CN115785283A - PAG-Tn5 mutant and application thereof - Google Patents
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Abstract
The invention discloses a PAG-Tn5 transposase mutant and application thereof, wherein the PAG-Tn5 transposase mutant is obtained by mutating amino acids at 669 and 670 sites of PAG-Tn5 transposase, cysteine at 669 site is mutated into tryptophan, glutamine at 670 site is mutated into lysine, and compared with wild PAG-Tn5 transposase, the characteristic peak of nucleosome of a library constructed by the mutated PAG-Tn5 transposase is more obvious. Sequencing shows that the fragmentation sites of the PAG-Tn5 transposase mutant basically have no preference and no host pollution, and better meets the requirements of CUT & Tag technology.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a PAG-Tn5 transposase mutant and a preparation method and application thereof.
Background
CUT & Tag is a novel technology for researching interaction of specific Protein and DNA at the whole genome level, and the principle is to fuse Protein G/A and Tn5 transposase to form novel pG-Tn5/pA-Tn5 fusion enzyme simultaneously having an antibody recognition function and transposase activity. During research, a specific antibody is firstly incubated with a nuclear target protein, then pG-Tn5/pA-Tn5 enzyme is added to recognize an antibody binding site, a tag sequence is inserted, and finally interaction between the specific protein and DNA is identified through high-throughput sequencing. The CUT & Tag can replace the traditional ChIP-seq to a certain extent, and has the advantages of low cell initiation amount, high signal-to-noise ratio and the like.
Protein A (Protein) A is a cell wall Protein of a strain of Staphylococcus aureus, which binds to mammalian IgG via an Fc region, contains 4 binding sites for Ig Fc regions, and is commonly used for immunoprecipitation of murine IgG2a, igG2b, igA, rabbit IgG, and human IgG1, igG2, and IgG4 antibodies. Protein G (Protein G) is a cell wall Protein from the streptococcal group G, which binds specifically to the Fc region of IgG and binds to all human and murine IgG antibody subtypes, whereas Protein A does not. Protein G has better binding capacity for IgG than Protein A.
The 3 XFlag tag polypeptide facilitates exposure of the antibody binding epitope to be bound by the antibody. In most cases, the FLAG tag is constructed at the N-terminal or C-terminal of the protein, and in most cases, the N-terminal or C-terminal of the protein is free on the surface of the protein, and the antibody can be bound.
Tn5 transposase can efficiently insert Tn5 transposon into a target sequence. The principle of building a library based on Tn5 enzyme specifically means that a sequencing joint is inserted into two ends of a fragmented DNA molecule while DNA is randomly interrupted through a transposition process of a Tn5 transposome, and finally, the construction of a DNA library containing a P5 end and a P7 end complete joint is completed through PCR and sequencing Index. The method greatly shortens the time of library construction, reduces the complex process of traditional library construction, and is widely used in the field of second-generation sequencing at present.
pAG-Tn5 Transposase (Transposase) is composed of Tn5 Transposase fused with Protein A, protein G and 3 XFLAG tag Protein, can better specifically identify antibody binding sites, accelerate Tn5 transposition efficiency, insert tag sequences, and finally identify interaction between specific Protein and DNA through high-throughput sequencing. However, pAG-Tn5 transposase has the problems of low enzyme activity, unobvious characteristic peaks of nucleosomes, host pollution and the like, and the application of the technology in CUT & Tag is greatly restricted.
Disclosure of Invention
In view of the problem that the enzyme activity of the current pAG-Tn5 transposase is not high, which causes the feature peak of nucleosome to be unobvious, the invention carries out site-directed mutation on the 669 th and 670 th amino acid sites of the pAG-Tn5 transposase, so that the mutated pAG-Tn5 transposase has higher enzyme activity and obvious feature peak of nucleosome, and the fragmentation site of the mutated pAG-Tn5 transposase has no preference basically and no host pollution, thereby better meeting the requirements of CUT & Tag technology.
On one hand, the invention protects the pAG-Tn5 transposase mutant, the mutant is obtained by mutating the amino acids 669 and 670 of the pAG-Tn5 transposase, the cysteine at the 669 is mutated into tryptophan, the glutamine at the 670 is mutated into lysine, the amino acid sequence is shown as SEQ ID NO.1, and the nucleotide sequence of the gene for coding the pAG-Tn5 transposase mutant is shown as SEQ ID NO. 2.
