Semi-synthetic nanopore, preparation method thereof and protein sequencing method

Abstract

The invention belongs to the technical field of biology, and relates to a semi-synthetic nanopore, a preparation method thereof and a protein sequencing method. The semi-synthetic nanopore comprises a polypeptide binding protein and a construction body of a nanopore channel, and the polypeptide binding protein can unfold protein into linear polypeptide binding protein; in addition, the polypeptide binding protein is combined with the nanopore channel through an amido bond; preferably, the binding by amido bonds comprises the binding by transpeptidase properties or isopeptide bonds; preferably, the polypeptide binding protein is a folding enzyme; preferably, the nano pore channel is a nano pore channel protein. The current characteristic spectrum with the time sequence when the protein full-length sequence penetrates through the nanopore is obtained, and the problem that the reading length of existing nanopore protein sequencing is short is solved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a semi-synthetic nanopore and a preparation method thereof and a protein sequencing method. Background Art

[0002] Protein is the material basis of life activities. Proteins with different sequences and structures determine their functions in life activities. Changing the sequence of proteins may change cell signaling pathways and material metabolism pathways, thereby inducing various diseases. Therefore, it is necessary to develop a simple and reliable method to detect proteins or achieve protein sequencing.

[0003] At present, DNA sequencing based on single-molecule nanopore technology has been widely used in many fields. Its principle is: double-stranded DNA is unwound into single-stranded DNA under the action of motor protein, and passes through the nanopore under the action of electric field force; different deoxyribonucleotides produce different current blocking effects in the nanopore, and machine learning is used to obtain a one-to-one mapping relationship between current changes and base sequences. Similarly, a polypeptide composed of amino acids passes through a nanopore under the action of an electric field force, and the composition of the polypeptide sequence can be analyzed by analyzing the current changes caused by different amino acids passing through the pore.

[0004] Precise manipulation of peptides or proteins is a key challenge to improve the accuracy of nanopore sequencing. Around this key challenge, several research teams have proposed corresponding solutions. (1) Dekker, Cees team proposed a protein sequencing principle based on Hel308 speed control (Brinkerhoff, Henry, et al. "Multiple re ads of single proteins at single-amino acid resolution using nanopores." Science 374.6574 (2021): 1509-1513.). First, the peptide and nucleic acid are coupled by chemical coupling to form a peptide-nucleic acid complex. Incubated with Hel308 to form a peptide-DNA-hel308 complex; after the complex is captured by the nanopore under the action of the electric field force, the complementary DNA chain separates; the DNA moves from the bottom to the top of the membrane under the action of Hel308; when the peptide is located in the sensor area of ​​the pore, the sequence is read. The key point of this method is to use the translocation activity of Hel308 to achieve the purpose of speed control. (2) Huang Shuo's team at Nanjing University proposed a peptide sequencing principle using Phi29 DNA polymerase to control the speed. (Yan, Shuanghong, et al. "Single molecule ratcheting motion of peptides in a Mycobacterium smegmatis Porin A (MspA) nanopore." Nano letters 21.15 (2021): 6703-6710.). Phi29 DNA polymerase is incubated with a synthetic ssDNA-peptide complex to form a complex. The polymerase is used to amplify DNA to indirectly control the speed of polypeptide movement. (3) Oxford Nanopore Technologies Limited (WO2021 / 111125A1) discloses a protein sequencing principle based on helicase speed control. Its design is to first synthesize an adaptor-peptide-dsDNA tail complex, then partially anneal the single-stranded DNA to form a double-stranded DNA, and then combine it with the oligonucleotide control protein to read the electrical signal through the nanopore.(4) Bai Jingwei's team (Chen, Zhijie, et al. "Controlled movement of ssDNA conjugated peptide through Mycobacterium smegmatis porin A (MspA) nanopore by ahelicase motor for peptide sequencing application." Chemical science 12.47 (2021): 15750-15756.) proposed a protein sequencing scheme based on MTA helicase speed control. However, the above schemes (1)-(4) have great limitations in terms of protein sequencing read length. The read length depends on the distance from the motor protein complex to the nanopore detection area, which generally does not exceed 30 amino acids. At the same time, the measured polypeptide must be coupled with a single-stranded nucleic acid to form a complex, that is, the terminal amino acid needs to be further modified to achieve coupling; it is more difficult. But most proteins consist of far more than 30 amino acids; and most of them are natural amino acids.

