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WO2019184044A1 - Protéine de fusion d'une protéine se liant au transposase-anticorps, préparation et utilisation associées - Google Patents

Protéine de fusion d'une protéine se liant au transposase-anticorps, préparation et utilisation associées Download PDF

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WO2019184044A1
WO2019184044A1 PCT/CN2018/084711 CN2018084711W WO2019184044A1 WO 2019184044 A1 WO2019184044 A1 WO 2019184044A1 CN 2018084711 W CN2018084711 W CN 2018084711W WO 2019184044 A1 WO2019184044 A1 WO 2019184044A1
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protein
fusion protein
transposase
domain
seq
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朱化星
王米
李科
何翼
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SHANGHAI SINOBIO BIOTECHNOLOGY CO Ltd
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SHANGHAI SINOBIO BIOTECHNOLOGY CO Ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/705—Fusion polypeptide containing domain for protein-protein interaction containing a protein-A fusion

Definitions

  • the invention belongs to the fields of molecular biology, genomics and biotechnology, and particularly relates to a fusion protein of a transposase-antibody binding protein, and preparation and application thereof.
  • the transposon sequence can be inserted and integrated into the random location of the genome under the action of a transposase. Due to the nature of random insertion of DNA, transposases are often used in mutant library construction and sequencing library construction. In the library construction of high-throughput sequencing, the transposase can randomly break the sequence to be tested, and add a linker at both ends of the fragmented sequence. After PCR amplification, the next step can be directly sequenced, which is compared with the conventional ultrasonic disruption. The law has great advantages, and fewer steps save money and time.
  • Antibody binding proteins such as ProteinA, ProteinG or ProteinL can bind to mammalian IgG via the Fc region.
  • the binding strength of the antibody binding protein to IgG is highly dependent on the species and subtype of the antibody, and the recombinant antibody binding protein has a stronger binding ability than the natural antibody binding protein.
  • Antibody binding proteins are often used in experiments such as immunoassays, antibody immunoprecipitation, and the like.
  • ChIP Chromatin-immunoprecipitation
  • the principle of ChIP is to crosslink the DNA and protein in the cell, cut the chromatin into small fragments by sonication, add IgG to specifically bind to the antigen protein, and precipitate the DNA fragment bound to the target protein in antibody binding. On the protein beads, this will enrich the DNA associated with the protein of interest.
  • the application of ChIP ranges from studying the relationship between the target protein and the known target sequence to the study of the interaction between the target protein and the unknown sequence; from studying the relationship between a target protein and DNA, to studying the mutual binding of two proteins and DNA.
  • ChIP Role; from the study of histone modifications in the promoter region to the study of protein complexes bound to DNA sequences. ChIP is a relatively mature technology, but there are still some technical difficulties. For example, ChIP experiments involve many steps, the results are less reproducible, and require a large amount of starting materials. For nerve cells and stem cells, it is often difficult to culture, it is difficult to obtain a large number of cells, and it is difficult to distinguish between individual cells and whole cells. Phenotype; chromatin immunoprecipitation often results in a large number of DNA, including a large number of non-specifically bound false positive binding sequences.
  • ChIP-Seq which combines ChIP with Next-generation sequencing technology, is capable of detecting DNA segments that interact with proteins across the genome.
  • ChIP-Seq will be purified and library constructed by ChIP-specific DNA fragments that bind to the protein of interest, and then high-throughput sequencing of these fragments. Sequence information for interaction with the protein of interest can be obtained and compared to the genome-wide map to accurately map these sequences to the genome.
  • ChIP-Seq inherits the same technical difficulties as ChIP, and additionally fills the end of the DNA fragment used in the library construction-sequencing step, then adds A' at the end, and then adds a Y-type connector at A'.
  • the method of sequencing is also cumbersome and has many steps, and each step will lose valuable samples and lose the final sequencing information. Due to the loss, the amount of sample DNA required must also be high, which is a big limitation for some studies that are difficult to obtain a large number of samples, such as some nerve cells that are difficult to cultivate, some need to study cell heterogeneity, and need to be directed to a single cell. Study of tumor cells.
  • a fusion protein comprising a first domain having a transposition function and a second domain having a function of binding an Fc fragment of an antibody.
  • the transposition function refers to a transposition insertion of a gene sequence function.
  • said binding antibody Fc segment function refers to binding to an Fc segment function in an IgG molecule.
  • the first domain is a transposase or a protein analog having a transposition function.
  • the protein analog is capable of carrying a DNA sequence and inserting the DNA sequence into another stretch of DNA.
  • the having a transposition function may mean having a transposase activity.