On the other hand, the invention protects the recombinant plasmid containing the coding gene of the pAG-Tn5 transposase mutant, the host cell expressing the pAG-Tn5 transposase mutant and the kit containing the pAG-Tn5 transposase mutant
The invention also protects the application of the pAG-Tn5 transposase mutant in the CUT & Tag technology, and compared with the wild pAG-Tn5, the library obtained by the mutant pAG-Tn5 transposase has more obvious nucleosome characteristic peaks, the transposase has higher enzyme activity and no host pollution. .
Compared with the prior art, the invention has the following beneficial effects:
the invention is based on a rational design method, amino acids at positions 669 and 670 of pAG-Tn5 transposase are mutated, and the obtained mutant enzyme has higher transposition insertion efficiency compared with wild pAG-Tn5 transposase, can obtain more obvious nucleosome characteristic peaks, increases the richness of a library, has no host pollution, and better meets the requirements of CUT & Tag technology.
Drawings
FIG. 1 is a PTXB1-pAG-Tn5 recombinant plasmid map
FIG. 2 shows the result of protein purification of pAG-Tn5 transposase mutant.
FIG. 3 is a nuclear map of porcine liver tissue after lysis.
FIG. 4 is a comparison of the enzymatic activity of mutant and wild-type pAG-Tn5 transposases.
FIG. 5 is a diagram showing the sequencing base content distribution of pAG-Tn5 transposase mutant after cutting a sample. The abscissa is the base position of the sequencing data and the ordinate is the ATCG base content at each position.
FIG. 6 is a graph showing the sequencing base mass distribution of PAG-Tn5 transposase mutant after cutting the sample. The abscissa is the base position of the sequencing data, the ordinate is the sequencing quality box map for each position, and the Y-axis divides the quality values into three parts: green (high quality), orange (medium quality) and red (low quality).
Detailed Description
Example 1: preparation of mutant pAG-Tn5 transposase
1. Acquisition of wild-type plasmid
The gene of the codon optimized wild pAG-Tn5 transposase (the nucleotide sequence of the gene is shown in SEQ ID NO. 3) is entrusted to the Scombrinology Limited company for whole gene synthesis, a PTXB1 plasmid is used as a cloning vector in the gene synthesis service to construct a recombinant plasmid PTXB1-pAG-Tn5, the plasmid map is shown in figure 1, and the plasmid is transferred into E.coli BL21 (DE 3) to obtain a recombinant bacterium.
2. Obtaining mutant PTXB1-pAG-Tn5 plasmid
Site-directed mutagenesis was performed on C669 and Q670 of Tn5 gene site in pAG-Tn5 transposase by site-directed mutagenesis PCR method, and the primers for site-directed mutagenesis are shown in Table 1.
TABLE 1 design of site-directed mutagenesis primers in pAG-Tn5 transposase
And performing site-directed PCR amplification by using the PTXB1-pAG-Tn5 plasmid as a template. The PCR amplification system was 50. Mu.L, comprising: prime STAR Max DNA Polymerase 25. Mu.L, 1. Mu.L of the forward primer (10. Mu.M), 1. Mu.L of the reverse primer (10. Mu.M), 1. Mu.L of the plasmid template (1 ng/. Mu.L), autoclaved ultrapure water to a total volume of 50. Mu.L.
The PCR amplification procedure was: after the denaturation at 98 ℃ for 1min, the amplification cycle is carried out, namely denaturation at 98 ℃ for 15s, annealing at 55 ℃ for 15s, extension at 72 ℃ for 3min, the cycle is 28 times totally, and finally extension at 72 ℃ for 5min. The PCR product is detected by electrophoresis, and the band is single and clear.
The obtained site-directed PCR reaction product is subjected to enzymolysis for 2h at 37 ℃ by using Dpn I to eliminate a male parent template, the enzymolysis product is transformed into a chemically competent cell E.coli DH 5 alpha by adopting a heat shock method, an LB solid plate containing ampicillin sodium (60 mu g/mL) is coated on the transformation liquid to obtain a site-directed mutagenesis library, and the site-directed mutagenesis library is cultured for 12h at 37 ℃.
