WO2024078621A1 - 一种pht纳米孔突变体蛋白及其应用 - Google Patents

一种pht纳米孔突变体蛋白及其应用 Download PDF

Info

Publication number
WO2024078621A1
WO2024078621A1 PCT/CN2023/124532 CN2023124532W WO2024078621A1 WO 2024078621 A1 WO2024078621 A1 WO 2024078621A1 CN 2023124532 W CN2023124532 W CN 2023124532W WO 2024078621 A1 WO2024078621 A1 WO 2024078621A1
Authority
WO
WIPO (PCT)
Prior art keywords
nanopore
pht
protein
wild
type
Prior art date
Application number
PCT/CN2023/124532
Other languages
English (en)
French (fr)
Inventor
张满丰
张宁
Original Assignee
北京普译生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 北京普译生物科技有限公司 filed Critical 北京普译生物科技有限公司
Publication of WO2024078621A1 publication Critical patent/WO2024078621A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention relates to a method for characterizing a target polynucleotide, in particular to a PHT nanopore mutant protein and application thereof, and belongs to the field of gene engineering and genetic engineering.
  • Nanopore sequencing technology is a new type of nucleic acid sequencing technology developed in recent years. According to the type of pore, it can be divided into solid-state pores and biological nanopores. Biological nanopores are pore proteins that allow substrates to pass through. The following nanopore sequencing refers only to biological nanopore sequencing technology. Under the action of electric field force, charged nucleic acid substrates can pass through biological nanopores. When nucleic acids pass through nanopores, they can hinder the current of nanopores and generate different current signals. By analyzing the current signals, the base information of nucleic acids can be obtained. Compared with other sequencing methods, it has the advantages of low equipment, simple sample preparation, and fast sequencing speed, and has begun to be widely used.
  • RNA can be directly measured. Based on the characteristics of nanopore sequencing, RNA no longer needs to be reverse transcribed into DNA for sequence analysis, so that the modification information on RNA can be preserved.
  • Porin is one of the key technologies that affect nanopore sequencing.
  • the characteristics of the constriction region (reading head) of the pore protein have a great influence on the acquisition of the original current signal.
  • the types of pore proteins are single, and the commonly used Escherichia coli CsgG mutant constriction region can accommodate 4-5 bases. Therefore, the current signal recorded at a single time is generated by the obstruction of 4-5 bases, making subsequent signal analysis difficult and making it difficult to achieve the accuracy level of second-generation sequencing. Therefore, it is necessary to explore pore proteins with new constriction regions. On the one hand, it can increase the selectable types of nanopores, and on the other hand, it can reduce the difficulty of current signal analysis and further improve the accuracy of sequencing.
  • the present invention aims at the disadvantage that the existing nanopore protein derived from Escherichia coli has a thick contraction zone, which leads to the synergistic effect of multiple continuous nucleotides in the current signal detection, and finds a PHT nanopore protein with a thinner contraction zone.
  • the contraction zone of the protein contains only one amino acid residue, thereby reducing the number of continuous polynucleotides that can be accommodated, reducing the difficulty of current signal decoding, and improving the base resolution efficiency.
  • the present invention uses the methods of systematic amino acid sequence alignment, selection, protein expression, purification, structural analysis, etc., and uses the amino acid mutation method to express and purify different mutant proteins of amino acids in the contraction region, and combines the phospholipid membrane current signal detection method to determine the membrane signal characteristics of different mutant proteins; select mutant proteins with less noise to determine their nucleic acid detection capabilities.
  • the present invention involves technologies including the construction of Escherichia coli membrane protein expression vectors, mutant protein vector construction; membrane protein expression and purification; cryo-electron microscopy sample preparation, data collection, structural analysis, structure construction, and structural analysis; nanopore protein phospholipid membrane embedding technology, current signal recording, processing, etc.
  • the present invention provides a PHT nanopore mutant protein, which mutates one or more amino acids of the wild-type pht nanopore protein shown in SEQ ID NO: 1 into other amino acids (for example, F87, F84, N83, A85, E235), or knocks out one or more amino acids of the wild-type pht nanopore protein shown in SEQ ID NO: 1 (for example, knocks out one or more of the amino acids at positions 80-86).
  • amino acids include natural amino acids and/or unnatural amino acids. Natural amino acids are the other 19 amino acids.
  • the PHT nanopore mutant protein is selected from:
  • the other 19 amino acids refer to the other 19 amino acids among the 20 common amino acids except the original amino acid.
  • the 20 common amino acids are: glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.
  • the PHT nanopore mutant protein is a wild-type pht nanopore protein whose contraction region amino acid F87 shown in SEQ ID NO: 1 is mutated into other 19 amino acids.
  • the PHT nanopore mutant protein is a wild-type pht nanopore protein contraction region amino acid F87 shown in SEQ ID NO: 1 mutated to: F87M, F87P, F87L, F87G, F87I, F87Q, F87E, F87C, F87R, F87H, F87K, F87T, F87A, F87S, F87Y, F87N, F87V or F87D.
  • the PHT nanopore mutant protein is a wild-type pht nanopore protein shown in SEQ ID NO: 1, in which the amino acid F87 in the contraction region is mutated to F87T, F87L, or F87N.
  • the PHT nanopore mutant protein is obtained by mutating amino acid N83 of the wild-type pht nanopore protein shown in SEQ ID NO: 1 into other 19 amino acids.
  • the PHT nanopore mutant protein is a wild-type pht nanopore protein shown in SEQ ID NO: 1 wherein the amino acid N83 is mutated to N83D, N83S. In some embodiments, the PHT nanopore mutant protein is a wild-type pht nanopore protein shown in SEQ ID NO: 1 wherein the amino acid F84 is mutated to other 19 amino acids.
  • the PHT nanopore mutant protein is a wild-type pht nanopore protein shown in SEQ ID NO: 1 wherein the amino acid F84 is mutated to F84S, F84N, F84K, F84H, F84D, F84W, F84R, F84S, F84E, F84P, F84L, F84A, F84V, F84G, F84M; preferably F84S, F84N, F84K.
  • the PHT nanopore mutant protein is obtained by mutating amino acid A85 of the wild-type pht nanopore protein shown in SEQ ID NO: 1 into other 19 amino acids.
  • the PHT nanopore mutant protein is obtained by mutating the amino acid A85 of the wild-type pht nanopore protein shown in SEQ ID NO: 1 to: A85F, A85N, A85H, A85Q, A85S, A85L, A85D, A85C, A85I, A85P, A85T, A85G, A85M, A85W, A85R; preferably A85I.
  • the PHT nanopore mutant protein is obtained by mutating the amino acid E235 of the wild-type pht nanopore protein shown in SEQ ID NO: 1 into other 19 amino acids.
  • the PHT nanopore mutant protein is a wild-type pht nanopore protein shown in SEQ ID NO: 1 in which the amino acid E235 is mutated to E235N.
  • the PHT nanopore mutant protein is a protein in which one or more (e.g., 1 or 2) of the 85th and 86th amino acids of the wild-type pht nanopore protein shown in SEQ ID NO: 1 are knocked out; for example, the 85th and 86th amino acids are knocked out.
  • the PHT nanopore mutant protein is a protein in which one or more (e.g., 1, 2, 3, 4) of the amino acids 80, 81, 82, and 83 of the wild-type pht nanopore protein shown in SEQ ID NO: 1 are knocked out; for example, amino acids 80 to 83 are knocked out.
  • the PHT nanopore mutant protein is a protein in which one or more (e.g., 1, 2, 3) of the amino acids 80, 81, and 82 of the wild-type pht nanopore protein shown in SEQ ID NO: 1 are knocked out; for example, amino acids 80 to 82 are knocked out.
  • the present invention provides a gene encoding the above-mentioned PHT nanopore mutant protein.
  • the encoding gene is selected from:
  • the homology is between 85%-99%.
  • the homology is 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%.
  • the present invention provides a recombinant vector comprising the above encoding gene.
  • the present invention provides a genetically engineered cell, wherein the cell contains the above-mentioned recombinant vector, or its genome contains the above-mentioned encoding gene.
  • Including the above encoding gene may be by integrating the above encoding gene or in other ways.
  • the present invention provides a product comprising the above-mentioned PHT nanopore mutant protein.
  • the product is a composition, a complex, or a kit.
  • the present invention provides a recombinant bacterium comprising the above-mentioned recombinant vector.
  • the present invention provides a method for preparing the above-mentioned PHT nanopore mutant protein, comprising the following steps:
  • the present invention provides a membrane layer, in which the above-mentioned PHT nanopore mutant protein is embedded.
  • the membrane layer is a phospholipid membrane layer.
  • the membrane layer is a phospholipid monolayer membrane layer.
  • the present invention provides a detection system, comprising the above-mentioned PHT nanopore mutant protein or the above-mentioned membrane layer.