  • the protein analog having a transposition function may be a protein analog having a transposase activity.
  • the first domain is a Tc1/Mariner, hobo, MITEs, hAT, PiggyBac (PB), TnA transposase family having a transposition function; or other protein analog having a transposition function.
  • the protein analog is capable of carrying a DNA sequence and inserting it into another piece of DNA.
  • the having a transposition function may mean having a transposase activity.
  • the protein analog having a transposition function may be a protein analog having a transposase activity.
  • the first domain is a family of TnA transposases.
  • the TnA transposase family is selected from the group consisting of Tn1, Tn2, Tn3, Tn4, Tn5, Tn6, Tn7, Tn8, Tn9 or Tn10.
  • the first domain is a Tn5 transposase or a Tn10 transposase.
  • the Tn5 transposase is selected from the group consisting of a full-length Tn5 transposase, a partial functional domain of a Tn5 transposase, a Tn5 transposase mutant, a tagged full-length Tn5 transposase, and a portion of a tagged Tn5 transposase Functional domain or tagged Tn5 transposase mutant.
  • the Tn10 transposase is selected from the group consisting of a full-length Tn10 transposase, a partial domain of a Tn10 transposase, a Tn10 transposase mutant, a tagged full-length Tn10 transposase, a portion of a tagged Tn10 transposase Functional domain or tagged Tn10 transposase mutant.
  • the label is selected from the group consisting of HHHHHH, DYKDDDDK, YPYDVPDYA, GGLLISGGAL.
  • the Tn5 transposase mutant is selected from the group consisting of: R30Q, K40Q, Y41H, T47P, E54K/V, M56A, R62Q, D97A, E110K, D188A, Y319A, R322A/K/Q, E326A, K330A/R, K333A, R342A, E344A, E345K, N348A, L372P, S438A, K439A, S445A, G462D, A466D.
  • amino acid sequence of the full-length Tn5 transposase is as shown in SEQ ID NO.
  • the full length Tn10 transposase amino acid sequence is set forth in SEQ ID NO.
  • the second domain is S. aureus Protein A (Protein A), Streptococcal Protein G (Protein G), Streptococcal Protein L (Protein L) or other protein analog having the function of binding to the Fc segment of the antibody.
  • the protein analog can bind to the Fc segment of the antibody.
  • the second domain is a full-length Staphylococcus aureus protein A, a partial functional domain of S. aureus A protein, a S. aureus A protein mutant, a full-length streptococcal protein G protein, a streptococcus G protein a partial functional domain, a Streptococcus G protein mutant, a tagged full-length S. aureus protein A, a partial functional domain of a tagged S. aureus protein A, a labeled S. aureus A protein mutant, Labeled full-length Streptococcal protein G, a partial functional domain of the tagged Streptococcus G protein or a labeled Streptococcus G protein mutant.
  • amino acid sequence of the full-length S. aureus A protein is as shown in SEQ ID NO.
  • amino acid sequence of the full-length Streptococcus G protein is shown in SEQ ID NO.
  • the first domain and the second domain are joined by a linker.
  • the present invention has no particular requirements for the order of connection as long as the object of the present invention is not limited.
  • the C-terminus of the first domain can be joined to the N-terminus of the second domain.
  • the C-terminus of the second domain may be joined to the N-terminus of the first domain.
  • the fusion protein has the general formula: a first domain-linker fragment-second domain or a second domain-linker fragment-first domain.
  • the linker has the structural formula of (GS) a (GGS) b (GGGS) c (GGGGS) d , wherein a, b, c, and d are each an integer greater than or equal to zero.
  • amino acid sequence of the ligated fragment can be selected from the following:
  • the ligated fragment may be Linker1, and the amino acid sequence of the ligated linker Linker1 is as shown in SEQ ID NO. 10, specifically: GGGGS.
  • the ligated fragment may be Linker2, and the amino acid sequence of the ligated linker Linker2 is as shown in SEQ ID NO. 11, specifically: GGGSGGGGS.
  • fusion proteins such as Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, and Tn5-ProteinA-5 are listed.
  • the amino acid sequence of the Tn5-Protein A-1 is shown in SEQ ID NO.
  • the amino acid sequence of the Tn5-ProteinG-2 is shown in SEQ ID NO.
  • the amino acid sequence of the Tn10-Protein A-3 is shown in SEQ ID NO.
  • the amino acid sequence of the Tn10-ProteinG-4 is shown in SEQ ID NO.
  • the amino acid sequence of the Tn5-Protein A-5 is shown in SEQ ID NO.
  • amino acid sequence of the fusion protein of the invention may be as set forth in any one of SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. However, it is not limited to the specific forms listed in the preferred cases of the present invention.