[0005] Therefore, how to achieve full-length protein sequence reading is crucial. The Maglia team achieved active regulation of natural protein unfolding by designing nanopores with unfolding activity functions (Zhang S, Huang G, Versloot RCA, Bruininks BMH, de Souza PCT, Marrink SJ, et al. Bottom-up fabrication of a proteasome-nanopore that unravels and processes single prote ins. Nat Chem 2021, 13 (12): 1192-1199). A semi-synthetic nanopore (900 kDa) formed by 42 protein components was constructed, including three parts: a β-structured barrel protein, a heptamer proteasome activator 28α, and a 20S core proteasome; after the GFP protein containing the ssrA tag was captured by the nanopore, the GFP protein was unfolded and enzymatically hydrolyzed into multiple fragments under the action of the proteasome, and then passed through the β-structured barrel protein detection area in sequence. After enzymatic hydrolysis, the signal of some polypeptides was not captured by the device because the perforation speed was too fast. Although all the polypeptides pass through the nanopore, it is impossible to obtain a current characteristic spectrum with a time series, and it is even more impossible to obtain sequence-related information of the polypeptide. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention provides a semi-synthetic nanopore and a preparation method thereof and a protein sequencing method, so as to obtain a current characteristic spectrum with a time series when the full-length sequence of a protein passes through the nanopore, and solve the problem of short read length of the existing nanopore protein sequencing.

[0007] To this end, the first aspect of the present invention provides a semi-synthetic nanopore, comprising a construct of a polypeptide binding protein and a nanopore, wherein the polypeptide binding protein is a polypeptide binding protein capable of unfolding a protein into a linear form; and the polypeptide binding protein is bound to the nanopore via an amide bond.

[0008] In some embodiments of the invention, the binding via an amide bond comprises connection via a transpeptidase property or an isopeptide bond.

[0009] In some embodiments of the invention, the polypeptide binding protein is an unfolding enzyme.

[0010] In some embodiments of the invention, the nanopore is a nanopore protein.

[0011] In some embodiments of the invention, the unfolding enzyme comprises ClpX, NTP-driven unfolding enzyme, ATP-dependent proteases, Mycobacterium tuberculosis ClpC1 unfolding enzyme, Msp1, ClpA unfolding enzyme, HsIU unfolding enzyme, Valosin-containing protein / p97, 26S proteasome or Lon protease.

[0012] In some embodiments of the invention, the unfolding enzyme comprises ClpX.

[0013] In some embodiments of the invention, the nanopore protein comprises MspA porin, Alpha-hemolysin, Frac, ClyA, PA63, CsgG, GspD, XcpQ, Wza, Phi29 connector, T7 connector, SPP1 connector, T3 connector, T4 connector or aerolysin.

[0014] In some embodiments of the invention, the nanopore protein comprises an MspA porin.

[0015] The second aspect of the present invention provides a method for preparing a semi-synthetic nanopore as described in the first aspect of the present invention, comprising: connecting a polypeptide binding protein and a nanopore via an amide bond to form a semi-synthetic nanopore, preferably, the amide bond bonding comprises connecting via transferase properties or isopeptide bonds.

[0016] In some embodiments of the present invention, the unfolding enzyme sequence is a sequence in which 60 amino acids at the N-terminus are deleted, and includes C169S and K408E amino acid mutations.

[0017] In some embodiments of the present invention, the transpeptidase includes: transpeptidase A (Staphylococcusaureus sortase A), SaSrtA (Staphylococcus aureus), SrtB (Bacilli, Listeria and S. aureus), SrtC (Actinomyces, Corynebacteria, Enterococci and Streptococci), SrtD (sporulating Gram-positive bacteria), BaSrtA, SavSrtE, CdSrtA, peptidyl asparaginyl ligases, Butelase-1 or OaAEP1.

[0018] In some embodiments of the invention, the transferase comprises transferase A.

[0019] In some embodiments of the present invention, the nanopore protein sequence is wild type and includes amino acid mutations D90N, D91N, D93N, D118R, E139K and D134R.

[0020] In some embodiments of the present invention, the isopeptide bond comprises: a SpyCatcher / SpyTag pair, a SpyTag002 / SpyCatcher002 pair, a SpyTag003 / SpyCatcher003 pair, an isopeptag-N / pilin-N pair, an isopeptag / pilin-C pair or a SnoopTag / SnoopCatcher pair.

[0021] In some embodiments of the invention, the isopeptide bond comprises a SpyTag003 / SpyCatcher003 pair.

[0022] In some embodiments of the present invention, the preparation method comprises the following steps:

[0023] S1: Use the nanopore to express and purify the tagged recombinant protein;

[0024] S2: Express and purify the peptide-binding protein;

[0025] S3: co-incubating the purified nanopore obtained by expressing the tagged recombinant protein obtained in step S1 with the purified polypeptide binding protein obtained in step S2 or adding an amide bond to co-incubate, to obtain a semi-synthetic nanopore;

[0026] Preferably, the tags in step S1 and step S2 include His, strep, MBP, FLAG, HA, Myc, V5, OVA, Spycatcher or SpyTag;

[0027] or,

[0028] Preferably, the tag in step G1 includes Spycatcher.