  • the present invention provides a device comprising the above-mentioned PHT nanopore mutant protein, the above-mentioned membrane layer or the above-mentioned detection system.
  • the present invention provides use of the above-mentioned PHT nanopore mutant protein, the above-mentioned membrane layer or the above-mentioned detection system in a device for preparing sample sequencing and/or detection.
  • the sample is one or more of nucleotides, nucleic acids, amino acids, oligopeptides, polypeptides, and proteins.
  • the present invention provides the use of the above-mentioned PHT nanopore mutant protein, the above-mentioned membrane layer, the above-mentioned detection system or the above-mentioned device in sample sequencing and/or detection.
  • the sample is one or more of nucleotides, nucleic acids, amino acids, oligopeptides, polypeptides, and proteins.
  • the present invention uses cryo-electron microscopy technology to analyze the high-resolution three-dimensional structure of PHT nanopores Structural analysis revealed that the width of the contraction region of the pht nanopore protein is The thickness is determined by only one amino acid (F87).
  • F87 one amino acid
  • the present invention found that the wild-type PHT has high noise on the phospholipid membrane and cannot be used for nucleic acid substrate detection. Further mutation experiments found that changing the type of amino acid at this position does not affect the expression of the nanopore protein, but the different types of amino acids after mutation have a greater impact on its current signal.
  • the present invention provides a PHT nanopore mutant protein, wherein after amino acid mutation or knockout, the current property and current signal of the mutant protein are significantly improved compared with the wild-type protein.
  • the F87T, E235N, F87L, F87N, A85I, F84S, F84N and F84K mutant proteins have better effects and can be used for sequencing and/or detection of samples such as DNA.
  • Figure 1 is a diagram of wild-type pht protein expression and purification
  • FIG2 is a molecular screening result diagram of wild-type pht protein
  • Figure 3 is a cryo-EM map of wild-type pht protein
  • FIG4 is a diagram showing the overall structure of a wild-type PHT nanopore
  • Figure 5 is a diagram of the overall conformation of the wild-type PHT nanopore contraction region
  • Figure 6 is a diagram of the monomer conformation of the wild-type PHT nanopore contraction region
  • FIG. 7 is a diagram of protein expression and purification of the F87 site PHT nanopore mutant
  • FIG8 is a diagram of protein expression and purification of a knockout PHT nanopore mutant
  • FIG9 is a diagram of protein expression and purification of the E235 site PHT nanopore mutant
  • FIG10 is a diagram of protein expression and purification of the N83 site PHT nanopore mutant
  • FIG11 is a diagram of protein expression and purification of the F84 site PHT nanopore mutant
  • FIG12 is a diagram of protein expression and purification of the A85 site PHT nanopore mutant
  • FIG13 is a graph showing the current properties of wild-type pht nanopores
  • FIG14 is a graph showing the nanopore current properties of the PHT nanopore mutant protein at the F87 and E235 sites;
  • FIG15 is a graph showing the nanopore current properties of a PHT nanopore mutant protein at the F87 site
  • FIG16 is a graph showing the nanopore current properties of a site-knockout PHT nanopore mutant protein
  • FIG17 is a graph showing the nanopore current properties of a PHT nanopore mutant protein at the N83 site
  • FIG18 is a graph showing the nanopore current properties of a PHT nanopore mutant protein at the F84 site
  • FIG. 19 is a graph showing the nanopore current properties of the A85 site PHT nanopore mutant protein.
  • the wild-type pht nanopore protein is derived from Nitratireductor pacificus pht-3B (ACCESSION: WP_008595533, SEQ ID NO: 1).
  • the protein expression gene was obtained by artificial synthesis, and the synthesis process was codon optimized for Escherichia coli expression. After synthesis, the gene was constructed into the pET-28a vector by seamless cloning, and 6 ⁇ his was added to the C-terminus of the protein as an affinity purification tag.
  • primer F GGGCTAACAGGAGGAATTAACCATGTCATCACGCTCACATCA primer
  • R TCAATGATGATGATGATGATGCTGCTGAACAGGGTTAGGTTC
  • the PCR system is as follows (20 ⁇ L):
  • the PCR program is as follows:
  • the seamless connection system (10 ⁇ L) is as follows: 2 ⁇ Seamless Ligation Buffer 5 ⁇ L Target fragment (50ng/ ⁇ L) 3 ⁇ L Linearized vector (10ng/ ⁇ L) 2 ⁇ L
  • Lysis buffer 20 mM Tris-HCl, 150 mM NaCl, pH 8.0.
  • Dissolution buffer 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, 1% LDAO (dodecyl dimethylamine oxide).
  • Washing solution 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, 0.5% LDAO, 50 mM imidazole.
  • Molecular sieve buffer 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, 0.06% LDAO.
  • Example 2 Atomic structure of wild-type pht nanopore protein
  • cryo-electron microscopy was used to analyze its resolution atomic level structure.
  • the holes in the map that can be used for data collection can be selected offline, which can save some time;
  • the electron microscope can be adjusted to prepare for data collection. This mainly includes the electron microscope axis alignment, background subtraction and basic data collection parameters (defocus -1.5 ⁇ m to -2.5 ⁇ m, electronic measurement Photo frame number 32 frames and pixel size )setting;
  • FIG. 3 After subsequent data processing and structural analysis, its electron density was obtained, as shown in Figure 3.
  • the overall map is a 9-mer structure, and the positions of the monomers are shown in the figure (the left picture of Figure 3 is a top view, and the right picture is a top view).
  • the right picture marks the position of the transmembrane region of the nanopore protein.
  • the atomic coordinates of the amino acids of the wild-type pht nanopore protein were obtained through homology modeling, structure construction, and refinement ( Figure 4, the left picture is a top view, and the right picture is a top view).
  • the contraction region of the pht nanopore protein is a single-layer structure, which is different from the three-layer structure of the Escherichia coli nanopore protein.
  • the contraction region is determined by a single amino acid, which is phenylalanine (Phe, F) at position 87, and the width of the contraction region is (Figure 5), the monomer structure analysis shows that only the F87 side chain points to the inner side of the pore ( Figure 6).
  • the contraction area is thin ( Figure 5), only
  • Example 3 Determination of current properties of wild-type and PHT nanopore mutant proteins on artificial membranes
  • F87 F, phenylalanine Phe: F87M (methionine Met), F87P (proline Pro), F87L (leucine Leu), F87G (glycine Gly), F87I (isoleucine Ile), F87Q (glutamine Gln), F87E (glutamate Glu), F87C (cysteine Cys), F87R (arginine Arg), F87H (histidine His), F87K (lysine Lys), F87T (threonine Thr), F87A (alanine Ala), F87S (serine Ser), F87Y (tyrosine Tyr), F87N (asparagine Asn), F87V (valine Val), F87D (aspartic acid Asp).
  • F87M methionine Met
  • F87P proline Pro
  • F87L leucine Leu
  • F87G glycine Gly
  • F87I isoleucine Ile
  • F87Q glutamine
  • E235 E, glutamic acid Glu
  • E235N asparagine Asn
  • N83 N, asparagine Asn
  • N83D amino acid Asp
  • N83S serine Ser
  • F84 F, phenylalanine Phe: F84S (serine Ser), F84N (asparagine Asn), F84K (lysine Lys), F84H (histidine His), F84D (aspartic acid Asp), F84W (tryptophan Trp), F84R (arginine Arg), F84S (serine Ser), F84E (glutamic acid Glu), F84P (proline Pro), F84L (leucine Leu), F84A (alanine Ala), F84V (valine Val), F84G (glycine Gly), F84M (methionine Met).
  • A85F phenylalanine Phe
  • A85N asparagine Asn
  • A85H histidine His
  • A85Q glucose Gln
  • A85S serine Ser
  • A85L leucine Leu
  • A85D aspartic acid Asp
  • A85C cyste Cys
  • A85I isoleucine Ile
  • A85P proline Pro
  • A85T threonine Thr
  • A85G glycine Gly
  • A85M methionine Met
  • A85W tryptophan Trp
  • A85R arginine Arg
  • mutant protein was expressed and purified in the same manner as the wild-type pht protein (described in Example 1);
  • the reverse primers of F87G, F87R, F87D, F87Y, F87T, F87S, F87E, F87K, F87H, F87L, F87I, F87V, F87P, F87M, F87C, F87Q, F87N, and F87A are all F87-r.
  • D4 primer information :
  • the reverse primers of N83D and N83S are both N83-r.
  • the reverse primers of F84S, F84N, F84K, F84H, F84D, F84W, F84R, F84S, F84E, F84P, F84L, F84A, F84V, F84G, and F84M are all F84-r.
  • the reverse primers of A85F, A85N, A85H, A85Q, A85S, A85L, A85D, A85C, A85I, A85P, A85T, A85G, A85M, A85W, and A85R are all A85-r.
  • PCR procedure and PCR system were as described in Example 1, and the PCR template was the wild-type pht nanopore protein plasmid. After PCR, the gel was recovered and directly transferred into DH5 ⁇ for positive clone screening. Single colonies were selected for sequencing, and after being correct, they were frozen at -20°C for future use.
  • the steps for embedding the nanopore are as follows:
  • the wild-type pht current has a large signal noise and spontaneous blocking. At 150mV, the entire current fluctuates between 0.3-0.4nA and is chaotic.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Plant Pathology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种PHT纳米孔突变体蛋白及其应用，PHT纳米孔突变体蛋白包括将SEQ ID NO：1所示的野生型pht纳米孔蛋白的一个或多个氨基酸突变为其他氨基酸，或者敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的一个或多个氨基酸。本发明提供的PHT纳米孔突变体蛋白能够用于核酸的测序检测。