3. Expression of the mutant enzyme
Randomly picking 1-3 single colonies from the site-directed mutagenesis library, culturing and extracting plasmids, sending a sample to the Scirpus technologies GmbH to determine a nucleotide sequence to determine whether to introduce expected mutagenesis, wherein the sequencing primer is a T7 universal primer, and the nucleotide sequence for coding the pAG-Tn5 transposase mutant is shown as SEQ ID NO. 1. Plasmids into which the desired mutations were introduced were transformed into E.coli BL21 (DE 3), and a single colony was picked up and inoculated into a test tube containing 5mL of LB liquid medium and cultured overnight at 37 ℃ at 200 r/min. Inoculating the cultured bacterial solution at an inoculation amount of 1% (volume ratio) into 100mL LB medium (tryptone 10g, yeast powder 5g, sodium chloride 10g, pH adjusted 7.0) containing 60. Mu.g/mL ampicillin sodium, culturing at 37 deg.C and 220r/min to OD 600 When the value is 0.4-0.6, adding IPTG (final concentration is 1 mmol/L) with appropriate volume, and inducing culture at 16 deg.C and 200r/minCulturing for 18h, and collecting thallus.
4. Purification of mutant enzymes
The collected cells were washed twice with phosphate buffer, resuspended in 10% broth volume lysis buffer (20 mM Tris-HCl,0.8M NaCl,1mM EDTA,1mM DTT,10% glycerol, pH = 7.5), and the cells were disrupted by sonication under the following conditions: the power is 300W, the work time is 3s, the pause time is 6s, and the ultrasound time is 8 minutes. And (3) centrifuging the cell disruption solution at the temperature of 4 ℃ for 30min at the speed of 10 000r/min, collecting supernatant, adding PEI with the final concentration of 1%, centrifuging at high speed (10 000r/min,30 min), collecting supernatant, and storing at the temperature of 4 ℃ to obtain crude enzyme solution containing the mutant pAG-Tn5 transposase.
Mutant pAG-Tn5 will undergo two-step protein purification: purifying a Chitin Resin medium in the first step, purifying a Focure 200PG medium in the second step, and finally dialyzing overnight to obtain a mutant pAG-Tn5 transposase pure enzyme solution, wherein the method comprises the following specific steps:
the first step is as follows: chitin Resin medium purification
The crude enzyme solution was combined with the Chitin Resin medium equilibrated with 5 column volumes of lysis buffer, the Chitin Resin medium was washed with 5 column volumes of lysis buffer, 3 column volumes of cleavage solution (20 mM Tris-HCl,0.8M NaCl,1mM EDTA,1mM DTT,10% glycerol,100mM DTT, pH = 7.5) was added, and the mutant pAG-Tn5 transposase was eluted after overnight cleavage and collected.
The second step is that: focurose 200PG media purification
The cleavage product was subjected to the second protein purification step, the column was equilibrated with a buffer (20 mM Tris-HCl,50mM NaCl,0.1mM EDTA,1mM DTT,10% glycerol, pH = 7.5), and the sample was introduced at a flow rate of 0.5mL/min, and the objective protein was collected according to the UV detection value and electrophoretically detected (the results are shown in FIG. 2). The buffer was dialyzed overnight at 4 ℃ in dialysis buffer (50 mM Tris-HCl,0.1M NaCl,0.1mM EDTA,1mM DTT,50% glycerol, pH = 7.5). The solution after dialysis is pure enzyme and is preserved at-20 ℃.
Example 2: application of pAG-Tn5 transposase mutant
The mutant pAG-Tn5 transposase after protein purification is carried out as follows:
(1) Preparation of linker-containing pAG-Tn5 transposase mutant
The wild type and mutant pAG-Tn5 transposase obtained in example 1 was added to assembly buffer (50 mM Tris-HCl,0.1M NaCl,0.1mM EDTA,1mM DTT,0.1% Triton X-100, 50% glycerol, pH = 7.5), 2. Mu.g of the wild type and mutant pAG-Tn5 transposase, the adaptor primer (10. Mu.M) after annealing, and the total volume was 7.5uL, reaction conditions: pAG-Tn5 mutant transposase with a linker was obtained at 30 ℃ for 60 min.
