Tn5 transposase mutant as well as preparation method and application thereof

Abstract

The invention discloses a Tn5 transposase mutant as well as a preparation method and application thereof. The invention discloses a Tn5 transposase mutant with an amino acid sequence as shown in SEQ ID NO.1 at first. The invention further discloses a preparation method and application of the Tn5 transposase mutant. Compared with the known Tn5 transposase EKLP, the Tn5 transposase mutant has the advantages that two point mutations A185S and E344Q are introduced, the Tn5 transposase mutant can be over-expressed in a prokaryotic host and purified through affinity chromatography and ion-exchange chromatography to obtain a large amount of protein, and a reliable technical scheme is provided for large-scale production of the Tn5 transposase mutant. The Tn5 transposase mutant Tn5Mut disclosed by the invention has the advantage of high activity, and can be applied to the fields of DNA library establishment and sequencing.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and DNA library construction. More specifically, it relates to a Tn5 transposase mutant, its preparation method, and its applications. Background Technology

[0002] Tn5 transposase is a enzyme derived from Escherichia coli (E. coli). Escherichia coli Tn5 transposases are DDE-type DNA transposases that mediate random DNA insertion into the genome via a "cut-and-paste" mechanism. Wild-type Tn5 transposases suffer from inherent defects such as low activity, strong sequence bias, and dependence on host factor activation. Tn5 transposases primarily recognize a specific 19bp Mosaic End (ME) sequence (5'-AGATGTGTATAAGAGACAG-3'), limiting their efficiency in in vitro applications and making them unsuitable for high-throughput sequencing. To overcome these technical bottlenecks, directed evolutionary modification of Tn5 transposases was employed, resulting in the development of a highly active mutant, EKLP (E54K / L372P). These mutations significantly improve transposition efficiency and reduce sequence bias by enhancing DNA binding capacity (E54K) and stabilizing the protein catalytic core conformation (L372P).

[0003] The core technological advantage of Tn5 transposase lies in its unique integrated "fragmentation-linker addition" function, which revolutionizes the traditional DNA library construction process. Specifically, Tn5 transposase in Mg 2+ Under suitable conditions, Tn5 transposase can simultaneously perform random DNA cleavage and sequencing adapter insertion, integrating the multi-step reactions of traditional library preparation, such as fragmentation, end repair, A-terminal addition, and adapter ligation, into a single step. Under optimized conditions, pre-assembled Tn5 transposase-adaptor complexes only require 5-15 minutes of incubation with genomic DNA to obtain uniform fragmented products (200-500 bp), significantly improving library preparation efficiency. Based on these characteristics, Tn5 transposase has become a core tool enzyme for high-throughput sequencing (NGS), single-cell sequencing (scATAC-seq), and functional genomics research. However, current technologies still suffer from uneven fragment size distribution, residual GC bias, and insufficient enzyme stability, necessitating the introduction of novel mutant combinations to further enhance the activity of Tn5 transposase. Summary of the Invention

[0004] One object of the present invention is to provide a highly active Tn5 transposase mutant.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned Tn5 transposase mutant and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: The present invention first provides a Tn5 transposase mutant, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] This invention introduces point mutations A185S and E344Q into the known Tn5 transposase EKLP. These point mutations are represented by a triplet: letter-number-letter, where the number indicates the position of the mutated amino acid, the letter before the number corresponds to the amino acid involved in the mutation, and the letter after the number indicates the amino acid used to replace the amino acid preceding the number. The known Tn5 transposase EKLP is a transposase derived from *E. coli*, and its amino acid sequence is shown in SEQ ID NO. 3.

[0008] The polynucleotides encoding the above-mentioned Tn5 transposase mutant are also within the scope of protection of this invention.

[0009] In a specific embodiment of the present invention, the polynucleotide encoding the above-mentioned Tn5 transposase mutant is shown in SEQ ID NO.2.

[0010] Recombinant expression plasmids or recombinant cells containing the above-mentioned polynucleotides are also within the scope of protection of this invention.

[0011] In a specific embodiment of the present invention, the recombinant expression plasmid is pET28a-Tn5Mut, which is obtained by inserting a polynucleotide encoding a Tn5 transposase mutant, as shown in SEQ ID NO.2, between NdeI and BamHI of pET28a, while keeping the other sequences of pET28a unchanged.