Description

一种PHT纳米孔突变体蛋白及其应用
本申请要求享有2022年10月14日向中国国家知识产权局提交的，专利申请号为202211262290.8，发明名称为“一种PHT纳米孔突变体蛋白及其应用”的在先申请的优先权权益。所述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明涉及一种表征目标多核苷酸的方法，特别涉及一种PHT纳米孔突变体蛋白及其应用，属于基因工程和遗传工程领域。
背景技术
纳米孔测序技术是近年被开发出来的新型核酸测序技术。依据孔道类型可分为固态孔和生物纳米孔，生物纳米孔即能够允许底物通过的孔道蛋白，以下纳米孔测序单指生物纳米孔测序技术。在电场力的作用下，带电核酸底物能够通过生物纳米孔。当核酸通过纳米孔时，能够阻碍纳米孔的电流，产生不同的电流信号，通过对电流信号的解析，可以获得核酸的碱基信息。和其它测序方法相比，它具有设备低廉，样品准备简便，测序速度快等优势，已经开始得到广泛应用。具体优势如下：在无需扩增的情况下，即可简便的建库；信号读取速度快，通常能达到200-300bp/s；读取长度长，通常可以达到数千个碱基；并且可以直接检测DNA上存在的修饰；可以直接测RNA，基于纳米孔测序的特点，RNA不再需要逆转录为DNA进行序列分析，由此便可以保存RNA上的修饰信息。
孔道蛋白是影响纳米孔测序的关键技术之一，孔道蛋白的收缩区(读取头)特征对于获取原始电流信号具有较大影响。目前孔道蛋白种类单一，常用的大肠杆菌CsgG突变体收缩区能够容纳4-5个碱基，因此单次记录的电流信号由4-5个碱基的阻碍作用产生，造成后续信号分析难度较大，造成准确率难以达到二代测序的水平。因此，需要发掘具有新型收缩区的孔道蛋白，一方面能够增加纳米孔的可选择种类，另一方面减少电流信号解析难度，进一步提高测序的准确率。
发明内容
本发明针对现有来源于大肠杆菌的纳米孔蛋白收缩区较厚，导致电流信号检测涉及连续多个核苷酸协同作用的缺点，寻找到一种收缩区厚度较薄的pht纳米孔蛋白，该蛋白收缩区仅包含一个氨基酸残基，从而减少所能容纳的连续多核苷酸的数量，降低电流信号解码难度，提高碱基分辨效率。同时，目前能够用于测序的孔蛋白种类比较少，所以，本发明提供了一种新型纳米孔蛋白，该蛋白野生型不能够直接用于测序，并证明经过收缩区氨基酸突变改造后，该蛋白具有核苷酸测序能力。
本发明通过系统的氨基酸序列比对、选择，蛋白表达、纯化，结构解析等手段，并通过氨基酸突变手段，分别表达、纯化了收缩区氨基酸不同突变体蛋白，结合磷脂膜电流信号检测手段，确定了不同突变体蛋白的膜信号特征；选取噪音较小的突变体蛋白，确定了其核酸检测能力。本发明涉及技术包括，大肠杆菌膜蛋白表达载体的构建、突变体蛋白的载体构建；膜蛋白的表达、纯化；冷冻电镜样品制备、数据收集、结构解析、结构搭建、结构分析；纳米孔蛋白磷脂膜嵌入技术、电流信号记录、处理等。
第一方面，本发明提供一种PHT纳米孔突变体蛋白，所述PHT纳米孔突变体蛋白将SEQ ID NO：1所示的野生型pht纳米孔蛋白的一个或多个氨基酸突变为其他氨基酸(例如F87、F84、N83、A85、E235)，或者敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的一个或多个氨基酸(例如敲除第80-86位氨基酸中的一个或多个)。
其他氨基酸包括天然氨基酸和/或非天然氨基酸。天然氨基酸为其他19种氨基酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白选自：
1)将SEQ ID NO：1所示的野生型pht纳米孔蛋白的F87、F84、N83、A85氨基酸中的一个或多个突变为其他19种氨基酸；
2)将SEQ ID NO：1所示的野生型pht纳米孔蛋白的E235氨基酸突变为其他19种氨基酸；
3)敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第80、81、82、83、84、85、86位氨基酸中的一个或多个(例如1个、2个、3个、4个、5个、6个、7个等)。
其他19种氨基酸指的是除原位点氨基酸外的20种常见氨基酸中的其他19种氨基酸。20种常见氨基酸为：甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸(蛋氨酸)、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天门冬氨酸、谷氨酸、赖氨酸、精氨酸和组氨酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白收缩区氨基酸F87突变为其他19种氨基酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白收缩区氨基酸F87突变为：F87M、F87P、F87L、F87G、F87I、F87Q、F87E、F87C、F87R、F87H、F87K、F87T、F87A、F87S、F87Y、F87N、F87V或F87D。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白收缩区氨基酸F87突变为F87T、F87L、F87N。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸N83突变为其他19种氨基酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸N83突变为：N83D、N83S。在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F84突变为其他19种氨基酸。在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F84突变为：F84S、F84N、F84K、F84H、F84D、F84W、F84R、F84S、F84E、F84P、F84L、F84A、F84V、F84G、F84M；优选为F84S、F84N、F84K。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸A85突变为其他19种氨基酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸A85突变为：A85F、A85N、A85H、A85Q、A85S、A85L、A85D、A85C、A85I、A85P、A85T、A85G、A85M、A85W、A85R；优选为A85I。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸E235突变为其他19种氨基酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸E235突变为：E235N。
