CN108913712B

CN108913712B - Expression and purification method of recombinant Tn5 transposase

Info

Publication number: CN108913712B
Application number: CN201810782084.7A
Authority: CN
Inventors: 崔伟斌; 叶国栋; 陈茂立; 许剑雄; 韩大雄; 郭奇伟; 肖剑萍; 蔡逸民; 杨燕燕; 李顺杰; 董康梅; 朱莎莎; 张丽芳; 宋丹
Original assignee: Xiamen Lifeint Technology Co ltd
Current assignee: Xiamen Lifeint Technology Co ltd
Priority date: 2018-07-17
Filing date: 2018-07-17
Publication date: 2021-04-02
Anticipated expiration: 2038-07-17
Also published as: CN108913712A

Abstract

The invention discloses an expression and purification method of recombinant Tn5 transposase. The method comprises the following steps: preparing a recombinant vector containing Tn5 transposase; the recombinant vector also has Strep tag II and SUMO tag; inducing expression; purifying the fusion protein by using Strep tag II and SUMO tag to obtain high-purity Tn5 enzyme protein, and enabling the Tn5 transposase protein to be correctly assembled and folded by depending on the characteristics of the SUMO tag without the occurrence of protein band degradation.

Description

Expression and purification method of recombinant Tn5 transposase

Technical Field

The invention relates to the field of genetic engineering, in particular to an expression and purification method of recombinant Tn5 transposase.

Background

Transposons are DNA fragments capable of moving and autonomous replication in genomes, and with the continuous deepening of the molecular level of recognition of the outcome and function of transposons, many transposons have been modified as tools for genetic analysis for gene function analysis, gene transformation, and gene therapy. Transposase is an important factor for realizing transposon function, and since the activity of natural transposase is low and the expression level is small, the transposability of transposon is not high.

Tn5 transposase is an important tool in transposase series for high-throughput library construction in next generation sequencing. The wild type Tn5 transposase has a low expression level and a low activity, and thus, it is an obstacle to the development and utilization of the second-generation sequencing. In recent years, scientists have used a method of combining bioinformatics and protein engineering to construct transposases with high activity, and have constructed chimeras of transposases by a protein fusion technology, so that the activity of the transposases is remarkably improved.

In the existing method for expressing and purifying Tn5 transposase, the phenomenon that the purified Tn5 enzyme has degradation and hybrid protein often occurs, and meanwhile, the obtained Tn5 transposase has the adverse results of low enzyme quantity, wrong protein conformation, low enzyme activity and the like. Therefore, the transposase is obviously different from the theory in actual production and application after amino acid optimization.

Disclosure of Invention

The invention provides a purification method of a recombinant Tn5 transposase. The purified Tn5 transposase was highly pure and no degradation of the protein band occurred.

In order to achieve the above object, the present invention provides a method for purifying Tn5 transposase, comprising the steps of:

preparing a recombinant vector containing Tn5 transposase; the recombinant vector also has Strep tag II and SUMO tag;

inducing expression;

and (3) purification: purifying the fusion protein by using Strep tag II and SUMO tag to obtain high-purity Tn5 enzyme protein.

Further, the sequence of Strep tag II is SEQ ID NO 5; the sequence of the SUMO tag is SEQ ID NO 6; the sequence of the Tn5 transposase is SEQ ID NO. 7.

Further, the induction medium for inducing expression is peptone with the weight and volume percentage of 1 percent; 0.5% by weight volume of yeast extract; 0.5% by volume of glycerol; 0.5% by weight volume lactose; 0.05% by weight volume of glucose; 0.05% MgSO by weight/volume₄·7H₂O；6.25mmol/LKH₂PO₄；31mmol/LK₂HPO₄·3H₂O；25mmol/L(NH₄)₂SO₄。

Further, the conditions for inducing expression were 30 ℃ and shaking at 250rpm overnight.

Further, the purification comprises the steps of cracking thalli, extracting fusion protein, cutting the fusion protein and purifying the fusion protein.

Further, the step of cracking the thallus is that the bacteria liquid obtained by induction expression is centrifuged for the first time to collect the thallus, after the thallus is fully resuspended by a precooled lysine buffer, the thallus is ultrasonically crushed in ice water bath, and then centrifuged for the second time, the supernatant is taken and filtered, and the filtrate is added with DNase I for incubation;

preferably, the conditions of the first centrifugation are 4000-;

optionally, the second centrifugation is performed at 800-;

optionally, the crushing power is 30%, the frequency is 5s ON/5s OFF, and the time is 25-35 min.