  • an isolated polynucleotide i.e., a DNA molecule
  • a DNA molecule encoding the aforementioned fusion protein
  • the polynucleotide encoding the fusion protein of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the polynucleotide encoding the fusion protein of the present invention can be prepared by any suitable technique well known to those skilled in the art. Such techniques are described in the general description of the art, such as the Guide to Molecular Cloning (J. Sambrook et al., Science Press, 1995). Methods including, but not limited to, recombinant DNA techniques, chemical synthesis, and the like; for example, overlapping extension PCR.
  • the nucleotide sequence encoding the Tn5 transposase is optimized as shown in SEQ ID NO.
  • the nucleotide sequence encoding the Tn10 transposase encoding is optimized as shown in SEQ ID NO.
  • the nucleotide sequence encoding the S. aureus Protein A is optimized as shown in SEQ ID NO.
  • the encoded nucleotide sequence encoding the Streptococcus G protein is optimized as set forth in SEQ ID NO.
  • the nucleotide sequence encoding the linker Linker1 is optimized as shown in SEQ ID NO. 16, specifically: GGTGGTGGTGGTTCT.
  • the nucleotide sequence encoding the linker Linker2 is shown in SEQ ID NO. 17, specifically: GGTGGTGGTTCTGGTGGTGGTGGTTCT.
  • nucleotide sequence encoding the fusion protein Tn5-ProteinA-1 is optimized as shown in SEQ ID NO.
  • nucleotide sequence encoding the fusion protein Tn5-ProteinG-2 is shown in SEQ ID NO.
  • nucleotide sequence encoding the fusion protein Tn10-Protein A-3 is shown in SEQ ID NO.
  • nucleotide sequence encoding the fusion protein Tn10-ProteinG-4 is shown in SEQ ID NO.
  • nucleotide sequence encoding the fusion protein Tn5-ProteinA-5 is shown in SEQ ID NO.
  • a cloning vector and an expression vector comprising the aforementioned polynucleotide are provided.
  • the expression vector of the present invention contains a polynucleotide encoding the fusion protein.
  • Methods well known to those skilled in the art can be used to construct the expression vector. These methods include recombinant DNA techniques, DNA synthesis techniques, and the like.
  • the DNA encoding the fusion protein can be operably linked to a multiple cloning site in the vector to direct mRNA synthesis to express the protein, or for homologous recombination.
  • the cloning vector can be used as Easy Cloning T-vector (Novoprotein, T003-01B), and the expression vector can be pET21a.
  • a host cell which is transformed with the aforementioned expression vector or cloning vector.
  • the host cell can employ BL21 (DE3) or Rosetta pLysS.
  • a method for preparing the aforementioned fusion protein comprises the steps of:
  • Synthesizing or cloning the DNA sequence of interest constructing a cloning vector containing the DNA sequence of interest, constructing an expression vector containing the DNA sequence of interest, transforming the expression vector containing the DNA sequence of interest into a prokaryotic host cell, and screening for a high-yielding cell highly expressed in the growth medium.
  • the strain is cultured and the highly expressed cell strain is cultured and the fusion protein is expressed, and the fusion protein is purified from the expression product.
  • the expression vector may employ pET21a.
  • the host cell may employ BL21 (DE3) or Rosetta pLysS.
  • a reagent combination comprising the aforementioned fusion protein and other components thereof for use.
  • components that are used correspondingly include the corresponding Buffer and other components used in combination.
  • Other components that are suitable for use may be in the form of a solid, a liquid or a material adsorbed on a special material.
  • An antibody-binding transposome can be obtained by ligating a linker in the first domain of the fusion protein. Further, the antibody binds to a transposome as a dimer.
  • the antibody-binding transposome has the function of cleaving a DNA duplex at a random position and inserting a linker at the cleavage site.
  • the antibody binding transposome can also bind to the Fc portion of the antibody and form a complex with the antibody.
  • an antibody-binding transposome comprising the fusion protein and a linker linked to the first domain of the fusion protein is provided.
  • the antibody binds to a transposome as a dimer.
  • the antibody-binding transposome has the function of cleaving a DNA duplex and inserting a linker at the cleavage site.
  • the antibody binding transposome can also bind to the Fc portion of the antibody and form a complex with the antibody.
  • the use of the antibody-binding transposome to construct a sequencing library is provided.
  • the use of the antibody-binding transposome for studying protein-chromatin interactions is provided.