在一些实施方案中，所述PHT纳米孔突变体蛋白是敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第85、86位氨基酸中的一个或多个(例如1个、2个)；例如敲除第85和第86位氨基酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白是敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第80、81、82、83位氨基酸中的一个或多个(例如1个、2个、3个、4个)；例如敲除第80至第83位氨基酸。
在一些实施方案中，所述PHT纳米孔突变体蛋白是敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第80、81、82位氨基酸中的一个或多个(例如1个、2个、3个)；例如敲除第80至第82位氨基酸。
第二方面，本发明提供编码上述PHT纳米孔突变体蛋白的编码基因。
在一些实施方案中，所述编码基因选自：
1)编码上述PHT纳米孔突变体蛋白的核酸序列；
2)与1)中的核酸序列至少具有85％的同源性，且编码上述PHT纳米孔突变体蛋白的核酸；
3)与1)或2)互补的核酸。
在一些实施方案中，同源性在85％-99％之间。
在一些实施方案中，同源性为85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％。
第三方面，本发明提供包含上述编码基因的重组载体。
第四方面，本发明提供一种基因工程化的细胞，所述的细胞含有上述重组载体，或者其基因组中包含上述编码基因。
包含上述编码基因可以为整合上述编码基因或者其他方式。
第五方面，本发明提供包含上述PHT纳米孔突变体蛋白的产品。
在一些实施方案中，所述产品是组合物、复合物、试剂盒。
第六方面，本发明提供包含上述重组载体的重组菌。
第七方面，本发明提供上述PHT纳米孔突变体蛋白的制备方法，包括以下步骤：
1)构建PHT纳米孔突变体蛋白载体；
2)PHT纳米孔突变体蛋白表达、纯化。
第八方面，本发明提供一种膜层，所述膜层中嵌入上述PHT纳米孔突变体蛋白。
在一些实施方案中，所述膜层是磷脂膜层。
在一些实施方案中，所述膜层是磷脂单层膜层。
第九方面，本发明提供一种检测系统，包括上述PHT纳米孔突变体蛋白或者上述膜层。
第十方面，本发明提供一种装置，包括上述PHT纳米孔突变体蛋白、上述膜层或者上述检测系统。
第十一方面，本发明提供上述PHT纳米孔突变体蛋白、上述膜层或上述检测系统在制备样品测序和/或检测的装置中的应用。
在一些实施方案中，所述样品为核苷酸、核酸、氨基酸、寡聚肽、多肽、蛋白质中的一种或多种。
第十二方面，本发明提供上述PHT纳米孔突变体蛋白、上述膜层、上述检测系统或上述装置在样品测序和/或检测中的应用。
在一些实施方案中，所述样品为核苷酸、核酸、氨基酸、寡聚肽、多肽、蛋白质中的一种或多种。
有益效果
本发明通过冷冻电镜技术，解析了高分辨率pht纳米孔三维结构结构分析发现pht纳米孔蛋白收缩区宽度为厚度仅由一个氨基酸(F87)决定。同时，本发明发现，野生型的pht在磷脂膜上具有较高的噪音，不能用于核酸底物检测。进一步通过突变实验发现，改变该位置氨基酸的种类，不影响纳米孔蛋白的表达，但突变后的不同氨基酸种类对其电流信号有较大影响。
本发明提供一种PHT纳米孔突变体蛋白，所述突变体蛋白经氨基酸的突变或敲除后，相比于野生型蛋白，其电流性质、电流信号得到显著改善。其中，F87T、E235N、F87L、F87N、A85I、F84S、F84N和F84K突变体蛋白的效果更优，可用于DNA等样品的测序和/或检测。
附图说明
图1是野生型pht蛋白表达纯化图；
图2是野生型pht蛋白分子筛结果图；
图3是野生型pht蛋白冷冻电镜map图；
图4是野生型pht纳米孔整体结构图；
图5是野生型pht纳米孔收缩区整体构象图；
图6是野生型pht纳米孔收缩区单体构象图；
图7是F87位点PHT纳米孔突变体蛋白表达纯化图；
图8是敲除PHT纳米孔突变体蛋白表达纯化图；
图9是E235位点PHT纳米孔突变体蛋白表达纯化图；
图10是N83位点PHT纳米孔突变体蛋白表达纯化图；
图11是F84位点PHT纳米孔突变体蛋白表达纯化图；
图12是A85位点PHT纳米孔突变体蛋白表达纯化图；
图13是野生型pht纳米孔电流性质图；
图14是F87、E235位点PHT纳米孔突变体蛋白纳米孔电流性质图；
图15是F87位点PHT纳米孔突变体蛋白纳米孔电流性质图；
图16是位点敲除的PHT纳米孔突变体蛋白纳米孔电流性质图；
图17是N83位点PHT纳米孔突变体蛋白纳米孔电流性质图；
图18是F84位点PHT纳米孔突变体蛋白纳米孔电流性质图；
图19是A85位点PHT纳米孔突变体蛋白纳米孔电流性质图。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1野生型pht纳米孔蛋白表达载体构建与蛋白的制备
1.野生型pht纳米孔蛋白载体的构建
野生型pht纳米孔蛋白来源于Nitratireductor pacificus pht-3B(ACCESSION：WP_008595533，SEQ ID NO：1)，该蛋白表达基因通过人工合成的方式获得，合成过程进行适用于大肠杆菌表达的密码子优化。合成后基因通过无缝克隆的方式构建到pET-28a载体，蛋白C端添加6×his，作为亲和纯化标签。
载体构建步骤如下：
以合成基因为模板，使用正反向引物(primer F：GGGCTAACAGGAGGAATTAACCATGTCATCACGCTCACATCA primer R：TCAATGATGATGATGATGATGCTGCTGAACAGGGTTAGGTTC)
PCR扩增目的基因片段，胶回收后与线性化的pET-28a载体连接，后转入DH5α感受态细胞进行阳性克隆的筛选。挑取2个克隆进行测序，测序正确后保存构建质粒于-20℃备用。
PCR体系如下(20μL)：
PCR程序如下：
无缝连接体系(10μL)如下：
2×无缝连接缓冲液      5μL
目的片段(50ng/μL)     3μL
线性化载体(10ng/μL)   2μL
50℃反应15min后取2μL转入DH5α细胞，挑点测序。
2.野生型pht纳米孔蛋白表达、纯化
野生型pht纳米孔蛋白经过Ni柱亲和层析、分子筛纯化后，蛋白纯度较高(附图1)，蛋白性质均一(附图2)。且在SDS-PAGE上看，加热产生单体蛋白(分子量约为30kDa)；不加热时，蛋白为寡聚状态(即成孔状态，分子量大于marker最大条带180kDa)。
表达纯化步骤如下：
1)野生型pht载体测序正确后，转入BL21(DE3)，进行表达。37℃ 200rpm获取种子液1mL后转入1L LB培养基，37℃ 200rpm至OD600为1.2，降温至26℃0.2mM IPTG(异丙基硫代半乳糖苷)诱导过夜；
2)4000rpm收集菌体，使用裂解缓冲液重悬(20mL/L菌)，高压破碎后18000rpm 4℃离心1小时，收集沉淀膜组分；
3)使用溶膜缓冲液(10mL/L菌)重悬膜组分，4℃磁力搅拌1小时进行膜蛋白抽提，18000rpm 4℃离心1小时，收集上清膜蛋白组分；