Optionally, inducing expression of the resulting bacterial fluid: the volume ratio of lysine buffer is 100 (30-40), preferably 100: 35.

Optionally, the filtrate: the volume ratio of DNase I is 9000-11000, preferably 10000: 1.

Further, the extraction step of the fusion protein is to add BeaverBeads^TMInverting and mixing Magrose Strep-Tactin, centrifuging and removing supernatant; adding Wash buffer for washing, centrifuging, and discarding the supernatant; the washing step can be repeated for 1-2 times;

then adding Elution Buffer for Elution, and centrifuging to obtain a supernatant; repeatedly eluting for 2-6 times, and collecting all eluates; dialyze against Lysisbuffer.

Further, the fusion protein is cut by adding SUMO protease for enzyme digestion to obtain an enzyme digestion product.

Further, the purification of the fusion protein is to add BeaverBeads^TMMixing Magrose Strep-Tactin and NiSepharose 6FF, centrifuging to obtain supernatant, and dialyzing with Tn5 transposase stock solution.

The recombinant vector containing the Tn5 transposase is prepared by ingenious design of the applicant, so that the purification of the Tn5 transposase is conveniently and quickly carried out, the purified Tn5 transposase has high purity, no protein band degradation occurs, and the Tn5 transposase protein is correctly assembled and folded depending on the characteristics of a SUMOtag. The Tn5 gene expresses a high enzyme activity product, the dissolving is promoted in the purification process, the Tn5 transposase yield is increased, and the enzyme digestion of the tag protein is depended on SUMO protease. The product with the same purity as the natural Tn5 transposase is obtained.

The invention reduces the traditional IPTG induction process by using the self-induction culture medium, combines the bacteria expansion process and the induction process, and has more convenient expression process. Through the characteristics of high resolution of Strep tag II label and dissolution promotion expression of sumo tag, and being capable of being used as molecular chaperone, Tn5 enzyme is correctly folded, meanwhile, sumo proteolytic enzyme is used for cutting tag protein from fusion protein, and recombinant protein which is the same as natural protein is obtained through affinity chromatography. Improving the purification effect of Tn5 enzyme and ensuring the functional activity.

The invention comprises the following steps:

two tags are constructed on a pET22b vector before Tn5 transposase expression genes.

② since it is constructed on pET22b vector, it can be used for colony culture using self-induction medium. Simplifying culture operation, enabling the thalli to grow rapidly, improving the Tn5 expression level and reducing the culture cost.

And thirdly, reducing the protein conformation error rate in the Tn5 transposase expression process through the constructed Strep tag II tag property. At the same time, rely on BeaverBeads^TMAnd (3) extracting the fusion protein with higher purity from the high-resolution of the Magrose Strep-Tactin to the Strep tag II tag protein.

And fourthly, carrying out enzyme digestion and purification on the fusion protein with Strep tag II, sumo tag protein and Tn5 enzyme through sumo protease, wherein the purified Tn5 transposase does not have tag protein, the conformation and the enzyme activity of the Tn5 transposase are not influenced by the tag protein, and a product with the same purity as the natural Tn5 transposase is obtained. In the using process, the Tn5 enzyme is different from the Tn5 enzyme with 6 × His tag protein in the general method, and influences the protein conformation and the enzyme activity, thereby influencing the functional activity of the Tn5 enzyme.

A purification mode: in the general method, the fusion protein is extracted by 6 × His tag protein, and then the fusion protein is eluted and quantitatively diluted. The purification method of the invention extracts the fusion protein through Strep tag II tag protein, then elutes the fusion protein, uses sumo enzyme to carry out enzyme digestion, and uses Beaver beads mixed according to proportion^TMMagrose Strep-Tactin and Ni Sepharose 6FF, two tag proteins and sumo enzyme were removed from the product to obtain high purity Tn5 enzyme.

Drawings

FIG. 1 is a map of the expression vector pET22b-Strep-SUMO-Tn5 of the present invention.

FIG. 2 is a photograph showing the result of PAGE-SDS electrophoresis of Tn5 transposase expression purified by the present invention and general method.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

SEQ ID NOs 1 to 7 of the following examples were synthesized by Xiamengtai Biotechnology, Inc. Wherein the sequence of Strep tag II gene is shown in SEQ ID NO. 5; the sequence of the SUMO tag gene is shown as SEQ ID NO. 6; the sequence of the Tn5 transposase gene is shown in SEQ ID NO. 7.