  • a ninth aspect of the invention there is provided a method of studying protein-chromatin interaction, comprising the steps of:
  • the present invention uniquely found that in the step (3), the antibody-binding transposome will be linked to the antibody via the second domain, and the antibody can specifically bind to the chromatin transcription factor ( On TranscriptionFactor, TF) or histone (Histone), the antibody-binding transposome-antibody-TF-DNA will be joined together to form a complex. Limited by this complex, the effect of the antibody binding to the transposome to cleave DNA will be limited to the location of the DNA where the TF is located, and the DNA at a further position will not be cleaved.
  • TF On TranscriptionFactor
  • Histone histone
  • the position to be cleaved is introduced into the linker, and these linkers are used as a part of the primer for PCR amplification, and a DNA fragment having TF binding position information is obtained. Moreover, these fragments have been connected to the sequencing linker in the PCR, and after the magnetic bead screening, the sequencing library construction is completed, and the next high-throughput sequencing can be directly performed.
  • the above method for studying protein-chromatin interaction of the present invention omits co-immunoprecipitation-elution, reduces the operation steps, and greatly reduces sample loss, and directly constructs a sequencing library, reduces the operation steps, and greatly facilitates subsequent sequencing. Work, so that the requirements for the starting sample are reduced, the loss information is less, and the repeatability and credibility are greatly improved.
  • the present invention has the following beneficial effects:
  • the present invention provides a novel fusion protein which has both a transposon insertion gene sequence function and an Fc segment function in an IgG molecule.
  • the fusion protein can be used to prepare an antibody-binding transposome for experiments on mutant library construction, high-throughput sequencing library construction, immunoassay, IgG purification, and the like.
  • Using the antibody-binding transposome to study protein-chromatin interactions, which is simpler than ChIP-Seq, is more efficient, more economical, has better repeatability, and requires less sample size. The amount of sample DNA required is greatly reduced, and the sample and data lost in the middle are greatly reduced. This is of great significance for the study of protein-DNA interaction.
  • FIG. 1 Electrophoresis pattern of Tn5-ProteinA-1 fusion protein expressed after IPTG induction, wherein Lane A: non-induced crude; Lane B: Induced crude; LaneB1-B4: Induced crude; Lane C: Supernatant of lysate; Lane D : Precipitation of lysate; MK: Molecular weight marker.
  • FIG. 2 Electrophoresis pattern of Tn5-ProteinG-2 fusion protein expressed after IPTG induction, wherein Lane A: non-induced crude; Lane B: Induced crude; LaneB1-B2: Induced crude; Lane C: Supernatant of lysate; Lane D : Precipitation of lysate; MK: Molecular weight marker.
  • FIG. 3 Electrophoresis pattern of Tn10-ProteinA-3 fusion protein expressed after IPTG induction, wherein Lane A: non-induced crude; Lane B: Induced crude; LaneB1-B4: Induced crude; Lane C: Supernatant of lysate; Lane D : Precipitation of lysate; MK: Molecular weight marker.
  • FIG. 4 Electrophoresis pattern of Tn10-ProteinG-4 fusion protein expressed after IPTG induction, wherein Lane A: non-induced crude; Lane B: Induced crude; LaneB1-B4: Induced crude; Lane C: Supernatant of lysate; Lane D : Precipitation of lysate; MK: Molecular weight marker.
  • FIG. 5 Electrophoresis pattern of Tn5-ProteinA-5 fusion protein expressed after IPTG induction, wherein Lane A: non-induced crude; Lane B: Induced crude; LaneB1-B4: Induced crude; Lane C: Supernatant of lysate; Lane D : Precipitation of lysate; MK: Molecular weight marker.
  • FIG. 6 Electronization map of Tn5-ProteinA-1 amplification expression, wherein Lane A: non-induced crude; Lane B: Induced crude; Lane C: Supernatant of lysate; Lane D: Precipitation of lysate; MK: Molecular weight marker.
  • Figure 7 Amplified expression electrophoresis pattern of Tn5-ProteinG-2, wherein Lane A: non-induced crude; Lane B: Induced crude; Lane C: Supernatant of lysate; Lane D: Precipitation of lysate; M: Molecular weight marker.
  • Figure 8 Amplified expression electrophoresis pattern of Tn10-ProteinA-3, wherein Lane A: non-induced crude; Lane B: Induced crude; Lane C: Supernatant of lysate; Lane D: Precipitation of lysate; M: Molecular weight marker.
  • Figure 9 Amplified expression electrophoresis pattern of Tn10-ProteinG-4, wherein Lane A: non-induced crude; Lane B: Induced crude; Lane C: Supernatant of lysate; Lane D: Precipitation of lysate; M: Molecular weight marker.
