CN115785283B - 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, the 669 th cysteine is mutated into tryptophan, the 670 th glutamine is mutated into lysine, and compared with wild PAG-Tn5 transposase, the characteristic peak of library nucleosome constructed by the mutated PAG-Tn5 transposase is more obvious. Sequencing shows that the PAG-Tn5 transposase mutant fragmentation site is basically non-preferential, has 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 between specific proteins and DNA at the whole genome level, and the principle is that Protein G/A and Tn5 transposase are fused to form a novel pG-Tn5/pA-Tn5 fusion enzyme with antibody recognition function and transposase activity. During research, specific antibody is first used to incubate with nuclear target protein, then pG-Tn5/pA-Tn5 enzyme is added to recognize the antibody binding site and insert the label sequence, and finally the interaction between the specific protein and DNA is identified through high flux sequencing. CUT & Tag can replace the traditional ChIP-seq to a certain extent, and has the advantages of low initial cell quantity, high signal-to-noise ratio and the like.
Protein a (Protein) a is a cell wall Protein of one strain of staphylococcus aureus, which binds to mammalian IgG through the Fc region, contains 4 Ig Fc region binding sites, and is commonly used to immunoprecipitate murine IgG2a, igG2b, igA, rabbit IgG, and several antibodies of human IgG1, igG2, and IgG 4. Protein G (Protein G) is a cell wall Protein from the group G of Streptococcus, which binds specifically to the Fc region of IgG and binds all human and murine IgG antibody subtypes, whereas Protein A is not. Protein G has better binding capacity to IgG than Protein A.
The 3 xflag tag polypeptide facilitates exposure of the antibody binding epitope, and thus is bound by the antibody. In most cases, the FLAG tag will be built up at the N-terminus or C-terminus of the protein, and in most cases the N-terminus or C-terminus of the protein is free on the surface of the protein, and antibodies will bind.
Tn5 transposase can insert Tn5 transposons into a sequence of interest with high efficiency. The principle of Tn5 enzyme library construction specifically refers to that DNA is randomly interrupted by the transposition process of Tn5 transposomes, meanwhile, sequencing joints are inserted into two ends of fragmented DNA molecules, and finally, the construction of a DNA library containing complete joints of P5 ends and P7 ends is completed by adding sequencing indexes into PCR. The method greatly shortens the time for library construction and reduces the complex flow of the traditional library construction, so that the method is widely used in the field of second-generation sequencing at present.
PAG-Tn5 transposase (Transposase) consists of Tn5 transposase fusion Protein A, protein G and 3×FLAG tag proteins, can better specifically identify antibody binding sites, accelerate Tn5 transposition efficiency, insert tag sequences, and finally identify specific Protein and DNA interactions 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 greatly restricts the application of CUT & Tag technology.
Disclosure of Invention
In view of the problem that the characteristic peak of nucleosome is not obvious due to low enzymatic activity of the current pAG-Tn5 transposase, the invention better meets the requirements of CUT & Tag technology by carrying out site-directed mutation on amino acid 669 and 670 sites of the pAG-Tn5 transposase, wherein the enzymatic activity of the mutated pAG-Tn5 transposase is higher, the characteristic peak of nucleosome is obvious, and the fragmentation site of the mutated pAG-Tn5 transposase is basically free from preference and host pollution.
The invention discloses a pAG-Tn5 transposase mutant, which is characterized in that amino acids 669 and 670 of pAG-Tn5 transposase are mutated, cysteine 669 is mutated to tryptophan, glutamine 670 is mutated to lysine, the amino acid sequence of the mutant is shown as SEQ ID NO.1, and the nucleotide sequence of a gene encoding the mutant is shown as SEQ ID NO. 2.
In another aspect, the invention provides recombinant plasmids containing the gene encoding the pAG-Tn5 transposase mutant, host cells expressing the pAG-Tn5 transposase mutant, and kits containing the pAG-Tn5 transposase mutant.
The invention also protects the application of pAG-Tn5 transposase mutant in CUT & Tag technology, and compared with wild pAG-Tn5, the library obtained by mutant pAG-Tn5 transposase has more obvious characteristic peaks of nucleosomes, and 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 rational design method, mutate 669 and 670 site amino acids of pAG-Tn5 transposase, the mutant enzyme obtained has higher efficiency of transposition insertion than wild pAG-Tn5 transposase, can obtain more obvious characteristic peak of nucleosome, the library richness is increased, and has no host pollution, and better meets the requirements of CUT & Tag technology.
Drawings
FIG. 1 shows a recombinant plasmid map of PTXB1-pAG-Tn5
FIG. 2 shows the result of pAG-Tn5 transposase mutant protein purification.