4)上清中加入终浓度30mM的咪唑后与溶膜缓冲液平衡后的Ni beads进行孵育，4℃结合1小时后进行亲和纯化；
5)将上清和Ni beads导入柱子中，重力自然流穿；10mL洗涤液清洗，5mL洗脱液洗脱蛋白；
6)目的蛋白进行分子筛纯化后SDS-PAGE检测纯度，结果如附图1所示，分子筛结果如附图2所示。
裂解缓冲液：20mM Tris-HCl，150mM NaCl，pH 8.0。
溶膜缓冲液：20mM Tris-HCl，150mM NaCl，pH 8.0，1％LDAO(十二烷基二甲基氧化胺)。
洗涤液：20mM Tris-HCl，150mM NaCl，pH 8.0，0.5％LDAO，50mM咪唑。
洗脱液：20mM Tris-HCl，150mM NaCl，pH 8.0，0.1％LDAO，200mM咪唑。
分子筛缓冲液：20mM Tris-HCl，150mM NaCl，pH 8.0，0.06％LDAO。
实施例2：野生型pht纳米孔蛋白原子水平结构
得到纯度和均一度较高的野生型pht纳米孔蛋白后(实施例1)，利用冷冻电镜技术，解析了其分辨率为的原子水平结构。
冷冻电镜结构解析步骤如下：
1)准备筛好的样品，并在相同的冻样条件下制备几个备用样品，选取8个合适的样品上到Talos Arctica 200kV高端电镜中；
2)上样后等待真空和温度稳定后，开启镜筒在低倍下选择合适的方形孔。这一步和冷冻样品的筛选类似，其目的使选择合适的方形孔用于后面数据的收集；
3)根据电镜的机时空余以及每个方形孔能够采集的照片数量，计算大概需要选择的方形孔数量，选择完成后进行地图的拍摄；
4)地图拍摄过程中，可以离线选择地图中能够用于数据收集的孔，这样可以节省一部分的时间；
5)待地图拍摄完成后既可以对电镜进行调机，以备数据的收集工作。主要包括电镜的合轴、背底扣除及数据收集基本参数(欠焦量-1.5μm到-2.5μm，电子计量拍照帧数32帧和像素尺寸)的设置；
后续经过数据处理，结构解析，得到了其电子密度，整体如附图3所示，整体map为9聚体结构，单体位置如图所示(附图3左图为顶视图，右图为俯视图)，右图标注该纳米孔蛋白跨膜区位置。随后，通过同源建模，结构搭建、精修获得了野生型pht纳米孔蛋白氨基酸的原子坐标(附图4，左图为顶视图，右图为俯视图)。结构分析可见，pht纳米孔蛋白收缩区为单层结构，区别于大肠杆菌纳米孔蛋白的三层结构。其中，收缩区由单个氨基酸决定，为第87位苯丙氨酸(Phe，F)，收缩区宽度为(附图5)，单体结构分析只有F87侧链指向孔道内测(附图6)。收缩区厚度薄(附图5)，仅为
实施例3：野生型以及PHT纳米孔突变体蛋白在人工膜上电流性质的确定
检测野生型pht纳米孔蛋白在人工膜上的孔道电流性质。同时，针对其收缩区决定氨基酸(F87)及其附近的氨基酸N83、F84、A85以及E235、80-83、85-86等，进行突变分析。
F87(F，苯丙氨酸Phe)的突变：F87M(蛋氨酸Met)、F87P(脯氨酸Pro)、F87L(亮氨酸Leu)、F87G(甘氨酸Gly)、F87I(异亮氨酸Ile)、F87Q(谷氨酰胺Gln)、F87E(谷氨酸Glu)、F87C(半胱氨酸Cys)、F87R(精氨酸Arg)、F87H(组氨酸His)、F87K(赖氨酸Lys)、F87T(苏氨酸Thr)、F87A(丙氨酸Ala)、F87S(丝氨酸Ser)、F87Y(酪氨酸Tyr)、F87N(天冬酰胺Asn)、F87V(缬氨酸Val)、F87D(天冬氨酸Asp)。
敲除部分氨基酸：D2(敲除氨基酸85-86)、D3(敲除氨基酸80-82)、D4(敲除氨基酸80-83)。
E235(E，谷氨酸Glu)的突变：E235N(天冬酰胺Asn)。
N83(N，天冬酰胺Asn)的突变：N83D(天冬氨酸Asp)、N83S(丝氨酸Ser)。
F84(F，苯丙氨酸Phe)的突变：F84S(丝氨酸Ser)、F84N(天冬酰胺Asn)、F84K(赖氨酸Lys)、F84H(组氨酸His)、F84D(天冬氨酸Asp)、F84W(色氨酸Trp)、F84R(精氨酸Arg)、F84S(丝氨酸Ser)、F84E(谷氨酸Glu)、F84P(脯氨酸Pro)、F84L(亮氨酸Leu)、F84A(丙氨酸Ala)、F84V(缬氨酸Val)、F84G(甘氨酸Gly)、F84M(蛋氨酸Met)。
A85(A，丙氨酸Ala)的突变：A85F(苯丙氨酸Phe)、A85N(天冬酰胺Asn)、A85H(组氨酸His)、A85Q(谷氨酰胺Gln)、A85S(丝氨酸Ser)、A85L(亮氨酸Leu)、A85D(天冬氨酸Asp)、A85C(半胱氨酸Cys)、A85I(异亮氨酸Ile)、A85P(脯氨酸Pro)、A85T(苏氨酸Thr)、A85G(甘氨酸Gly)、A85M(蛋氨酸Met)、A85W(色氨酸Trp)、A85R(精氨酸Arg)。
经SDS-PAGE检测(附图7-12)，突变后蛋白寡聚状态没有受到影响。在不加热时含有寡聚状态的蛋白(Marker 180kDa上方)，加热后产生单体大小条带。与野生型pht相比，上述突变蛋白，从表达量、寡聚状态、热稳定性上均没有明显变差，由此可以说明，突变没有造成蛋白的生化性质发生明显变化。
涉及材料与方法如下：
1.突变蛋白载体构建与蛋白表达纯化
突变蛋白与野生型pht蛋白的表达纯化方式相同(实施例1中所述)；
1)引物如下：f：正向引物r：反向引物
F87引物信息：
F87-r CTGAGCAAAGTTATCTTCAGGT
F87G-f GATAACTTTGCTCAGggcTCCAAAGCGGTCTCACAGG
F87R-f GATAACTTTGCTCAGcgcTCCAAAGCGGTCTCACAGG
F87D-f GATAACTTTGCTCAGgatTCCAAAGCGGTCTCACAGG
F87Y-f GATAACTTTGCTCAGtatTCCAAAGCGGTCTCACAGG
F87T-f GATAACTTTGCTCAGacaTCCAAAGCGGTCTCACAGG
F87S-f GATAACTTTGCTCAGtcaTCCAAAGCGGTCTCACAGG
F87E-f GATAACTTTGCTCAGgaaTCCAAAGCGGTCTCACAGG
F87K-f GATAACTTTGCTCAGaaaTCCAAAGCGGTCTCACAGG
F87H-f GATAACTTTGCTCAGcatTCCAAAGCGGTCTCACAGG
F87L-f GATAACTTTGCTCAGttaTCCAAAGCGGTCTCACAGG
F87I-f GATAACTTTGCTCAGattTCCAAAGCGGTCTCACAGG
F87V-f GATAACTTTGCTCAGgttTCCAAAGCGGTCTCACAGG
F87P-f GATAACTTTGCTCAGcctTCCAAAGCGGTCTCACAGG
F87M-f GATAACTTTGCTCAGatgTCCAAAGCGGTCTCACAGG
F87C-f GATAACTTTGCTCAGtgtTCCAAAGCGGTCTCACAGG
F87Q-f GATAACTTTGCTCAGcaaTCCAAAGCGGTCTCACAGG