[0029] Preferably, the tag in step G2 includes SpyTag.

[0030] In some embodiments of the present invention, the nanopore protein sequence is wild type and includes amino acid mutations D90N, D91N, D93N, D118R, E139K and D134R.

[0031] In some embodiments of the present invention, the unfolding enzyme sequence is a sequence in which 60 amino acids at the N-terminus are deleted, and includes C169S and K408E amino acid mutations.

[0032] The third aspect of the present invention provides a semi-synthetic nanopore biosensor, comprising the semi-synthetic nanopore as described in the first aspect of the present invention or the semi-synthetic nanopore prepared by the preparation method as described in the second aspect of the present invention.

[0033] In some embodiments of the present invention, the semisynthetic nanopore is placed in a membrane. The membrane usually separates two volumes of aqueous solution, and the membrane resists the flow of current between the volumes. The nanopore inserted into the membrane selectively allows ions to pass through the membrane, which can be recorded as an electrical signal detected by electrodes in the two volumes of aqueous solution.

[0034] In some embodiments of the present invention, suitable membranes are well known in the art, and the membrane is preferably an amphiphilic layer or a solid layer. The amphiphilic layer is a layer formed by amphiphilic molecules, and the amphiphilic molecules have both hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic (e.g., block copolymers) or naturally occurring (e.g., phospholipids).

[0035] In some embodiments of the invention, the membrane is an amphiphilic layer, such as a lipid bilayer (eg, a phospholipid bilayer) or a high molecular polymer membrane (eg, di-block, tri-block).

[0036] A fourth aspect of the present invention provides a protein sequencing method, the protein sequencing method comprising the following steps:

[0037] M1: contacting the protein analyte to be tested with the above nanopore biosensor or the nanopore biosensor prepared by the above preparation method, so that the protein analyte to be tested is unfolded into a chain under the action of the unfolding enzyme and passes through the nanopore channel, and unfolding and sequencing are performed simultaneously in the same system;

[0038] M2: When the protein analyte to be detected moves in the nanopore, the measurement values ​​of the entire time series of the protein analyte to be detected are obtained.

[0039] In other embodiments of the present invention, one end of the protein analyte to be detected contains a tag sequence that can be specifically recognized by an unfolding enzyme, such as an ssrA tag.

[0040] Beneficial effects of the present invention:

[0041] (1) The present invention provides a semi-synthetic nanopore, in which an unfolding enzyme and a nanopore protein are constructed into a complex by utilizing the properties of a transpeptidase or isopeptide bonds. After a single protein analyte to be tested is captured under the action of an electric field force, it is unfolded into a chain and perforated under the action of an unfolding enzyme, thereby achieving simultaneous regulation of the unfolding and translocation of a single natural protein analyte by an enzyme, i.e., "sequencing while unfolding", which can realize the reading of the full-length sequence of a protein and solve the problem of short read length (generally less than 30 amino acids) of protein nanopore sequencing in current technology.

[0042] (2) The present invention provides a method for preparing a semi-synthetic nanopore, which not only makes the construction of the semi-synthetic nanopore simpler, but also allows the constructed semi-synthetic nanopore to obtain a perforation signal of the full-length sequence of a protein (i.e., a characteristic current change spectrum with a time series).

[0043] (3) The present invention provides a protein sequencing method, which uses the nanopore biosensor provided by the present invention to perform protein sequencing. The operation is simple, and the unfolding and translocation of natural proteins are regulated by unfolding enzymes, thereby obtaining a current characteristic spectrum with a time series of the full-length sequence of the protein passing through the nanopore, thereby solving the problem of short read length in existing nanopore protein sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1Schematic diagram of the design of the nanopore protein and unfolding enzyme sequence of Example 1 of the present invention; wherein A-nanopore protein sequence design, B-unfolding enzyme sequence design, C-semi-synthetic nanopore sequence after connecting the nanopore and unfolding enzyme using transpeptidase.

[0045] Figure 2 Schematic diagram of the design of the nanopore protein and unfolding enzyme sequence of Example 2 of the present invention; wherein A-nanopore protein monomer sequence design, B-spycatcher-containing nanopore protein monomer sequence design, C-unfolding enzyme sequence design.

[0046] Figure 3 This is a schematic diagram of the principle of protein sequencing by the semi-synthetic nanopore biosensor provided by the present invention; wherein, 1-nanopore protein, 2-unfolding enzyme, 3-protein analyte to be tested, and 4-phospholipid bilayer.