[0012] In a specific embodiment of the present invention, the host cell of the recombinant cells is a modified BL21 Escherichia coli. In a preferred embodiment of the present invention, the host cell is a BL21(DE3) competent cell.

[0013] This invention further discloses a method for preparing the above-mentioned Tn5 transposase mutant, the method comprising the following steps: a1) Construct a recombinant expression plasmid containing the polynucleotide shown in SEQ ID NO.2; a2) Transform the recombinant expression plasmid into host cells, induce expression, and obtain bacterial cells; a3) Disrupt the bacterial cells, centrifuge to obtain the supernatant, precipitate the protein, and obtain the crude protein solution; a4) The Tn5 transposase mutant was obtained after purifying the crude protein solution.

[0014] In a specific embodiment of the present invention, the recombinant expression plasmid is pET28a-Tn5Mut; the host cell is BL21(DE3) competent cells.

[0015] In a specific embodiment of the present invention, the protein precipitation is ammonium sulfate precipitation; the purification includes Ni column purification, Q column purification and SP column purification.

[0016] The present invention further discloses the application of the above-mentioned Tn5 transposase mutant, and / or, the above-mentioned polynucleotide, and / or, the above-mentioned recombinant expression plasmid, and / or, the above-mentioned recombinant cells in DNA library construction and / or in a kit for preparing DNA libraries.

[0017] In a specific embodiment of the present invention, the application can be any of the following: b1) Applications in epigenetic research; b2) Applications in single-cell sequencing; b3) Applications in long-fragment sequencing; b4) Applications in synthetic biology.

[0018] The present invention further discloses a DNA library construction kit, the kit comprising the above-mentioned Tn5 transposase mutant.

[0019] The beneficial effects of this invention are as follows: Compared to the known Tn5 transposase EKLP, the Tn5 transposase mutant of this invention introduces two point mutations, A185S and E344Q. This Tn5 transposase mutant can be overexpressed in prokaryotic hosts and purified in large quantities by affinity chromatography and ion exchange chromatography, providing a reliable technical solution for its large-scale production. The Tn5 transposase mutant Tn5Mut of this invention has the advantage of high activity and can be applied in the fields of DNA library construction and sequencing. Attached Figure Description

[0020] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0021] Figure 1 This is an SDS-PAGE gel image of the Tn5 transposase mutant of the present invention.

[0022] Figure 2 This figure shows the fragment distribution results of the Tn5 transposase mutant of this invention and the known Tn5 transposase mutant EKLP in a 50 ng human genome library. Detailed Implementation

[0023] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments and accompanying drawings, further explains the invention. Similar components in the drawings are indicated by the same reference numerals. Those skilled in the art should understand that the specific description below is illustrative rather than restrictive and should not be construed as limiting the scope of protection of the present invention.

[0024] Example 1: Preparation of Tn5 transposase mutant I. Design of the Tn5 transposase mutant Tn5Mut Based on the known highly active Tn5 transposase mutant EKLP (Tn5 EKLP, whose amino acid sequence is shown in SEQ ID NO. 3 and polynucleotide sequence in SEQ ID NO. 4), two key site mutations, A185S and E344Q, were rationally designed to further optimize its catalytic performance. The A185S mutation introduces a serine hydroxyl group to form a new hydrogen bond network, stabilizing the active conformation required for the transposition reaction and thus improving catalytic efficiency. The E344Q mutation, a consensus sequence mutation, replaces the negatively charged glutamate with neutral glutamine, eliminating the potentially unfavorable electrostatic repulsion at this site, optimizing the local electrostatic environment for enzyme-DNA substrate binding, and enhancing hydrogen bond formation, further improving the substrate binding and catalytic microenvironment. These two mutations work synergistically to further enhance the activity of the Tn5 transposase, providing a superior tool for library construction using low-starting-volume, high-difficulty samples.

[0025] The final amino acid sequence of the Tn5 transposase mutant is shown in SEQ ID NO.1, and the polynucleotide encoding the Tn5 transposase mutant is shown in SEQ ID NO.2.

[0026] II. Construction of Tn5 transposase mutant recombinant expression plasmid The Tn5 transposase mutant recombinant expression plasmid pET28a-Tn5Mut was synthesized from the whole genome. pET28a-Tn5Mut was obtained by inserting a polynucleotide encoding the Tn5 transposase mutant as shown in SEQ ID NO.2 between NdeI and BamHI of pET28a, while keeping the other sequences of pET28a unchanged.