F87N-f GATAACTTTGCTCAGaatTCCAAAGCGGTCTCACAGG
F87A-f GATAACTTTGCTCAGgctTCCAAAGCGGTCTCACAGG
其中，F87G、F87R、F87D、F87Y、F87T、F87S、F87E、F87K、F87H、F87L、F87I、F87V、F87P、F87M、F87C、F87Q、F87N、F87A的反向引物均为F87-r。
D2引物信息：
D2-f ATAACTTTTTCTCCAAAGCGGTCTCAC
D2-r TGGAGAAAAAGTTATCTTCAGGTTTCTGCT。
D3引物信息：
D3-f GGACAGCAGAAAAACTTTGCTCAGTTCTCCAAAG
D3-r AAAGTTTTTCTGCTGTCCGGTCTTATCA。D4引物信息：
D4-f CAGCAGAAATTTGCTCAGTTCTCCAAAGCG
D4-r CTGAGCAAATTTCTGCTGTCCGGTCTTA。
E235N引物信息：
E235N-f AGGCTaacGCTGGCTTTACACGCAAC
E235N-r AGCCAGCgttAGCCTGAAGAATTTCATCAAT。N83引物信息：
N83-r ATCTTCAGGTTTCTGCTGTCC
N83D-f CAGAAACCTGAAGATgatTTTGCTCAGTTCTCCAAAGCG
N83S-f CAGAAACCTGAAGATtctTTTGCTCAGTTCTCCAAAGCG
其中，N83D、N83S的反向引物均为N83-r。
F84引物信息：
F84-r ATCTTCAGGTTTCTGCTGTCC
F84S-f CAGAAACCTGAAGATtctTTTGCTCAGTTCTCCAAAGCG
F84N-f CAGAAACCTGAAGATaatTTTGCTCAGTTCTCCAAAGCG
F84K-f CAGAAACCTGAAGATaaaTTTGCTCAGTTCTCCAAAGCG
F84H-f CAGAAACCTGAAGATcatTTTGCTCAGTTCTCCAAAGCG
F84D-f CAGAAACCTGAAGATgatTTTGCTCAGTTCTCCAAAGCG
F84W-f CAGAAACCTGAAGATtggTTTGCTCAGTTCTCCAAAGCG
F84R-f CAGAAACCTGAAGATc gtTTTGCTCAGTTCTCCAAAGCG
F84S-f CAGAAACCTGAAGATtctTTTGCTCAGTTCTCCAAAGCG
F84E-f CAGAAACCTGAAGATgaaTTTGCTCAGTTCTCCAAAGCG
F84P-f CAGAAACCTGAAGATcctTTTGCTCAGTTCTCCAAAGCG
F84L-f CAGAAACCTGAAGATttaTTTGCTCAGTTCTCCAAAGCG
F84A-f CAGAAACCTGAAGATgctTTTGCTCAGTTCTCCAAAGCG
F84V-f CAGAAACCTGAAGATgttTTTGCTCAGTTCTCCAAAGCG
F84G-f CAGAAACCTGAAGATggtTTTGCTCAGTTCTCCAAAGCG
F84M-f CAGAAACCTGAAGATatgTTTGCTCAGTTCTCCAAAGCG
其中，F84S、F84N、F84K、F84H、F84D、F84W、F84R、F84S、F84E、F84P、F84L、F84A、F84V、F84G、F84M的反向引物均为F84-r。
A85引物信息：
A85-r AAAGTTATCTTCAGGTTTCTGCTG
A85F-f CCTGAAGATAACTTTtttCAGTTCTCCAAAGCGGTC
A85N-f CCTGAAGATAACTTTaatCAGTTCTCCAAAGCGGTC
A85H-f CCTGAAGATAACTTTcatCAGTTCTCCAAAGCGGTC
A85Q-f CCTGAAGATAACTTTcaaCAGTTCTCCAAAGCGGTC
A85Y-f CCTGAAGATAACTTTtatCAGTTCTCCAAAGCGGTC
A85L-f CCTGAAGATAACTTTttaCAGTTCTCCAAAGCGGTC
A85D-f CCTGAAGATAACTTTgatCAGTTCTCCAAAGCGGTC
A85C-f CCTGAAGATAACTTTtgtCAGTTCTCCAAAGCGGTC
A85I-f CCTGAAGATAACTTTattCAGTTCTCCAAAGCGGTC
A85P-f CCTGAAGATAACTTTcctCAGTTCTCCAAAGCGGTC
A85T-f CCTGAAGATAACTTTactCAGTTCTCCAAAGCGGTC
A85G-f CCTGAAGATAACTTTggtCAGTTCTCCAAAGCGGTC
A85M-f CCTGAAGATAACTTTatgCAGTTCTCCAAAGCGGTC
A85W-f CCTGAAGATAACTTTtggCAGTTCTCCAAAGCGGTC
A85R-f CCTGAAGATAACTTTcgtCAGTTCTCCAAAGCGGTC
其中，A85F、A85N、A85H、A85Q、A85S、A85L、A85D、A85C、A85I、A85P、A85T、A85G、A85M、A85W、A85R的反向引物均为A85-r。
PCR程序与PCR体系如实施例1所述，PCR模板为野生型pht纳米孔蛋白质粒。PCR后胶回收，直接转入DH5α中进行阳性克隆筛选。挑取单菌落测序，正确后于-20℃冻存备用。
2)突变体蛋白采用实施例1蛋白纯化步骤，获取蛋白。
2.膜上电流性质检测
在获得野生型和PHT纳米孔突变体蛋白之后，对这些蛋白的电流性质进行检测。采用人工形成磷脂单层膜(DPhPC)，再嵌入单个纳米孔蛋白，后在150mV或180mV电压下记录电流变化。
嵌入纳米孔步骤如下：
在缓冲液(600mM KCl，75mM K3[Fe(CN)6，25mM K4[Fe(CN)6]·3H2O，100mM Hepes，pH 8.0)中，由嵌入到DPhPC磷脂双分子层的纳米孔获得电信号测量值。在实现单孔插入磷脂双分子层后，用2mL缓冲液(600mM KCl，75mM K3[Fe(CN)6，25mM K4[Fe(CN)6]·3H2O，100mM Hepes，pH 8.0)流过系统以除去残留的过量纳米孔。分别记录野生型和PHT纳米孔突变体蛋白在磷脂膜上的电流信号，记录结果如图所示(附图13-19)。
如附图13所示，野生型pht电流大小，信号噪音较大存在自发封阻现象。150mV下，整个电流在0.3-0.4nA之间波动，杂乱。
比较附图13和附图14-19可知，相比于野生型pht，突变体pht蛋白的电流信号噪音和自发封阻现象均显著减少，电流性质、电流信号得到显著改善。其中，F87T、E235N、F87L、F87N、A85I、F84S、F84N和F84K突变体蛋白的效果更优，电流性质稳定，自发封堵的情况很少，电流信号宽度更窄(上下幅度)、毛刺更少，可用于DNA等样品的测序和/或检测。
序列信息