[0047] Figure 4 The nanopore protein electrophoresis results in Example 2 of the present invention. Among them, A-MspA (D90N / D91N / D93N / D118R / E139K / D134R, corresponding to Figure 2 A- is the protein electrophoresis results of the nanopore channel (10% SDS-PAGE, lane M: protein molecular weight marker; lane S: supernatant after bacterial lysis; lane P: precipitate after bacterial lysis; lane 2: nickel column loading flow-through; lanes 4-5: low concentration imidazole washing solution; lanes 11-16: 100-500mM imidazole linear elution solution); B- is (Spycatcher) 8-MspA (corresponding to Figure 2 (B design) Protein electrophoresis results of the nanopore (10% SDS-PAGE, lane M: protein molecular weight marker; lane S: supernatant after bacterial lysis; lane P: precipitate after bacterial lysis; lane 2: nickel column load flow-through; lanes 4-5: low concentration imidazole washing solution; lanes 12, 13, 16, 17, 26-28: 100-500mM imidazole linear elution solution).

[0048] Figure 5The nanopore protein electrophoresis results of Spycatcher-MspA (MspA) 7 in Example 2 of the present invention. A- Electrophoresis of MspA protein containing Spycatcher (10% SDS-PAGE, lane M: protein molecular weight marker; lanes 25-36: MspA protein mixture containing different Spycatchers). B- Electrophoresis of MspA (i.e. Spycatcher-MspA (MspA) 7) protein after gel recovery (10% SDS-PAGE), wherein the band in the frame of lane 2 is Spycatcher-MspA (MspA) 7, lane 1 is MspA (D90N / D91N / D93N / D118R / E139K / D134R), and lane 3 is (Spycatcher) 8-MspA.

[0049] Figure 6 This is a current characterization diagram of a single MspA (D90N / D91N / D93N / D118R / E139K / D134R) nanopore protein inserted into a phospholipid membrane in Example 2 of the present invention. Voltage: 150 mV, buffer: 0.5 M KCL, 20 mM HEPES, pH 8.

[0050] Figure 7 The current characterization diagram of a single (Spycatcher) 8-MspA nanopore protein at different voltages (±50 mV, ±100 mV) after being inserted into a phospholipid membrane. Buffer: 0.15 M KCL, 10 mM HEPES, 10 mM MgCL2, pH 8.

[0051] Figure 8 The current characterization diagram of Spycatcher-MspA (MspA)7 nanopore protein inserted into phospholipid membrane at different voltages. Voltage: ±50mV, ±100mV, flushing solution: 0.15M KCL, 10mM HEPES, 5mM ATP, 10mM MgCL2, pH 8.

[0052] Fig. 9 The current characteristic diagram of the semisynthetic nanopore Spycatcher-MspA (MspA) 7 sensor based on the mCherry test protein analyte containing the ssrA tag. The buffer contains ATP, and the semisynthetic nanopore can exert the unfolding enzyme activity. A and B are the current characteristic diagrams of randomly selected different time periods. Voltage: 100mV. Buffer: 0.15M KCL, 10mM HEPES, 5mM ATP, 10mM MgCL2, pH 8.

[0053] Fig.10The current characteristic diagram of the semisynthetic nanopore Spycatcher-MspA (MspA) 7 sensor based on the mCherry test protein analyte containing the ssrA tag. The buffer contains ATP, and the semisynthetic nanopore can exert the unfolding enzyme activity. A and B are the current characteristic diagrams of different time periods randomly selected. Voltage: 100mV. Buffer: 0.15M KCL, 10mM HEPES, 10mM ATP, 20mM MgCL2, pH 8.

[0054] Fig.11 The current characteristic diagram of the semisynthetic nanopore Spycatcher-MspA (MspA) 7 sensor based on the mCherry test protein analyte containing the ssrA tag. The buffer does not contain ATP, and the semisynthetic nanopore cannot exert the unfolding enzyme activity. A and B are the current characteristic diagrams of randomly selected different time periods, and C is the enlarged current characteristic diagram of the frame area in Figure B. Voltage: 100mV. Buffer: 0.15M KCL, 10mM HEPES, 10mM MgCL2, pH 8.

[0055] Fig.12 The current characteristic diagram of the mCherry protein analyte containing the ssrA tag based on the (Spycatcher) 8-MspA nanopore sensor. Voltage: 100mV. Buffer: 0.15M KCL, 10mM HEPES, 5mM ATP, 20mM MgCL2, pH 8. DETAILED DESCRIPTION

[0056] In order to make the present invention more easily understood, the present invention will be described in detail below in conjunction with embodiments. These embodiments are only for illustration and are not intended to limit the application scope of the present invention.