  • Figure 10 Amplified expression electrophoresis pattern of Tn5-ProteinA-5, wherein Lane A: non-induced crude; Lane B: Induced crude; Lane C: Supernatant of lysate; Lane D: Precipitation of lysate; MK: Molecular weight marker.
  • FIG. 11 Transposase formed by a fusion protein of a transposase-antibody binding protein and a adaptor, which can fragment a double-stranded DNA and add a linker at both ends of the fragmented DNA.
  • Figure 12 Fusion protein 1-5, Tn5 transposome digestion 50 ng human genomic DNA, wherein MK: DNA Marker; 1: Tn5-Protein A-1; 2: Tn5-ProteinG-2; 3: Tn10-ProteinA- 3; 4: Tn10-Protein G-4; 5: Tn5-Protein A-5; 6: Tn5; 7: untreated genomic DNA.
  • MK DNA Marker
  • 1 Tn5-Protein A-1
  • 2 Tn5-ProteinG-2
  • 3 Tn10-ProteinA- 3
  • 4 Tn10-Protein G-4
  • Figure 13 DNA fragments sorted by magnetic beads after PCR amplification, 1-7 is the difference in the amount of magnetic beads added twice, and sorted into DNA of different fragment sizes.
  • Figure 14 Detection of fragment size using an Agilent 2100 high-sensitivity DNA chip, 1: unsorted DNA fragments; 2-6: DNA fragments of different lengths after sorting.
  • Figure 15 The antibody binding protein portion of the fusion protein can bind to the Fc portion of IgG.
  • Figure 16 Standard curve of ProteinA protein.
  • FIG. 17 ChT-Seq method to study the interaction between protein and genomic DNA.
  • the transposase can only cleave adjacent DNA sequences under the restriction of the Transposase-ProteinA/G-IgG-TF complex and introduce a sequencing linker.
  • FIG. 19 Genomic DNA electrophoresis map, 1, 2 is 1*10 6 Hela cells extracted genomic DNA for ChIP-Seq experiment; 3, 4 is 2*10 5 Hela cells extracted genomic DNA, used In the ChT-Seq experiment, it can be seen that the genomic DNA used in ChIP-Seq is more than the DNA used in ChT-Seq.
  • FIG. 20 Electrophoresis map of the library, M is DNA Marker; 1, 2 starts with about 10 ug of genomic DNA, and is subjected to PCR amplification by ChIP-Seq method ultrasonication-immunoprecipitation-complementing-addition of A-plus linker; 3, 4 start about 2 ug of genomic DNA, ChT-Seq method transposome one-step cleavage of the genome and ligation of the linker, the library obtained after PCR amplification. As can be seen from the figure, ChT-Seq obtained more libraries with fewer starting templates, with much less loss in the middle.
  • FIG. 21 Qubit, Nanodrop detection library quality, ordinate is DNA concentration (ng / ul).
  • ChIP-Seq represents a sequencing library constructed using the ChIP-Seq method for the initial 10 ug DNA
  • ChT-Seq represents a sequencing library constructed with the ChT-Seq method for the initial 2 ug DNA
  • blue is the Qubit test result
  • red is the Nanodrop test result.
  • ChT-Seq is able to obtain more sequencing libraries than ChIP-Seq with less initial amount of DNA.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the prior literature, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin ( PMWassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • the fusion protein of the present embodiment includes a first domain having a transposition function and a second domain having a function of binding an antibody Fc segment.
  • the transposition function refers to the function of transposition insertion of a gene sequence.
  • the binding antibody Fc segment function refers to binding to the Fc segment function in an IgG molecule.
  • the first domain and the second domain are connected by a linker, ie, a Linker. Further, the first domain may be a transposase protein and the second domain may be an antibody binding protein.
  • the specific construction scheme of the fusion protein is that the transposase protein and the antibody binding protein are linked by a fragment, that is, the structural formula is (GS) a (GGS) b (GGGS) c (GGGGS) d (where a, b, c, d Linker connections that are all integers greater than or equal to 0).
  • the present invention is directed to a transposase protein, an antibody binding protein, and a ligation fragment (GS) a (GGS) b (GGGS) c (GGGGS) d (where a, b, c, Sequences in which d is an integer greater than or equal to 0 are optimized.
  • This example exemplarily prepared fusion proteins of five differently constructed transposase-antibody binding proteins, named Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, respectively. , Tn5-ProteinA-5.
  • a fusion protein designated Tn5-ProteinA-1 the first domain of which is a Tn5 transposase, the second domain thereof is a S. aureus A protein, and the first domain and the second domain are linked by Fragment Linker1 connection.