以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种PHT纳米孔突变体蛋白，其特征在于，所述PHT纳米孔突变体蛋白将SEQ ID NO：1所示的野生型pht纳米孔蛋白的一个或多个氨基酸突变为其他氨基酸，或者敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白中的一个或多个氨基酸。
  2. 根据权利要求1所述的纳米孔蛋白，其特征在于，所述PHT纳米孔突变体蛋白选自：
    1)将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F87、F84、N83、A85中的一个或多个突变为其他19种氨基酸；
    2)将SEQ ID NO：1所示的野生型pht纳米孔蛋白的E235氨基酸突变为其他19种氨基酸；
    3)敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第80、81、82、83、84、85、86位氨基酸中的一个或多个；
    优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F87突变为其他19种氨基酸；优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F87突变为：F87M、F87P、F87L、F87G、F87I、F87Q、F87E、F87C、F87R、F87H、F87K、F87T、F87A、F87S、F87Y、F87N、F87V或F87D；优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F87突变为F87T、F87L或F87N；
    优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸N83突变为其他19种氨基酸；优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸N83突变为：N83D或N83S；
    优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F84突变为其他19种氨基酸；优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸F84突变为：F84S、F84N、F84K、F84H、F84D、F84W、F84R、F84S、F84E、F84P、F84L、F84A、F84V、F84G或F84M；优选为F84S、F84N或F84K；
    优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸A85突变为其他19种氨基酸；优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸A85突变为：A85F、A85N、A85H、A85Q、A85S、A85L、A85D、A85C、A85I、A85P、A85T、A85G、A85M、A85W或A85R；优选为A85I；
    优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸E235突变为其他19种氨基酸；优选地，所述PHT纳米孔突变体蛋白是将SEQ ID NO：1所示的野生型pht纳米孔蛋白的氨基酸E235突变为：E235N；
    优选地，所述PHT纳米孔突变体蛋白是敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第85、86位氨基酸中的一个或多个；优选为敲除第85和第86位氨基酸；
    优选地，所述PHT纳米孔突变体蛋白是敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第80、81、82、83位氨基酸中的一个或多个；优选为敲除第80至第83位氨基酸；
    优选地，所述PHT纳米孔突变体蛋白是敲除SEQ ID NO：1所示的野生型pht纳米孔蛋白的第80、81、82位氨基酸中的一个或多个；优选为敲除第80至第82位氨基酸。
  3. 一种编码权利要求1或2所述的PHT纳米孔突变体蛋白的编码基因；
    优选地，所述编码基因选自：
    1)编码权利要求1或2所述的PHT纳米孔突变体蛋白的核酸序列；
    2)与1)中的核酸序列至少具有85％的同源性，且编码权利要求1或2所述的PHT纳米孔突变体蛋白的核酸；
    3)与1)或2)互补的核酸；
    优选地，同源性在85％-99％之间。
  4. 一种包含如权利要求3所述的编码基因的重组载体；或，一种包含如所述重组载体的重组菌；或，一种包含如权利要求1或2所述的PHT纳米孔突变体蛋白的产品；优选地，所述产品是组合物、复合物、试剂盒。
  5. 一种基因工程化的细胞，其特征在于，所述细胞包含权利要求4所述的重组载体，或者所述细胞的基因组中包含权利要求3所述的编码基因。
  6. 一种如权利要求1或2所述的PHT纳米孔突变体蛋白的制备方法，其特征在于，包括以下步骤：
    1)构建PHT纳米孔突变体蛋白载体；
    2)PHT纳米孔突变体蛋白表达、纯化。
  7. 一种膜层，其特征在于，所述膜层中嵌入权利要求1或2所述的PHT纳米孔突变体蛋白；优选地，所述膜层是磷脂膜层；优选地，所述膜层是磷脂单层膜层。
  8. 一种检测系统，其特征在于，包括权利要求1或2所述的PHT纳米孔突变体蛋白或者权利要求7所述的膜层。
  9. 一种装置，其特征在于，包括权利要求1或2所述的PHT纳米孔突变体蛋白、权利要求7所述的膜层或者权利要求8所述的检测系统。
  10. 权利要求1或2所述的PHT纳米孔突变体蛋白、权利要求7所述的膜层、权利要求8所述的检测系统或权利要求9所述的装置在样品测序和/或检测中的应用；
    优选地，所述样品为核苷酸、核酸、氨基酸、寡聚肽、多肽、蛋白质中的一种或多种。
PCT/CN2023/124532 2022-10-14 2023-10-13 一种pht纳米孔突变体蛋白及其应用 WO2024078621A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211262290 2022-10-14
CN202211262290.8 2022-10-14