[0057] Example 1

[0058] This embodiment provides a method for preparing a semi-synthetic nanopore biosensor, the structure of which is as follows Figure 3 As shown, specifically including:

[0059] S1: Expression and purification of His-MspA recombinant protein

[0060] The MspA sequence is wild type, and the MspA gene has 6 amino acid mutations (D90N / D91N / D93N / D118R / E139K / D134R), as shown in the amino acid sequence of SEQ ID NO.1. Figure 1The schematic diagram shown in A, each gene fragment is designed and synthesized, Histag has an amino acid sequence as shown in SEQ ID NO.2, the His-MspA gene fragment is cloned into the pET-21a(+) expression vector, and transformed into BL21(DE3) competent cells; a single colony is picked, inoculated into 5 mL of LB medium containing kanamycin, and cultured at 37°C with shaking overnight, and then transferred to 1 L of LB medium and cultured at 37°C with shaking until OD 600 =0.6-0.8, cooling to 16°C, adding isopropylthiogalactoside (IPTG) at a final concentration of 500 μM to induce expression overnight, collecting the cells and breaking them; firstly purifying by chromatography column, then eluting by imidazole concentration gradient of nickel column (imidazole concentration 25 mM-250 mM) and ion exchange column by salt ion concentration gradient elution, and finally separating by molecular sieve to obtain His-Ms pA;

[0061] S2: His-ClpX protein expression and purification

[0062] The sequence of ClpX is ClpXΔN, i.e., the N-terminal 60 amino acid sequence is deleted, and there are two amino acid mutations (C169S / K408E), as shown in the amino acid sequence of SEQ ID NO. 3. According to literature reports (Am or AJ, Schmitz KR, Sello JK, Baker TA, Sauer RT. Highly Dynamic Interactions Maintain Kinetic Stability of the ClpXP Protease During the ATP-Fueled Mechanical Cycle. ACS Chem Biol 2016, 11(6): 1552-1560), His-ClpX was expressed and purified, and the following was designed and synthesized: Figure 1 The gene fragment shown in B was cloned into the expression vector and transformed into BL21(DE3) competent cells; a single colony was picked and inoculated into 5 mL of LB medium, cultured at 37°C overnight with shaking, and then transferred into 1 L of LB medium and cultured at 37°C with shaking until OD 600 =0.7, add IPTG with a final concentration of 1 mM to induce expression for 4 h, collect the bacteria and then break them; firstly perform nickel column affinity chromatography purification, then use PD-10 (GE Healthcare) desalting column to replace the buffer (25 mM HEPES, pH 7.5, 50 mM KCl, 10% glycerol), and further purify and elute through ion column and molecular sieve Superdex-200 to obtain His-ClpX; SDS-PAGE verifies the protein molecular weight;

[0063] S3: Semisynthetic Nanopore Construction

[0064] The His-MspA prepared in step S1 and the His-ClpX prepared in step S2 were co-incubated with a transpeptidase at 4°C for 60 minutes, the transpeptidase recognition sequence was the amino acid sequence shown in SEQ ID NO.4 (recognizing LPETG at the C-terminus of one protein and GGGGG at the N-terminus of another protein, respectively), the co-incubation buffer was: 50mM Tris-HCl, pH 7.5, 150mM NaCl, 10mM CaCl2, and then further purified by molecular sieve to obtain His-MspA-ClpX semi-synthetic nanopores; the product was identified by polyacrylamide gel electrophoresis;

[0065] S4: Semisynthetic nanopore biosensor construction and single-channel nanopore experiments

[0066] 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids) phospholipids were dissolved in hexane and decane to final concentrations of 2 mg / mL and 20 mg / mL, respectively, to form the pretreatment solution and membrane-forming solution of the phospholipid membrane; the Teflon film divided the sample tank into two chambers, the cis chamber and the trans chamber, and a pair of Ag / AgCl electrodes were placed in different chambers, 1 μL of the pretreatment solution was dripped into the small hole (diameter 100 μm) of the Teflon film until it was completely evaporated, 0.5 mL of buffer was added to the two chambers, a small amount of 20 mg / mL of the membrane-forming solution was drawn into the small hole with a glass capillary, and the small hole was repeatedly blown with air bubbles until a phospholipid membrane was formed (the capacitance reached 50-80 pF); the other end of the Ag / AgCl electrode was connected to the probe working electrode and the patch clamp reference electrode of the instrument, and the patch clamp system included a microcurrent amplifier (Axopatch 200B, Molecular Devices) and a digital-to-analog converter (Axon Digidata 1550B1, Molecular Devices); an appropriate amount of semi-synthetic nanopores were added to the cis chamber, a voltage of 180 mV was applied and a period of time was waited until a single channel was inserted; after the single channel was formed, an appropriate amount of the protein analyte to be tested was added, and the voltage was applied according to different experimental purposes to start the experiment; pCLAMP 11.2 was used to record and save data (abf format), and Clampfit 11.2 was used for data viewing and current trajectory extraction.