FIG. 3 is a diagram of nuclei after lysis of porcine liver tissue.
FIG. 4 is a comparison of the enzymatic activities of mutant and wild-type pAG-Tn5 transposase.
FIG. 5 is a graph showing the sequence base content profile of pAG-Tn5 transposase mutants after cleavage of samples. The abscissa indicates the base position of the sequencing data, and the ordinate indicates the ATCG base content at each position.
FIG. 6 is a graph showing the sequencing of the base mass distribution of PAG-Tn5 transposase mutants after cleavage of samples. The abscissa is the base position of the sequencing data, the ordinate is the sequencing mass box subgraph of each position, and the Y-axis divides the mass value 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 as SEQ ID NO. 3) is entrusted to complete gene synthesis by the biological science and technology Co., ltd, a PTXB1 plasmid is used as a cloning vector in gene synthesis service to construct a recombinant plasmid PTXB1-pAG-Tn5, the plasmid map is shown as figure 1, and the plasmid is transferred into E.coli BL21 (DE 3) to obtain recombinant bacteria.
2. Acquisition of mutant PTXB1-pAG-Tn5 plasmid
Site-directed mutagenesis PCR was performed on C669 and Q670 of Tn5 gene locus in pAG-Tn5 transposase respectively, and primers for site-directed mutagenesis are shown in Table 1.
TABLE 1 design of site-directed mutagenesis primer in pAG-Tn5 transposase
Site-directed PCR amplification was performed using the PTXB1-pAG-Tn5 plasmid as a template. The PCR amplification system was 50. Mu.L, comprising: PRIME STAR Max DNA Polymerase. Mu.L, 1. Mu.L of the upstream primer (10. Mu.M), 1. Mu.L of the downstream primer (10. Mu.M), 1. Mu.L of the plasmid template (1 ng/. Mu.L) and the total volume of the sterilized ultrapure water were made up to 50. Mu.L.
The PCR amplification procedure was: after denaturation at 98 ℃ for 1min, the amplification cycle is carried out, namely 15s denaturation at 98 ℃, 15s annealing at 55 ℃, 3min extension at 72 ℃, 28 times of total cycle and 5min extension at 72 ℃. The PCR product is detected by electrophoresis, and the band is single and clear.
The obtained fixed-point PCR reaction product is subjected to enzymolysis for 2 hours at 37 ℃ to eliminate a male parent template, the enzymolysis product is converted into chemically competent cells E.coli DH 5 alpha by a heat shock method, a LB solid plate containing ampicillin sodium (60 mug/mL) is coated with the conversion solution to obtain a fixed-point mutation library, and the fixed-point mutation library is cultured for 12 hours at 37 ℃.
3. Expression of mutant enzymes
Randomly picking 1-3 single colonies from the site-directed mutagenesis library, culturing and extracting plasmids, sample delivery to the field of the biological technology of the Optimum the nucleotide sequence was determined by the company Limited, to determine whether the desired mutation was introduced, the sequencing primer was a T7 universal primer, and the nucleotide sequence encoding pAG-Tn5 transposase mutant is shown in SEQ ID NO. 2. Plasmid with the desired mutation was transformed into E.coli BL21 (DE 3), and single colonies were picked and inoculated into a tube with 5mL of LB liquid medium, and cultured overnight at 37℃at 200 r/min. The cultured bacterial liquid is inoculated into 100mL LB culture medium (tryptone 10g, yeast powder 5g, sodium chloride 10g, pH 7.0) containing 60 mu g/mL ampicillin sodium in an inoculum size of 1 percent (volume ratio), when the bacterial liquid is cultured to an OD 600 value of 0.4-0.6 at 37 ℃ and 220r/min, a proper volume of IPTG (final concentration of 1 mmol/L) is added, and then the bacterial body is collected after induction culture for 18 hours at 16 ℃ and 200 r/min.
4. Purification of mutant enzymes
The collected cells were washed twice with phosphate buffer, then resuspended with 10% fermentation broth volume lysis buffer (20 mM Tris-HCl,0.8M NaCl,1mM EDTA,1mM DTT,10%glycerol,pH =7.5), sonicated and the sonicated conditions were: the power is 300W, the operation is 3s, the interval is 6s, and the ultrasonic treatment is carried out for 8 minutes. Centrifuging the cytoblast at 10 000r/min and 4 ℃ for 30min, collecting supernatant, adding PEI with a final concentration of 1%, centrifuging at high speed (10 000r/min and 30 min), collecting supernatant, and preserving at 4 ℃ to obtain crude enzyme solution containing mutant pAG-Tn5 transposase.