[0027] III. Induction, Expression, and Purification of Tn5 Transposase Mutants The Tn5 transposase mutant recombinant expression plasmid was transformed into host competent cells, and the expression of the target protein was induced. The specific steps are as follows: 1) Take BL21(DE3) competent cells and thaw them on ice.

[0028] 2) Take 1 µL of recombinant expression plasmid pET28a-Tn5Mut and add it to the thawed BL21(DE3) competent cells, and incubate on ice for 30 min.

[0029] 3) After the ice bath, competent cells were placed in a 42°C water bath for 45 seconds for heat shock, and then placed on ice for 2 minutes.

[0030] 4) Add 400 µL of LB liquid medium to the cells obtained in step 3) and incubate at 220 rpm for 45 min in a shaker at 37°C.

[0031] 5) Take 200 µL of cells incubated in step 4) and spread them on a kanamycin plate. Incubate at 37°C overnight.

[0032] 6) Pick one single clone from an overnight culture plate and add it to 20 mL of LB medium containing 50 µg / mL kanamycin. Incubate at 37°C and 220 rpm for 7 hours.

[0033] 7) Add 20 mL of the culture obtained in step 6) to 1 L LB medium containing 50 µg / mL kanamycin, and incubate at 37°C and 220 rpm for 2 hours. OD 600 When the concentration of the sample is approximately 0.6, add 200 µL of 0.5 M IPTG, adjust the temperature and speed of the shaker to 16℃ and 180 rpm, and induce expression overnight for approximately 15 hours.

[0034] 8) Centrifuge the bacterial culture after the induction expression in step 7) to collect the bacterial cells at 8000 rpm for 15 min at 4℃, and weigh the bacterial cells.

[0035] Following cell disruption and PEI and ammonium sulfate precipitation, the Tn5 transposase mutant was purified using Ni, Q, and SP column purification methods to obtain high-purity Tn5 transposase mutants. The specific steps are as follows: 1. Cell disruption and precipitation treatment 1) Resuspend the bacterial cells in 50 mL PBS buffer and centrifuge at 12000 rpm for 10 min at 4℃. Discard the supernatant to obtain the bacterial cells.

[0036] 2) Resuspend the bacterial cells in 50 ml of lysis buffer and add PMSF to a final concentration of 1 mM.

[0037] 3) Use a high-pressure homogenizer to break up the bacterial suspension added in step 2) of PMSF.

[0038] 4) Centrifuge the lysate from step 3) at 12,000 rpm for 30 min at 4°C, discard the precipitate and obtain the supernatant.

[0039] 5) Add 10% PEI (final concentration 0.3%) to the supernatant for nucleic acid precipitation for 30 min at 12000 rpm for 30 min at 4℃. Discard the precipitate to obtain the supernatant.

[0040] 6) Slowly add ammonium sulfate powder to the supernatant until the saturation concentration is 60%. After the ammonium sulfate is completely dissolved, let it stand in a refrigerator at 4°C for 1 hour to allow precipitation.

[0041] 7) Centrifuge the precipitated sample at 12,000 rpm for 30 min at 4°C, and discard the supernatant to obtain the protein precipitate.

[0042] 8) The protein precipitate was resuspended in 20 mL of Ni-binding buffer and filtered through a 0.45 µm filter to obtain the filtered protein sample (crude protein solution).

[0043] 2. NI column purification 1) Connect a 5 ml Ni pre-packed column to the AKTA system. Place the filtered and evacuated Ni-binding buffer and Ni-elution buffer into pumps A and B, respectively, and then pump the AKTA system.

[0044] 2) Equilibrate the Ni column with Ni-binding buffer at a flow rate of 2 mL / min.

[0045] 3) Load the filtered protein sample onto a Ni column at a flow rate of 2 mL / min.

[0046] 4) Wash with Ni-washing buffer at a flow rate of 2 mL / min for 50 min.

[0047] 5) Elute the target protein with Ni-elution buffer gradient (2 mL / min flow rate, 30 min elution time, 0-100% B pump gradient).

[0048] 6) Collect the target protein peak to obtain the protein sample eluted by the Ni column.

[0049] 3. Q-column purification 1) Connect 5 ml of the pre-packed Q column to the AKTA system. Place the filtered and degassed Q-binding buffer and Q-elution buffer into pumps A and B respectively, and then pump the AKTA system to wash.