[0067] Example 2

[0068] This embodiment provides a method for preparing a semi-synthetic nanopore biosensor, the structure of which is as follows Figure 3 As shown, specifically including:

[0069] S1: Spycatcher-MspA (MspA) 7 recombinant protein expression and purification

[0070] The MspA sequence is wild type, and the MspA gene has 6 amino acid mutations (D90N / D91N / D93N / D118R / E139K / D134R). Figure 2 A and 2B are shown in the schematic diagram, each gene fragment is designed and synthesized, Spycatcher003 has an amino acid sequence as shown in SEQ ID NO.5, Strep tag has an amino acid sequence as shown in SEQ ID NO.6, His-Spycatcher-MspA and His-MspA gene fragments are cloned into expression vectors with different resistances, respectively, and co-transformed into BL21 (DE3) competent cells; a single colony is picked, inoculated into 5 mL of LB medium containing two antibiotics, kanamycin and ampicillin, and cultured at 37°C overnight, and then transferred to 1 L of LB medium and cultured at 37°C until OD 600 =0.6-0.8, cooled to 16°C, isopropylthiogalactoside (IPTG) was added at a final concentration of 500 μM to induce expression overnight, and the bacteria were collected and broken; all heterologously assembled MspA proteins were eluted through an imidazole concentration gradient of a nickel column (imidazole concentration 5 mM-500 mM), and then gel electrophoresis was performed using 10% SDS-PAGE to identify and recover the nanopore protein Spycatcher-MspA (MspA)7 containing only one Spycatcher.

[0071] S2: Expression and purification of single-chain ClpX pseudo-hexamer Spytag-ClpX

[0072] The sequence of ClpX is ClpXΔN, i.e., the N-terminal 60 amino acid sequence is deleted, and there are two amino acid mutations (C169S / K408E). Spytag-ClpX was expressed and purified according to literature reports (Amor AJ, Schmitz KR, Sello JK, Baker TA, Sauer RT. Highly Dynamic Interactions Maintain Kinetic Stability of the Clp XP Protease During the ATP-Fueled Mechanical Cycle. ACS Chem Biol 2016, 11(6): 1552-1560), and the design and synthesis of Figure 2The gene fragment shown in C and the amino acid sequence of Spytag003 shown in SEQ ID NO.7 were cloned into the expression vector and transformed into BLR21 (DE3) competent cells; a single colony was picked and inoculated into 5 mL of LB medium, cultured at 37°C with shaking overnight, and then transferred into 1 L of LB medium and cultured at 37°C with shaking until OD 600 =0.7, add IPTG with a final concentration of 1 mM to induce expression for 4 h, collect the bacteria and then break them; perform nickel column affinity chromatography purification, and verify the protein molecular weight by SDS-PAGE;

[0073] S3: Semisynthetic Nanopore Construction

[0074] The Spytag-ClpX prepared in step S2 and the Spycatcher-MspA (MspA) 7 prepared in step S1 are co-incubated at room temperature to form a complex, i.e., a semi-synthetic nanopore; the product is further separated and identified using a molecular sieve;

[0075] S4: Semisynthetic nanopore biosensor construction and single-channel nanopore experiments

[0076] 1,2-Diphytanoyl-sn-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids) phospholipids were dissolved in hexane and decane to final concentrations of 2 mg / mL and 20 mg / mL, respectively, to form the pretreatment solution and membrane-forming solution of the phospholipid membrane; the Teflon film divided the sample tank into two chambers, the cis chamber and the trans chamber, and a pair of Ag / AgCl electrodes were placed in different chambers, 1 μL of the pretreatment solution was dripped into the small hole (diameter 100 μm) of the Teflon film until it was completely evaporated, 0.5 mL of buffer was added to the two chambers, a small amount of 20 mg / mL of the membrane-forming solution was drawn into the small hole with a glass capillary, and the small hole was repeatedly blown with air bubbles until a phospholipid membrane was formed (the capacitance reached 50-80 pF); the other end of the Ag / AgCl electrode was connected to the probe working electrode and the patch clamp reference electrode of the instrument, and the patch clamp system included a microcurrent amplifier (Axopatch200B, Molecular Devices) and a digital-to-analog converter (Axon Digidata 1550B1, Molecular Devices); add an appropriate amount of pore protein or enzyme-pore complex to the cis chamber, apply a voltage of 180 mV and wait for a while until a single channel is inserted; after the single channel is formed, add an appropriate amount of the protein analyte to be tested, and apply voltage to start the experiment according to different experimental purposes; use pCLAMP 11.2 to record and save data (abf format), and Clampfit 11.2 is used for data viewing and current trajectory extraction.