Mutant pAG-Tn5 will undergo two-step protein purification: the preparation method comprises the steps of medium purification in the first step CHITIN RESIN, medium purification in the second step Focurose PG, and finally dialysis overnight to obtain mutant pAG-Tn5 transposase pure enzyme solution, wherein the specific steps are as follows:
The first step: CHITIN RESIN Medium purification
The CHITIN RESIN volumes of lysis buffer were equilibrated with the crude enzyme solution, the CHITIN RESIN medium was washed with 5 volumes of lysis buffer, 3 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 after cleavage overnight mutant pAG-Tn5 transposase was eluted and collected.
And a second step of: focurose 200PG media purification
The cleaved product was subjected to a second step of protein purification, equilibrated with buffer (20 mM Tris-HCl,50mM NaCl,0.1mM EDTA,1mM DTT,10%glycerol,pH =7.5), injected at a flow rate of 0.5mL/min, the target protein was collected according to UV detection values, and electrophoretically detected (the result is 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 was subjected to the following procedures:
(1) Preparation of linker-containing pAG-Tn5 transposase mutants
To assembly buffer (50 mM Tris-HCl,0.1M NaCl,0.1mM EDTA,1mM DTT,0.1% Triton X-100, 50% glyciol, pH=7.5) was added 2. Mu.g of the wild-type and mutant pAG-Tn5 transposase obtained in example 1, annealed adaptor primer (10. Mu.M), total volume 7.5. Mu.L, reaction conditions: 30 ℃ and 60min to obtain pAG-Tn5 mutant transposase with a joint.
Transposase recognition sequence: AGATGTGTATAAGAGACAG; a first linker sequence: AATGATACGGCGACCACCGAGATCTACACXXXXXXXXTCGTCGGCAGCG TC; a second linker sequence: CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTC TCGTGGGCTCGG; wherein XXXXXXXX is an Index sequence.
(2) Sample processing and construction of fragmented DNA libraries
Fresh pig liver tissue is taken, liver cell nucleuses are separated and counted according to a frozen tissue cell nucleuses extraction flow, 1X 10 5 cell nucleuses are taken for a downstream CUT & Tag experiment, 1 mu L of primary antibody-Histone H3 is added for incubation, and 1 mu L of secondary antibody Goat pAb to Rb lgG is added for enhancing pAG-Tn5 targeting binding capacity. Wild-type and mutant pAG-Tn5 transposase were added separately for incubation. Mg 2+ was added at a final concentration of 1mM to activate the cleavage activity of the pAG-Tn5 transposase mutant, and the target protein-bound DNA sequence was fragmented. Extracting DNA by using a magnetic bead method, performing PCR amplification to construct a library, and purifying a PCR product by using magnetic bead separation to obtain the library. As shown in FIG. 4, by taking histone as an example, the peak diagram shows that compared with the wild pAG-Tn5, the library obtained by the mutant pAG-Tn5 transposase has more obvious characteristic peaks of nucleosomes, and after magnetic bead separation, the library yield can reach 900ng, thereby meeting the library building requirement, indicating that the mutant pAG-Tn5 transposase provided in example 1 has higher enzyme activity and can be well applied to CUT & Tag technology.
(3) GC preference, base quality and host pollution analysis of pAG-Tn5 transposase mutant
10G data is used for reading a DNA library constructed by mutant pAG-Tn5 transposase by using a Huada T7 sequencer, the sequencing result is shown in FIG. 5, and GC content and distribution result show that mutant pAG-Tn5 transposase fragmentation sites are basically unbiased; according to the characteristics of the sequencing technology, the base quality of the tail end of the sequencing fragment is generally lower than that of the front end, and the sequencing base quality distribution diagram of the PAG-Tn5 transposase mutant in FIG. 6 after cutting a sample shows that the sequencing data are normal. And (3) 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 shown according to Total mapped (%) data.
Table 1 sample data comparison results
Note that: total READS PAIR: CLEAN READS total READS PARI; total reads: CLEAN READS total number of sequences; total mapped (%): the total number and proportion of sequences aligned to the reference genome; properly mapped (%): the number of reads that correctly match to the reference sequence; nodup Mapped (%): properly MAPPED READS number removes the number of reads after PCR duplication.
Claims (7)
- PAG-Tn5 transposase mutant, wherein the amino acid sequence of the mutant is shown as SEQ ID NO. 1.
- 2. A gene encoding the PAG-Tn5 transposase mutant of claim 1, wherein the nucleotide sequence of the gene is shown in SEQ ID No. 2.
- 3. A recombinant plasmid comprising 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 as defined in claim 1 in DNA banking.
- 6. A kit comprising the PAG-Tn5 transposase mutant as defined in claim 1.
- 7. Use of a PAG-Tn5 transposase mutant of claim 1 or a kit of claim 6 in CUT & Tag technology.
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