  • the amino acid sequence of the Tn5 transposase is as shown in SEQ ID NO. 1, specifically:
  • the nucleotide sequence encoding the Tn5 transposase is as shown in SEQ ID NO. 12, specifically:
  • amino acid sequence of the S. aureus A protein is shown in SEQ ID NO. 3, specifically:
  • S. aureus A protein The nucleotide sequence encoding the S. aureus A protein is shown in SEQ ID NO. 14, specifically:
  • the amino acid sequence of the linker, Linker1 is set forth in SEQ ID NO. 10, specifically GGGGS.
  • the coding sequence of the ligated fragment, Linker1, is set forth in SEQ ID NO. 16, specifically: GGTGGTGGTGGTTCT.
  • amino acid sequence of the fusion protein Tn5-ProteinA-1 is shown in SEQ ID NO. 5, specifically:
  • the coding nucleotide sequence of the fusion protein Tn5-ProteinA-1 is shown in SEQ ID NO. 18, specifically:
  • a fusion protein designated Tn5-ProteinG-2 the first domain of which is a Tn5 transposase, the second domain of which is a Streptococcus G protein, and the first structure and the second domain are linked by a linker Linker1 .
  • the amino acid sequence of the Tn5 transposase is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the Tn5 transposase is set forth in SEQ ID NO.
  • the amino acid sequence of the Streptococcus G protein is as shown in SEQ ID NO. 4, specifically:
  • the nucleotide sequence encoding the Streptococcus G protein is as shown in SEQ ID NO. 15, specifically:
  • the amino acid sequence of the linker, Linker1, is set forth in SEQ ID NO.
  • Linker1 The coding sequence of the ligated fragment, Linker1, is set forth in SEQ ID NO.
  • Tn5-ProteinG-2 fusion protein The amino acid sequence of the Tn5-ProteinG-2 fusion protein is shown in SEQ ID NO. 6, specifically:
  • the nucleotide sequence encoding the Tn5-ProteinG-2 fusion protein is shown in SEQ ID NO. 19, specifically:
  • a fusion protein designated Tn10-ProteinA-3 has a first domain of Tn10 transposase and a second domain of S. aureus A protein, the first domain and the second domain being joined by a linker Linker1.
  • Tn10 transposase amino acid sequence is shown in SEQ ID NO. 2, specifically:
  • the Tn10 transposase encoding nucleotide sequence is SEQ ID NO. 13, specifically:
  • amino acid sequence of the S. aureus A protein is SEQ ID NO.
  • the nucleotide sequence encoding the S. aureus A protein is SEQ ID NO.
  • the amino acid sequence of the linker, Linker1, is set forth in SEQ ID NO.
  • Linker1 The coding sequence of the ligated fragment, Linker1, is set forth in SEQ ID NO.
  • Tn10-Protein A-3 fusion protein The amino acid sequence of the Tn10-Protein A-3 fusion protein is shown in SEQ ID NO. 7, specifically:
  • the coding nucleotide sequence of the Tn10-Protein A-3 fusion protein is shown in SEQ ID NO. 20, specifically:
  • a fusion protein designated Tn10-ProteinG-4 the first domain of which is a Tn10 transposase, the second domain of which is a Streptococcus G protein, and the first domain and the second domain are joined by a linker Linker1.
  • Tn10 transposase amino acid sequence is shown in SEQ ID NO.
  • Tn10 transposase encoding nucleotide sequence is set forth in SEQ ID NO.
  • the amino acid sequence of the Streptococcus G protein is shown in SEQ ID NO.
  • the nucleotide sequence encoding the Streptococcus G protein is shown in SEQ ID NO.
  • the amino acid sequence of the linker, Linker1, is set forth in SEQ ID NO.
  • Linker1 The coding sequence of the ligated fragment, Linker1, is set forth in SEQ ID NO.
  • Tn10-ProteinG-4 fusion protein The amino acid sequence of the Tn10-ProteinG-4 fusion protein is shown in SEQ ID NO. 8, specifically:
  • the nucleotide sequence encoding the Tn10-ProteinG-4 fusion protein is shown in SEQ ID NO. 21, specifically:
  • a fusion protein designated Tn5-ProteinA-5 the first domain of which is a Tn5 transposase, the second domain thereof is a S. aureus A protein, and the first domain and the second domain are linked by Fragment Linker2 connection.
  • the amino acid sequence of the Tn5 transposase is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the Tn5 transposase is set forth in SEQ ID NO.
  • amino acid sequence of the S. aureus A protein is shown in SEQ ID NO.
  • the nucleotide sequence encoding the S. aureus Protein A is set forth in SEQ ID NO.