KOD polymerase is manufactured by Toyobo Co., Ltd., Japan, cat # KOD-101, DNA polymerase.

Example 1: construction of pET22b-Strep-SUMO-Tn5 expression vector

The Tn5 transposase gene was amplified by PCR and amplified as SEQ ID: NO:1 and SEQ ID: NO: 2 as a primer, KOD polymerase (manufactured by Toyobo Co., Ltd., product No. KOD-101 as DNA polymerase) was added to a 50. mu.l reaction system (artificially synthesized SEQ ID NO:7, i.e., Tn5 transposase gene, as a reaction template). The amplification conditions were 94 ℃ for 30 s; 15s at 94 ℃; 15s at 58 ℃; 50s at 74 ℃; 3min at 74 ℃; for a total of 32 cycles. PCR product 1 was obtained.

The PCR product 1 obtained in the above step was recovered, electrophoresed in 1.5% (W/V) agarose gel, and the band of interest was excised for gel recovery.

Artificially synthesizing a target fragment with a Strep tag II tag (shown as SEQ ID: NO: 5).

Amplifying the SUMO tag gene by using a PCR technology, wherein the sequence is shown in SEQ ID: NO: 3. (ii) SEQ ID: NO: 4 as a primer, and KOD polymerase as DNA polymerase in a 50 μ l reaction system (with artificially synthesized SEQ ID NO:6, SUMO tag gene, as a reaction template). The amplification conditions were 94 ℃ for 30 s; 15s at 94 ℃; 15s at 58 ℃; 15s at 74 ℃; 1min at 74 ℃; for a total of 30 cycles. PCR product 2 was obtained.

The PCR product 2 obtained in the above step was recovered, electrophoresed in 1.5% (W/V) agarose gel, and the band of interest was excised for gel recovery.

After double digestion of 2. mu.g of pET22b vector (available from vast plasmid) with 10U of each of Nde I and Xho I restriction enzymes overnight, 1.5% (W/V) agarose gel was run, the desired band was excised and recovered to give cleaved product 3, which was transformed into DH 5. alpha. competence, plated overnight and observed for cleavage.

Using a Gibson assembly cloning kit (manufactured by New England Biolabs, cat. No. E5510S), the reaction system was as follows: 25 μ l of Gibson assembly premix, 600ng Tn5 transposase gene PCR product (i.e., PCR product 1), 600ng strep tag II (i.e., sequence shown in SEQ ID: NO: 5), 600ng SUMO tag-tagged target fragment (i.e., PCR product 2), 300ng double-digested pET22b vector (i.e., digested product 3), and water was added to make up to 50 μ l. Incubation was carried out at 50 ℃ for 25 min. And inserting the mixture back to the ice for standby.

100 μ lDH5 α was added to the Gibson ligation product, ice-cooled for 30min, heat-shocked at 42 ℃ for 45s, inserted back on ice for 2min, and 150 μ l of non-resistant LB medium was added. After pre-shaking at 37 ℃ and 220rpm for 45min, the plates were plated on LB ampicillin plate media overnight.

The monoclonal colonies were picked and shaken at 37 ℃ and 220rpm for 10 hours in 10ml of LB ampicillin medium. 1 μ l of the vector was used for PCR detection of bacterial suspension, and the vector was named pET22b-Strep-SUMO-Tn5 (see FIG. 1 for map structure diagram).

The pET22b-Strep-SUMO-Tn5 expression vector was extracted from the remaining bacterial liquid using a small and medium endotoxin-free plasmid extraction kit (purchased from Tiangen Biochemical technology Co., Ltd., product No. DP 118).

Example 2: expression purification of pET22b-Strep-SUMO-Tn5 vector

preparation of a Strain expressing vector pET22b-Strep-SUMO-Tn 5: 100ng of pET22b-Strep-SUMO-Tn5 expression vector obtained in example 1 was taken. Add to 100. mu. lDE3 competent cells, ice-wash for 30min, heat shock at 42 ℃ for 45s, insert back on ice for 2min, add 150. mu.l of non-resistant LB medium. After pre-shaking at 37 ℃ and 220rpm for 45min, the plates were plated with LB ampicillin and inverted overnight.