[0077] Experimental Results

[0078] Nanopore expression and purification

[0079] To further confirm the successful expression and purification of Spycatcher-MspA (MspA) 7, as well as the need to serve as a control group in the nanopore experiment, the inventors constructed, expressed and purified MspA (D90N / D91N / D93N / D118R / E139K / D134R and (Spycatcher) 8-MspA. MspA is an octameric protein, of which MspA (D90N / D91N / D93N / D118R / E139K / D134R is a nanopore protein without Spycatcher, and (Spyc atcher) 8-MspA is a nanopore protein in which each subunit contains Spycatcher. Figure 4 A is MspA (D90N / D91N / D93N / D118R / E139K / D134R, corresponding to Figure 2 A) Protein electrophoresis results of nanopores; Figure 4 B is (Spycatcher) 8-MspA (corresponding to Figure 2 (B) Protein electrophoresis results of the nanopore. According to the protein gel results, both proteins were successfully expressed and purified.

[0080] Figure 5 A is the electrophoresis result of the MspA nanopore protein containing Spycatcher in Example 2 of the present invention after affinity chromatography. The protein obtained at this time is a mixture of nanopore proteins containing different Spycatchers. In order to obtain a nanopore protein with higher purity, Spycatcher-MspA (MspA) 7, the inventors used gel separation and recovery to obtain a nanopore protein with higher purity and containing only one Spycatcher, namely Spycatcher-MspA (MspA) 7 ( Figure 5 B).

[0081] Nanopore sensor construction

[0082] The above-mentioned nanopore proteins were respectively inserted into phospholipid membranes to obtain the pore opening currents under different conductance buffers. Figure 6 This is a current characterization diagram of the insertion of a single MspA (D90N / D91N / D93N / D118R / E139K / D134R) nanopore protein into a phospholipid membrane in Example 2 of the present invention. Figure 7 This is a current characterization diagram of different voltages after a single (Spycatcher) 8-MspA nanopore protein is inserted into a phospholipid membrane. Figure 8The current characterization diagram of Spycatcher-MspA (MspA) 7 nanopore protein inserted into phospholipid membrane. The above results show that three different nanopore protein sensors were successfully constructed.

[0083] Experimental Example 3

[0084] Using the semi-synthetic nanopore biosensor constructed in Example 1 or Example 2, a protein containing an ssrA tag was added to the cis or trans chamber, and a voltage was applied to observe the current change; pCLAMP 11.2 was used to record and save data (abf format), and Clampfit 11.2 was used for data viewing and current trajectory extraction.

[0085] Experimental Results

[0086] This example uses a low-salt buffer (containing 0.15M KCL) to ensure enzyme activity. After a single Spycatcher-MspA (MspA) 7 is inserted into the phospholipid membrane, the opening current of a single nanopore is obtained; an appropriate amount of mCherry protein analyte to be tested is added to the cis-chamber, gently mixed, and the change in the electrical signal is observed under a voltage of 100mV. Depending on the purpose of the experiment, the buffer may contain fuel (ATP and MgCL2) for enzyme activity. Fig. 9 This is the current characteristic diagram of the semisynthetic nanopore Spycatcher-MspA (MspA) 7 sensor based on the mCherry protein containing the ssrA tag; the buffer contains ATP, and the semisynthetic nanopore can exert the unfolding enzyme activity. Buffer: 0.15M KCL, 10mM HEPES, 5mM ATP, 10mM MgCL2, pH 8. A and B are the current characteristic diagrams of randomly selected different time periods.

[0087] The inventors also tested the current characterization of mCherry protein under different concentrations of ATP and MgCL2. Fig.10 Current characteristics of the semisynthetic nanopore Spycatcher-MspA (MspA) 7 sensor containing ssrA-tagged mCherry protein. Buffer: 0.15M KCL, 10mM HEPES, 10mM ATP, 20mM MgCL2, pH 8. A and B are current characteristics of randomly selected different time periods.

[0088] In order to further illustrate that the mCherry protein analyte is captured and unfolded under the action of the semisynthetic nanopore Spycatcher-MspA (Msp A) 7, the inventors further tested the current characteristic graph of the mCherry protein in the absence of fuel ATP. In this experiment, except for slightly different buffer components, other experimental conditions remained the same. Fig.11 This is the current characteristic diagram of the semisynthetic nanopore Spycatcher-MspA (MspA) 7 sensor based on the mCherry protein containing the ssrA tag. The conductivity buffer does not contain ATP, and the semisynthetic nanopore cannot exert the unfolding enzyme activity. Buffer: 0.15M KCL, 10mM HEPES, 10mM MgCL2, pH 8. A and B are the current characteristic diagrams of randomly selected different time periods, and C is the enlarged current characteristic diagram of the frame area in Figure B. It can be clearly judged from the figure that the current characteristic diagram of the mCherry protein is significantly different in the buffer containing ATP and not containing ATP.