  • Linker 2 which is Linker 2 is shown in SEQ ID NO. 11, specifically: GGGSGGGGS.
  • linker ie, Linker2
  • SEQ ID NO. 17 The coding sequence of the linker, ie, Linker2, is represented by SEQ ID NO. 17, specifically:
  • the amino acid sequence of the fusion protein Tn5-ProteinA-5 is shown in SEQ ID NO. 9, specifically:
  • the coding nucleotide sequence of the fusion protein Tn5-ProteinA-5 is shown in SEQ ID NO. 22, specifically:
  • the nucleotide sequence encoding the above-mentioned optimized transposase-antibody-binding protein fusion protein was transferred into the expression vector pET21a, and the reaction system was 20 ⁇ L. The following components were added to a 0.2 mL EP tube:
  • the reaction was carried out at 37 degrees for 20 minutes to obtain a recombinant expression vector.
  • the kit used was NR001 from Novoprotein.
  • step 2 1) Add the DNA of interest to the competent cell suspension (i.e., the expression vector of step 1), gently rotate the tube to mix the contents, and let stand in an ice bath for 30 min.
  • the competent cell suspension i.e., the expression vector of step 1
  • the sample is subjected to SDS-PAGE to detect whether the target protein is expressed.
  • Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, and Tn5-ProteinA-5 were successfully expressed.
  • Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, and Tn5-ProteinA-5 were sequenced correctly, which were consistent with expectations.
  • Example 1 The function of the fusion protein of the transposase-antibody binding protein obtained in Example 1 was examined. First, the random disrupted genome of the fusion protein was verified, and the tag sequence was inserted, and the sequencing library function was constructed after PCR.
  • the principle and schematic diagram of the random insertion of the fusion protein is shown in Figure 11.
  • the transposome formed by the fusion protein and the linker can fragment the double-stranded DNA and add a linker at both ends of the fragmented DNA.
  • fusion protein 1-5 ie Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, Tn5-ProteinA-5) and Tn5 transposase (positive control), respectively Processing human genomic DNA, it is expected that short DNAs of different lengths and fragments will be fragmented, and linker sequences will be ligated at both ends of these fragmented DNA.
  • PCR can be used to amplify fragmentation using the linker sequence as a primer.
  • the DNA which thus constructed the sequenced library, also demonstrated the ability of the fusion protein to have the same random insertion of integrated DNA as the Tn5 transposase.
  • Fusion protein 1-5 (ie Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, Tn5-ProteinA-5) and Tn5 were respectively dissolved in a stock solution (50 mM HPCRES-KOH) pH 7.2, 0.1 M NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, 10% glycerol), quantified by BCA method, and the molar concentration was calculated.
  • the reaction system is configured as follows:
  • reaction conditions were: 30 ° C, 1 hour, -20 ° C preservation, respectively, to prepare fusion protein 1-5 (ie Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, Tn5 -ProteinA-5) Transposable body and Tn5 transposome.
  • fusion protein 1-5 ie Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, Tn5 -ProteinA-5) Transposable body and Tn5 transposome.
  • the 5* reaction buffer 50 mM TAPS-NaOH pH 8.5, 25 mM MgCl 2 . was thawed at room temperature, mixed upside down and set aside. The following 20 ul reaction system was placed in a sterile PCR tube, and a negative control without a transposome was set. Positive control added to the Tn5 transposome:
  • the components were thoroughly mixed by gently pipetting with a pipette; the PCR tube was placed in a PCR machine and reacted at 55 ° C for 10 min. The PCR tube was taken out from the PCR instrument, and several fragmented products were taken up and electrophoresed together with the control to observe the fragmentation effect.
  • the electrophoresis pattern is shown in Fig. 12, and the fusion protein 1-5 (i.e., Tn5-Protein A-1, Tn5-ProteinG- 2.
  • Tn10-ProteinA-3, Tn10-ProteinG-4, Tn5-ProteinA-5) successfully fragmented the human genome.
  • reaction tube is briefly centrifuged and placed in a magnetic frame to completely separate the magnetic beads from the liquid (about 5 minutes, after the solution is clarified), and the supernatant is carefully removed, and the reaction tube is kept on the magnetic frame;
  • reaction tube is briefly centrifuged and placed in a magnetic stand to completely separate the magnetic beads from the liquid (about 5 min, after the solution is clarified), and 14 ul of the supernatant is carefully pipetted into a new sterile PCR tube for PCR enrichment step;
  • the obtained PCR product was subjected to length sorting by magnetic bead sorting.
  • the magnetic beads are equilibrated to room temperature and shaken well, and the PCR system must be supplemented with 50 ⁇ l of sterile distilled water to avoid evaporation of the sample during the PCR process, resulting in the sorted fragments being inconsistent with the expected length.