Publications (1)

Publication Number Publication Date
WO2024078621A1 true WO2024078621A1 (zh) 2024-04-18

Family

ID=90640016

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/124532 WO2024078621A1 (zh) 2022-10-14 2023-10-13 一种pht纳米孔突变体蛋白及其应用

Country Status (2)

Country Link
CN (1) CN117886907A (zh)
WO (1) WO2024078621A1 (zh)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034018A2 (en) * 2008-09-22 2010-03-25 University Of Washington Msp nanopores and related methods
CN105801676A (zh) * 2016-04-13 2016-07-27 东南大学 一种突变MspA蛋白单体及其表达基因和应用
US20180209952A1 (en) * 2015-04-14 2018-07-26 Katholieke Universiteit Leuven Nanopores with internal protein adaptors
US20190079067A1 (en) * 2016-03-31 2019-03-14 Genia Technologies, Inc. Nanopore protein conjugates and uses thereof
US20190292235A1 (en) * 2016-07-12 2019-09-26 Rijksuniversiteit Groningen Biological Nanopores for Biopolymer Sensing and Sequencing Based on FRAC Actinoporin
CN111413383A (zh) * 2020-04-20 2020-07-14 深圳市梅丽纳米孔科技有限公司 一种构建双识别位点纳米孔的方法
WO2020155242A1 (zh) * 2019-01-30 2020-08-06 深圳市梅丽纳米孔科技有限公司 一种突变NfpAB纳米孔、测试系统及制作方法与应用
CN113480620A (zh) * 2021-08-18 2021-10-08 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010034018A2 (en) * 2008-09-22 2010-03-25 University Of Washington Msp nanopores and related methods
US20180209952A1 (en) * 2015-04-14 2018-07-26 Katholieke Universiteit Leuven Nanopores with internal protein adaptors
US20190079067A1 (en) * 2016-03-31 2019-03-14 Genia Technologies, Inc. Nanopore protein conjugates and uses thereof
CN105801676A (zh) * 2016-04-13 2016-07-27 东南大学 一种突变MspA蛋白单体及其表达基因和应用
US20190292235A1 (en) * 2016-07-12 2019-09-26 Rijksuniversiteit Groningen Biological Nanopores for Biopolymer Sensing and Sequencing Based on FRAC Actinoporin
WO2020155242A1 (zh) * 2019-01-30 2020-08-06 深圳市梅丽纳米孔科技有限公司 一种突变NfpAB纳米孔、测试系统及制作方法与应用
CN111413383A (zh) * 2020-04-20 2020-07-14 深圳市梅丽纳米孔科技有限公司 一种构建双识别位点纳米孔的方法
CN113480620A (zh) * 2021-08-18 2021-10-08 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用

Also Published As

Publication number Publication date
CN117886907A (zh) 2024-04-16

Similar Documents

Publication Publication Date Title
JP7282697B2 (ja) 新規タンパク質細孔
CN112375784B (zh) 制备重组新型冠状病毒Spike蛋白的方法
CN109627344B (zh) cAMP荧光探针及其应用
US20220412948A1 (en) Artificial nanopores and uses and methods relating thereto
WO2018126470A1 (zh) 重组dna聚合酶
WO2024078621A1 (zh) 一种pht纳米孔突变体蛋白及其应用
WO2024032020A1 (zh) 一种增强型单体StayGold蛋白及其应用
WO2000040707A1 (en) Modified forms of hepatitis c virus ns3 protease
US8981067B2 (en) Composition, method and kit for obtaining purified recombinant proteins
JPWO2020045472A5 (zh)
US20110287484A1 (en) Expression vector containing the major envelope protein p9 of cystovirus phi6 as a fusion partner, and process for producing a membrane protein using the same
CN117417418A (zh) 一种具有超高热稳定性的纳米孔突变体及其应用
CN117511978B (zh) 一种提高外源蛋白表达量的方法
CN117417421A (zh) 一种突变体膜蛋白复合物纳米孔及其应用
CN103172713A (zh) 一种链霉亲和素突变体及其制备方法
JPH0371111B2 (zh)
JPS6387981A (ja) 新規組換えプラスミドｐＢＳＦＯＬＥＫ１およびそれを含有する菌株
CN115746110A (zh) 一种转录调控因子LysG的突变体及其应用
JP6235815B2 (ja) 組換えヒトμ−カルパインの調製方法
CN116200363A (zh) Taq酶突变体、其制备方法和应用
EP1225181A1 (en) Protein expression and structure resolution using specific fusion vectors
JP2004535185A5 (zh)
JPS63102696A (ja) ジヒドロ葉酸還元酵素―ロイシンエンケファリン融合タンパクおよびその製造方法
KR20070098343A (ko) 잔토모나스속 균주의 엔도뉴클레아제인 Ｘｏｒ Ⅰ의 발현및 정제방법
JPH0354556B2 (zh)