[0050] 2) Equilibrate the Q column with Q-binding buffer at a flow rate of 2 mL / min.

[0051] 3) Load the protein sample eluted from the Ni column onto the Q column at a flow rate of 2 mL / min, collect the flow-through protein peak, and obtain the protein sample.

[0052] 4. SP column purification 1) Connect 5 ml of the pre-packed SP column to the AKTA system. Place the filtered and evacuated SP-binding buffer and SP-elution buffer into pumps A and B respectively, and then pump the AKTA system to wash.

[0053] 2) Equilibrate the SP column with SP-binding buffer at a flow rate of 2 mL / min.

[0054] 3) Load the protein sample that has flowed through the Q column onto the SP column at a flow rate of 2 mL / min, and continue equilibration for 50 min after loading.

[0055] 4) Elute the target protein with SP-elution buffer gradient (2 ml / min flow rate, 30 min elution time, 0-100% B pump gradient).

[0056] 5) Collect the target protein peak to obtain a protein sample.

[0057] Protein samples were dialyzed into storage buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton-X 100, 50% glycerol). SDS-PAGE gel analysis results are shown below. Figure 1 As shown in the figure, the protein sample obtained in this embodiment has a size of 55.4 kDa, which is consistent with the expected size of the Tn5 transposase mutant, indicating that the protein sample finally obtained in this embodiment is the Tn5 transposase mutant (abbreviated as Tn5Mut). Furthermore, sequencing confirmed its correctness.

[0058] Example 2: Construction of a short fragment library of Tn5 transposase mutants using the Illumina high-throughput sequencing platform 1. Joint Annealing Annealing reaction systems for joint A and joint R, and joint B and joint R were prepared according to Table 1, and the joint sequence is shown in Table 2. After preparing the systems, the joints were annealed according to the procedure in Table 3 to obtain joint A + joint R and joint B + joint R.

[0059] Table 1 Connector System

[0060] Table 2 Connector Sequence

[0061] Table 3 Joint Annealing Procedure

[0062] II. Preparation of Tn5 transposase complex containing linker Prepare the Tn5 transposase-adaptor complex system according to Table 4. Incubate the prepared system at 35°C for 2 hours (42°C with a hot cap). After incubation, dilute with Tn5 SB by one-fold to obtain the Tn5 transposase-adaptor complex.

[0063] Table 4. System for assembling adapters and Tn5 transposase

[0064] III. Connector Insertion and Fragmentation Prepare the library construction system on ice according to Table 5, mix gently, centrifuge briefly, and then place in a PCR instrument. Incubate at 55°C for 5 minutes. After the reaction is complete, immediately add 30 µL of 2×Stop Buffer and mix well. Incubate in the PCR instrument at 55°C for 5 minutes, then immediately place on ice to obtain the reaction solution.

[0065] Table 5 Database Construction System

[0066] IV. DNA Purification Add one volume of magnetic beads to the above reaction solution (60 µL). Mix thoroughly and incubate at room temperature for 5 minutes to allow DNA to bind to the magnetic beads. Place the reaction tube on a magnetic rack and allow it to stand until the solution becomes clear. Carefully remove the supernatant. Keeping the reaction tube on the magnetic rack, wash the magnetic beads twice with 200 µL of freshly prepared 80% ethanol, allowing it to stand for 30 seconds each time before removing the ethanol. Open the cap and allow the magnetic beads to air dry at room temperature for 3–5 minutes. Remove the tube from the magnetic rack, add 22 µL of 10 mM Tris-HCl (pH 8.0) elution buffer, and resuspend the magnetic beads thoroughly. Allow it to stand at room temperature for 2 minutes. Return the tube to the magnetic rack and, after the solution becomes clear, transfer 20 µL of the supernatant containing the purified DNA to a new PCR tube. This product is the adapter-bound fragmented DNA.

[0067] V. Library Expansion Prepare the PCR amplification system on ice according to Table 6, and perform the PCR amplification reaction according to the PCR amplification reaction procedure in Table 7 to obtain the PCR product.