Transposase recognition sequence: AGATGTGTATAAGAGACAG; a first linker sequence: AATGATATACGGCGACCGACAGTACXXXXXXTCGTCGGCAGCGTC; a second linker sequence: CAAGCAGAGACGGCATACGAGAGAGATACGAGATXXXXXXGTCTCGTGGGCTCGG; wherein XXXXXXXXX is an Index sequence.
(2) Sample processing and construction of fragmented DNA libraries
Collecting fresh pig liver tissue, separating liver cell nucleus according to frozen tissue cell nucleus extraction process, counting, collecting 1 × 10 5 Use of individual nuclei for downstream CUT&In Tag experiment, cell nucleus is shown in figure 3, 1 mu L of primary Anti-tissue H3 is added for incubation, and 1 mu L of secondary Anti-Goat pAb to Rb lgG is added to enhance the target binding capacity of pAG-Tn 5. Wild type and mutant pAG-Tn5 transposases were added for incubation, respectively. Mg was added to a final concentration of 1mM 2+ The pAG-Tn5 transposase mutant cleavage activity is activated to fragment the target protein-bound DNA sequence. Extracting DNA by a magnetic bead method, carrying out PCR amplification to construct a library, and carrying out PCR product purification by magnetic bead sorting to obtain the library. Library length distribution detection is carried out on the prepared library on a 4200Bioanalyzer, as shown in figure 4, by taking histone as an example, a peak diagram shows that the library obtained by the mutant pAG-Tn5 transposase has more obvious nucleosome characteristic peak compared with the wild pAG-Tn5, and after magnetic bead sorting, the yield of the library can reach 900ng, thereby meeting the requirement of library construction, and showing that the mutant pAG-Tn5 transposase provided by the embodiment 1 has higher enzyme activity and can be well applied to CUT&Tag technology.
(3) Analysis of GC bias, base quality and host contamination of pAG-Tn5 transposase mutant
Reading a DNA library constructed by the mutant pAG-Tn5 transposase by using 10G data through a Huada T7 sequencer, wherein the sequencing result is shown in figure 5, and the GC content and distribution result shows that the fragmentation sites of the mutant pAG-Tn5 transposase have no preference basically; according to the characteristics of sequencing technology, the base quality of the tail end of a sequencing fragment is generally lower than that of the front end, and a sequencing base quality distribution diagram of a PAG-Tn5 transposase mutant cutting sample in figure 6 shows that sequencing data are normal. Comparing Clean Reads with a reference genome to obtain comprehensive Reads distribution information, wherein the comparison result of sample data is shown in table 1, and no host pollution is displayed according to Total mapped (%) data.
TABLE 1 sample data comparison results
Note: total Reads pair: the total number of reads pari of clean reads; total reads: total number of sequences of clean reads; total mapped (%): comparing the total number and proportion of the sequences on the reference genome; properly mapped (%): the number of reads that correctly match to the reference sequence; nodup Mapped (%): the number of Property mapped reads minus the number of reads after PCR replication.
Claims (7)
- PAG-Tn5 transposase mutant, characterized in that the amino acid sequence of the mutant is shown in SEQ ID NO. 1.
- 2. A gene encoding the PAG-Tn5 transposase mutant according to claim 1, wherein the nucleotide sequence of the gene is set forth in SEQ ID No. 2.
- 3. A recombinant plasmid containing the gene of claim 2.
- 4. A host cell expressing the mutant of claim 1 or carrying the recombinant plasmid of claim 3.
- 5. Use of a PAG-Tn5 transposase mutant according to claim 1 for DNA pooling.
- 6. A kit comprising a PAG-Tn5 transposase mutant according to claim 1.
- 7. Use of a PAG-Tn5 transposase mutant as claimed in claim 1 or a kit as claimed in claim 6 in CUT & Tag technology.
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| CN115948363B (en) * | 2022-08-26 | 2024-02-27 | 武汉影子基因科技有限公司 | Tn5 transposase mutant and preparation method and application thereof |
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