Inducing expression: the above monoclonal colonies were picked and added to 100ml of an auto-induction medium (1% Tryptone (w/v); 0.5% Yeast extract (w/v); 0.5% Glycerol (v/v); 0.5% Lactose (w/v); 0.05% Gluc)ose(w/v)；0.05％MgSO₄·7H₂O(w/v)；6.25mmol/LKH₂PO₄；31mmol/LK₂HPO₄·3H₂O；25mmol/L(NH₄)₂SO₄) 30 ℃ and shaking the bacteria at 250rpm overnight.

And (3) purification:

cracking thalli: 100ml of the overnight suspension was dispensed into two 50ml centrifuge tubes and centrifuged at 4200rpm at 4 ℃ for 15min to collect the cells. The following steps were all performed ON ice, and the collected cells were thoroughly resuspended in a pre-cooled 35 mM lyss buffer (25mM Tris-HCl (pH 8.0); 50mM KCl; 0.1mM DTT; 20mM imidazole; 10% glycerol (v/v)), and then sonicated in an ice water bath at a power of 30% and a frequency of 5s ON/5s OFF for 30 min.

Then centrifuged again at 1000rpm for 30min at 4 ℃. The supernatant was filtered through a 0.22 μm filter in a 50ml tube. One-thousandth volume of DNase I was added and incubated for 3h at 4 ℃ with a homogenizer.

Purifying: adding 1ml of BeaverBeads^TMMagrose Strep-Tactin (purchased from beaver biomedical engineering, Suzhou, Inc., cat # 70808-5) was mixed by inversion for 1h using a 4 ℃ mixer. 500g, 10min, 4 ℃, soft centrifugation, supernatant removal. After washing with 50ml of Wash buffer (25mM Tris-HCl (pH 8.0); 50mM KCl; 0.1mM DTT; 20mM imidazole; 10% glycerol (v/v)) for 5min, 500g, 10min, 4 ℃ soft was centrifuged and the supernatant discarded. The above washing steps were repeated one more time.

BeaverBeads obtained by washing the above^TMThe Magrose Strep-Tactin was dispensed into two tubes, 500. mu.l each. Each tube was eluted with 1ml of Elution Buffer (25mM Tris-HCl (pH 8.0); 50mM KCl; 0.1mM DTT; 300mM imidazole; 10% glycerol (v/v)), and the supernatant was centrifuged 30 times by reversing the top and bottom, 500g, 2min, 4 ℃ and taking the supernatant by soft centrifugation. Elution was repeated 5 times and all eluates were collected. Dialyze overnight against lysine buffer and exchange the solution once.

The dialysis bag was recovered and digested with SUMO Protease (with poly-His tag, from Solebao, Inc., cat # P2070) (1ml digestion system: 1000. mu.g dialysis bag product protein; 20. mu.L 10 XSULMO Protease Buffer; 10U SUMO Protease; ddH)₂O to a constant volume of 1mL), 4 ℃ enzymeCutting overnight to obtain the enzyme digestion product.

Adding 1ml of BeaverBeads into the enzyme digestion product^TMAfter Magrose Strep-Tactin and 200. mu.l of Ni Sepharose 6FF (from Soilebo, cat. No. P2010), the mixture was mixed by inversion for 1h using a 4 ℃ mixer. The supernatant was centrifuged at soft f at 500g for 10min at 4 ℃. The supernatant was collected and dialyzed overnight against Tn5 transposase stock solution (50mM Tris-HCl (pH 7.5); 0.1M NaCl; 0.1mM EDTA; 1mM dithioreitol; 0.1% Triton X-100 (v/v); 50% glycerol (v/v)). The supernatant protein concentration was measured by BCA method (BCA protein quantification kit was purchased from Tiangen Biochemical technology Ltd., Cat. No. PA115), and then diluted as required and stored at-80 ℃ for a long period of time.

Comparative example: general expression and purification of Tn5 transposase

The Tn5 transposase expression vector pET28b was constructed by SLIC method: : tnpA, transformed into e.coli BL21(DE3), colony PCR and sequencing results (mansion ny, inc. biotechnology limited) verified the recombinant plasmid pET28 b: : tnpA was successfully constructed.

