S, T, N, Q.

7. The modified nanopore protein according to any one of claims 2 to 6, characterized in that, The amino acid sequence of the modified nanopore protein is modified, and the modification includes linking at least one of biotin, streptavidin, antibody, maleimide, cysteine, β-cyclodextrin and its derivatives, polypeptide, DNA, phenylboronic acid, nitrilotriacetic acid, and azide compound.

8. A nanopore protein, characterized in that, The nanopore protein includes a multimer composed of subunit polypeptides, and the amino acid sequence of the subunit polypeptide is the amino acid sequence of the transmembrane channel protein used as a nanopore as described in claim 1, or the amino acid sequence of the modified nanopore protein as described in any one of claims 2-7; Optionally, the multimer includes a 12-15 mer; Optionally, the multimer is a homomultimer or a heteromultimer.

9. A nucleic acid, characterized in that, The nucleic acid encodes the transmembrane channel protein used as a nanopore as described in claim 1, or the nucleic acid encodes the modified nanopore protein as described in any one of claims 2-7, or the nucleic acid encodes the nanopore protein as described in claim 8.

10. The transmembrane channel protein used as a nanopore as described in claim 1, or the modified nanopore protein as described in any one of claims 2-7, or the nanopore protein as described in claim 8 in the application for preparing a biochip or as an ion-selective transport channel; the biochip can detect the electrical and / or optical signals of the substance to be detected; Optionally, the substance to be detected includes at least one of nucleic acid, protein, polysaccharide, neurotransmitter, chiral compound, heavy metal, and toxin; Optionally, the ion-selective transport channel is used for the separation of metal ions or organic compounds; Optionally, the nanopore protein is used as a component of a selective filter membrane; Optionally, the modified nanopore protein is used as a substance transport machine between cells, cell-like structures, and vesicles.

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