[0089] The inventors also tested the changes in the current characteristics of the mCherry protein based on the (Spycatcher) 8-MspA nanopore sensor. The nanopore does not contain the unfolding enzyme ClpX. The inventors speculated that mCheery could not be effectively unfolded, but might be stretched and unfolded locally under the action of voltage. After a single (Spycatcher) 8-MspA is inserted into the phospholipid bilayer, a nanopore sensor is formed. The mCherry protein was added to the cis-chamber and different voltages were applied to observe. Fig.12 The current characteristic diagram of the (Spycatcher) 8-MspA nanopore sensor based on the mCherry protein containing the ssrA tag under the voltage condition of 100mV is shown. Its signal characteristics are significantly different from those of the semi-synthetic nanopore channel.

[0090] In summary, the inventors have successfully verified the construction of the semisynthetic nanopore Spycatcher-MspA (MspA) 7 sensor, as well as the capture and unfolding of the protein analyte based on the semisynthetic nanopore. This method has laid a certain foundation for the realization of natural protein sequencing.

[0091] The amino acid sequences involved in the present invention are shown in Table 1:

[0092] Table 1 Amino Acid Sequence Listing

[0093]

[0094] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation to the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A semi-synthetic nanopore, characterized in that: A construct comprising a polypeptide binding protein and a nanopore, wherein the polypeptide binding protein is a polypeptide binding protein capable of unfolding a protein into a linear form; and the polypeptide binding protein is bound to the nanopore via an amide bond; Preferably, the amide bond binding comprises linkage via transpeptidase properties or isopeptide bonds; Preferably, the polypeptide binding protein is an unfolding enzyme; Preferably, the nanopore is a nanopore protein.

2. The semi-synthetic nanopore according to claim 1, characterized in that The unfoldase comprises ClpX, NTP-driven unfoldase, ATP-dependent protease, Mycobacterium tuberculosis ClpC1 unfoldase, Msp1, ClpA unfoldase, HsIU unfoldase, valosin-containing protein, 26S proteasome or Lon protease; Preferably, the unfoldase comprises ClpX.

3. The semi-synthetic nanopore according to claim 1, characterized in that The nanopore protein includes MspA porin, Alpha-hemolysin, Frac, ClyA, PA63, CsgG, GspD, XcpQ, Wza, Phi29 connector, T7 connector, SPP1 connector, T3 connector, T4 connector or aerolysin; Preferably, the nanopore protein comprises an MspA porin.

4. A method for preparing a semi-synthetic nanopore according to any one of claims 1 to 3, characterized in that: include: The polypeptide binding protein and the nanopore are linked via an amide bond to form a semi-synthetic nanopore. Preferably, the amide bond binding includes linking via a transpeptidase property or an isopeptide bond.

5. The preparation method according to claim 4, characterized in that: The transpeptidase includes: transpeptidase A, SaSrtA, SrtB, SrtC, SrtD, BaSrtA, SavSrtE, CdSrtA, peptidyl asparagine ligase, Butelase-1 or OaAEP1; Preferably, the transferase comprises transferase A.

6. The preparation method according to claim 4, characterized in that: The isopeptide bonds include: SpyCatcher / SpyTag pair, SpyTag002 / SpyCatcher002 pair, SpyTag003 / SpyCatcher003 pair, isopeptag-N / pilin-N pair, isopeptag / pilin-C pair or SnoopTag / SnoopCatcher pair; Preferably, the isopeptide bond comprises a SpyTag003 / SpyCatcher003 pair.

7. The preparation method according to claim 4, characterized in that: The preparation method comprises the following steps: S1: Use the nanopore to express and purify the tagged recombinant protein; S2: Express and purify the peptide-binding protein; S3: co-incubating the purified nanopore obtained by expressing the tagged recombinant protein obtained in step S1 with the purified polypeptide binding protein obtained in step S2 or co-incubating with the addition of a transpeptidase to obtain a semi-synthetic nanopore; Preferably, the tags in step S1 and step S2 include His, strep, MBP, FLAG, HA, Myc, V5, OVA, Spycatcher or SpyTag; or, Preferably, the tag in step S1 includes Spycatcher. Preferably, the tag in step S2 includes SpyTag.

8. A semisynthetic nanopore biosensor, characterized in that: The invention comprises the semi-synthetic nanopore according to any one of claims 1 to 3 or the semi-synthetic nanopore prepared by the preparation method according to any one of claims 4 to 7.

9. A method for preparing a semi-synthetic nanopore biosensor as claimed in claim 8, characterized in that: include: Semisynthetic nanopores are placed in the membrane.

10. A protein sequencing method, characterized in that: The protein sequencing method comprises the following steps: M1: contacting the protein analyte to be tested with the nanopore biosensor according to claim 8 or the nanopore biosensor prepared by the preparation method according to claim 9, so that the protein analyte to be tested is unfolded into a chain under the action of the unfolding enzyme and passes through the nanopore channel, and unfolding and sequencing are performed simultaneously in the same system; M2: When the protein analyte to be detected moves in the nanopore, the measurement values ​​of the entire time series of the protein analyte to be detected are obtained.

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