Contains plasmid pET28 b: : the single colony of TnpA BL (DE3) was cultured overnight, and then transferred to 100mL liquid LB medium, where the cell growth OD was observed at 37 ℃₆₀₀0.6-0.8, 0.4mM IPTG, 25 ℃ for 3 hours. Centrifuging at 4 deg.C to collect thallus, ultrasonic breaking cell, and centrifuging to obtain supernatant containing Tn5 transposase. The hetero-proteins non-specifically bound to the nickel column were washed off with 1M NaCl-TEGX (20mM Tris-HCl, 1mM EDTA, 10% Glycerol (V/V), 0.1% Triton X-100(V/V), pH7.5) containing 20mM imidazole, Tn5 transposase was eluted with TEGX (20mM Tris-HCl, 1mM EDTA, 10% Glycerol (V/V), 0.1% Triton X-100, pH7.5) containing 250mM imidazole, then salt ions such as imidazole in Tn5 transposase were removed with a desalting column, Tn5 transposase was dissolved in 50mM Tris-HCl (pH7.5) containing 0.1mM EDTA, 0.1NaCl,1mM dithioreol, 0.1% Triton X-100, followed by addition of 50% Glycerol and storage at-80 ℃. Tn5 transposase was quantified by the BCA method.

After the Tn5 transposase products of example 2 and comparative example were quantified to 10mg/mL by BCA method, 5. mu.L of each was subjected to PAGE-SDS electrophoresis, and the results are shown in FIG. 2. Wherein lane 1 is the protein marker, lane 2 is the Tn5 transposase product of example 2, and lane 3 is the Tn5 transposase product of comparative example. As can be seen from fig. 2: the purified Tn5 transposase has no obvious degradation and impurity protein phenomena, and has higher purity; the Tn5 transposase purified by the general method contains foreign proteins and is obviously degraded.

Finally, it will be understood by those skilled in the art that various modifications and equivalent arrangements may be made without departing from the spirit and scope of the present invention and it is intended to cover the appended claims.

SEQUENCE LISTING

<110> Xiamen Life interconnection science and technology Co., Ltd

<120> expression and purification method of recombinant Tn5 transposase

<130> SMHL-18009-CNI

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 49

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<213> Artificial Synthesis

<400> 1

ggtggttccg gtacaggagg tggatccatg attaccagtg cactgcatc 49

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<212> DNA

<213> Artificial Synthesis

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tcagtggtgg tggtggtggt gctcgagcta gcagatttta atgccctgcg 50

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<212> DNA

<213> Artificial Synthesis

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ctggtgccgc gcggcagcgc tagcatgtcg gactcagaag tcaat 45

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<211> 48

<212> DNA

<213> Artificial Synthesis

<400> 4

aatcatggat ccacctcctg taccggaacc accaatctgt tctctgtg 48

<210> 5

<211> 69

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<213> Artificial Synthesis

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atgggcagca gctggagcca cccgcagttc gaaaaaggca gcggcctggt gccgcgcggc 60

agcgctagc 69

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atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60

gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120

aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180

gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240

ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggt 294

<210> 7

<211> 1431

<212> DNA

<213> Artificial Synthesis

<400> 7

atgattacca gtgcactgca tcgtgcggcg gattgggcga aaagcgtgtt ttctagtgct 60

gcgctgggtg atccgcgtcg taccgcgcgt ctggtgaatg ttgcggcgca actggccaaa 120

tatagcggca aaagcattac cattagcagc gaaggcagca aagccatgca ggaaggcgcg 180

tatcgtttta ttcgtaatcc gaacgtgagc gcggaagcga ttcgtaaagc gggtgccatg 240

cagaccgtga aactggccca ggaatttccg gaactgctgg caattgaaga taccacctct 300

ctgagctatc gtcatcaggt ggcggaagaa ctgggcaaac tgggtagcat tcaggataaa 360

agccgtggtt ggtgggtgca tagcgtgctg ctgctggaag cgaccacctt tcgtaccgtg 420

ggcctgctgc atcaagaatg gtggatgcgt ccggatgatc cggcggatgc ggatgaaaaa 480

gaaagcggca aatggctggc cgctgctgca acttcgcgtc tgagaatggg cagcatgatg 540

agcaacgtga ttgcggtgtg cgatcgtgaa gcggatattc atgcgtatct gcaagataaa 600

ctggcccata acgaacgttt tgtggtgcgt agcaaacatc cgcgtaaaga tgtggaaagc 660

ggcctgtatc tgtatgatca cctgaaaaac cagccggaac tgggcggcta tcagattagc 720

attccgcaga aaggcgtggt ggataaacgt ggcaaacgta aaaaccgtcc ggcgcgtaaa 780

gcgagcctga gcctgcgtag cggccgtatt accctgaaac agggcaacat taccctgaac 840

gcggtgctgg ccgaagaaat taatccgccg aaaggcgaaa ccccgctgaa atggctgctg 900

ctgaccagcg agccggtgga aagtctggcc caagcgctgc gtgtgattga tatttatacc 960

catcgttggc gcattgaaga atttcacaaa gcgtggaaaa cgggtgcggg tgcggaacgt 1020

cagcgtatgg aagaaccgga taacctggaa cgtatggtga gcattctgag ctttgtggcg 1080

gtgcgtctgc tgcaactgcg tgaatctttt actccgccgc aagcactgcg tgcgcagggc 1140

ctgctgaaag aagcggaaca cgttgaaagc cagagcgcgg aaaccgtgct gaccccggat 1200

gaatgccaac tgctgggcta tctggataaa ggcaaacgca aacgcaaaga aaaagcgggc 1260

agcctgcaat gggcgtatat ggcgattgcg cgtctgggcg gctttatgga tagcaaacgt 1320

accggcattg cgagctgggg tgcgctgtgg gaaggttggg aagcgctgca aagcaaactg 1380

gatggctttc tggccgcgaa agacctgatg gcgcagggca ttaaaatctg c 1431

Claims

1. A method for expressing and purifying Tn5 transposase is characterized by comprising the following steps:

preparing a recombinant vector containing Tn5 transposase; the recombinant vector also has Strep tag II and SUMO tag; the sequence of Strep tag II is SEQ ID NO 5; the sequence of the SUMO tag is SEQ ID NO 6; the sequence of the Tn5 transposase is SEQ ID NO. 7;

inducing expression: shaking bacteria at 250rpm at 30 ℃ overnight, wherein the induction culture medium is peptone with the weight and volume percentage of 1 percent; 0.5% by weight volume of yeast extract; 0.5% by volume of glycerol; 0.5% by weight volume lactose; 0.05% by weight volume of glucose; 0.05% MgSO by weight/volume₄·7H₂O；6.25mmol/LKH₂PO₄；31mmol/LK₂HPO₄·3H₂O；25mmol/L（NH₄）₂SO₄；

And (3) purification: purifying the fusion protein by using Strep tag II and SUMO tag, comprising the steps of cracking thallus, extracting the fusion protein, cutting the fusion protein and purifying the fusion protein to obtain high-purity Tn5 enzyme protein;

wherein the step of cracking the thallus comprises the steps of centrifuging the bacteria liquid obtained by induced expression for the first time at 4000-45000rpm and 4 ℃ for 12-18min to collect the thallus, fully suspending the thallus by using precooled lysate, and then carrying out ultrasonic crushing in an ice water bath, wherein the bacteria liquid obtained by induced expression comprises the following steps: the volume ratio of the lysis solution is 100 (30-40), the breaking power is 30%, the frequency is 5s ON/5s OFF, the time is 25-35min, the secondary centrifugation is carried out at 800-1200rpm and 4 ℃ for 25-35min, the supernatant is taken and filtered, the filtrate is added with DNase I and incubated for 3h at 4 ℃, and the filtrate: the volume ratio of DNase I is 9000-: 1;

the extraction step of the fusion protein is that BeaverBeads polypeptide-Tactin is added, and after inversion and uniform mixing, the mixture is centrifuged and the supernatant is discarded; adding cleaning solution for washing, centrifuging, and removing supernatant; the washing step can be repeated for 1-2 times; adding eluent for elution, centrifuging and taking supernatant; repeatedly eluting for 2-6 times, and collecting all eluates; dialyzing with lysate;

cutting the fusion protein, namely adding SUMO protease for enzyme digestion to obtain an enzyme digestion product;

the purification of the fusion protein comprises adding BeaverBeads ™ Magrose Strep-Tactin and Ni Sepharose 6FF, mixing uniformly, centrifuging to obtain supernatant, and dialyzing with Tn5 transposase stock solution, wherein the Tn5 transposase stock solution is 50mM Tris-HCl pH 7.5; 0.1M NaCl; 0.1mM EDTA; 1mM dithioritol; 0.1% TritonX-100; 50% of glycerol.

2. The method for expressing and purifying Tn5 transposase as claimed in claim 1, wherein in the step of lysing the bacterial cells, the bacterial solution obtained by induction expression: the volume ratio of the lysate was 100: 35.

3. The method for purifying Tn5 transposase as claimed in claim 1, wherein in the step of lysing the cells, the filtrate: the volume ratio of DNase I was 10000: 1.