[0068] Table 6 PCR Amplification System

[0069] Table 7 PCR Amplification Reaction Procedure

[0070] VI. Fragment sorting of library amplification products Add 0.6 times the volume of magnetic beads to the PCR product, mix gently, and incubate at room temperature for 5 minutes. Place the reaction tube on a magnetic rack, let it stand, and then carefully transfer the supernatant to a new EP tube (this step discards excessively long fragments and residual primer dimers). Add 0.15 times the volume of the original PCR product to the new tube containing the supernatant, mix gently, and incubate at room temperature for 5 minutes (this step discards excessively short fragments). Place the reaction tube on a magnetic rack, let it stand, and then carefully remove the supernatant. Keeping the reaction tube on the magnetic rack, wash the magnetic beads twice with 200 µL of 80% ethanol. After opening and drying the magnetic beads, resuspend and elute the DNA with 23 µL of 10 mM Tris-HCl (pH 8.0) elution buffer. After separation on a magnetic rack, transfer the supernatant to a new tube, which is the final purified sequencing library.

[0071] VII. Analysis of Sequencing Results Quality control: Use Qubit for precise quantification of the library. Analyze the fragment size distribution of the library using a bioanalyzer to ensure the main peak is within the target range (e.g., 300-500 bp).

[0072] Sequencing: Based on the library concentration, mix the libraries in appropriate proportions (if multiplexing is required) and perform normalized dilution. Then, perform cluster generation and paired-end sequencing on an Illumina sequencing platform (such as NovaSeq, NextSeq, MiSeq).

[0073] Fragment size distribution of the analysis library by the bioanalyzer, as shown in Figure 2 As shown, compared with the known Tn5 transposase mutant EKLP (represented as Tn5 EKLP in the figure), the specific activity of the Tn5 transposase mutant of the present invention (represented as Tn5Mut in the figure) is increased by approximately 4-fold. Specifically, under the same starting amount of 50 ng genomic DNA, the target fragment size of 300-500 bp and the total library yield obtained using 2.5 μM of the Tn5 transposase mutant of the present invention are basically consistent with the experimental results using 10 μM of the Tn5 transposase mutant EKLP. This indicates that, under the premise of achieving the same fragmentation effect and library construction quality, the amount of the Tn5 transposase mutant of the present invention is only 1 / 4 of that of the existing Tn5 transposase mutant EKLP, which can efficiently complete the cleavage and transposition functions of genomic DNA. Therefore, with the same number of protein moles, the Tn5 transposase mutant of the present invention has higher catalytic efficiency, or stronger genomic DNA cleavage and adapter ligation activity. This specific activity enhancement characteristic can not only significantly reduce the amount of enzyme used per unit reaction, which is beneficial for saving costs and reducing enzyme-related side reactions, but also provide more flexible optimization space for library construction of trace or low starting sample amounts.

[0074] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is impossible to exhaustively list all the implementation methods here. All obvious variations or modifications derived from the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A Tn5 transposase mutant, characterized in that, The amino acid sequence of the Tn5 transposase mutant is shown in SEQ ID NO.

1.

2. A polynucleotide encoding the Tn5 transposase mutant of claim 1.

3. The polynucleotide according to claim 2, characterized in that, The polynucleotide is shown in SEQ ID NO.

2.

4. A recombinant expression plasmid comprising the polynucleotide of claim 2 or 3.

5. Recombinant cells comprising the polynucleotide of claim 2 or 3 or the recombinant expression plasmid of claim 4.

6. The method for preparing the Tn5 transposase mutant according to claim 1, characterized in that, The preparation method includes the following steps: a1) Construct a recombinant expression plasmid containing the polynucleotide shown in SEQ ID NO.2; a2) Transform the recombinant expression plasmid into host cells, induce expression, and obtain bacterial cells; a3) Disrupt the bacterial cells, centrifuge to obtain the supernatant, precipitate the protein, and obtain the crude protein solution; a4) The Tn5 transposase mutant was obtained after purifying the crude protein solution.

7. The preparation method according to claim 6, characterized in that, The protein precipitate was an ammonium sulfate precipitate. Preferably, the purification includes Ni column purification, Q column purification and SP column purification.

8. The Tn5 transposase mutant of claim 1, and / or the polynucleotide of claim 2 or 3, and / or the recombinant expression plasmid of claim 4, and / or the recombinant cell of claim 5, in the application of DNA library construction.

9. The use of the Tn5 transposase mutant of claim 1, and / or the polynucleotide of claim 2 or 3, and / or the recombinant expression plasmid of claim 4, and / or the recombinant cells of claim 5 in a kit for preparing DNA libraries.

10. A DNA library construction kit, characterized in that, The kit includes the Tn5 transposase mutant as described in claim 1.

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