  • the sorting results are shown in Figure 13.
  • the fragmented DNA is sorted by different ratios of magnetic beads, leaving the DNA of the desired length, MK:DNA Marker, 1-6: different ratios of magnetic beads after sorting DNA fragment, 7: Negative control without sorting.
  • the product was assayed for fragment size using an Agilent 2100 high-sensitivity DNA chip (see Figure 14). Since the primers used were specific primers designed for the adaptor Adaptor 1, 2, it was found that the transposome successfully broke the target DNA and inserted the Adaptor linker sequence at the same time.
  • the library was sequenced by Illumina Hiseq XTM system, the sequencing strategy was PE150, the sequencing result data is shown in Table 6, and the sequencing data was compared with the reference genome sequence as follows:
  • the effective data can be more than 95%, and the Q20 and Q30 data are over 90. %, about 98% coverage can be achieved with a sequencing depth of about 13X.
  • the library construction process is fast, easy to operate, and requires a small amount of sample.
  • Example 2 The function of the fusion protein successfully combined with IgG obtained in Example 1 was examined.
  • FIG. 1 A schematic diagram of the binding of the fusion protein to IgG, as shown in FIG.
  • the ProteinA/G portion of the fusion protein can bind to the Fc portion of the IgG.
  • the plate was sealed with a cover plate and incubated at 37 ° C for 30 minutes.
  • Washing the plate Remove the cover film, discard the liquid in the plate hole, add 260 ul of 1 ⁇ washing solution to each well, soak for 30 seconds, discard the washing solution, and repeat the washing 4 times.
  • the color reaction time is affected by temperature.
  • the ideal reaction temperature is 20-25 ° C. When the temperature is low, the reaction time should be extended.
  • the OD value is plotted on the ordinate
  • the ProteinA standard protein concentration is plotted on the abscissa
  • a standard curve is drawn.
  • the content of the fusion protein in the sample is calculated according to the standard curve and converted into the binding efficiency of the fusion protein.
  • the fusion protein concentration was 1.8 ng/ml and the standard ProteinA concentration was 0.2 ng/ml, the molar concentration of the fusion protein and the standard was consistent. From the standard curve of Fig. 16, the standard ProteinA concentration was 0.2 ng/ml, and the OD450 was 0.318, which was similar to the OD450 value of the fusion protein. Elisa results showed that the fusion proteins Tn5-ProteinA-1, Tn5-ProteinG-2, Tn10-ProteinA-3, Tn10-ProteinG-4, Tn5-ProteinA-5 could bind to IgG and the binding ability was similar to that of the standard ProteinA.
  • ChIP-Seq The principle of the traditional ChIP-Seq method is to firstly enrich the DNA fragments of the target protein by chromatin immunoprecipitation (ChIP), and then purify and construct the fragments, and then perform high-throughput sequencing.
  • ChIP chromatin immunoprecipitation
  • step (2) ultrasonic interrupting inconvenience control, easy to interrupt excessive or insufficient, poor repeatability
  • step (3) immunoprecipitation experiments are not only complicated and cumbersome, but also A large number of samples will be lost
  • step (5) has a lot of steps in the construction of the library, and will also lose sample and DNA information; thus ChIP-Seq requires more starting samples and less repeatability of the experiment.
  • transposase-antibody-binding fusion protein of the present invention due to its unique function and characteristics, has been applied to protein-DNA interaction research, creating a new method ChT-Seq (Chromatin-Transposase-Sequencing). .
  • ChT-Seq Chroatin-Transposase-Sequencing
  • transposase-antibody binding protein transposome (2) eluting formaldehyde, adding a specific antibody of the target protein, a transposase-antibody binding protein transposome; the transposome will combine with the antibody and the target protein to form a complex, and the transposase functions as a transposition function and will be cut.
  • a sequence adjacent to the DNA site to which the target protein is ligated as shown in Figure 18;
  • ChT-Seq omits the co-immunoprecipitation-elution, reduces the operation steps, greatly reduces the sample loss, and directly connects the linker, which greatly simplifies the library construction work and facilitates sequencing.
  • the requirements for the initial sample are reduced, the loss information is less, and the repeatability and credibility are greatly improved.
  • Collect cells Hela cells, about 2 * 10 5 , traditional Chip-Seq about 1 * 10 6-7 ), add formaldehyde to a final concentration of 1%, gently shake and mix, react at room temperature for 10 min;
  • cell lysis according to the amount of cells added to the pre-cooled cell lysate and 5ul protease inhibitor, resuspended cells, can be divided into 300 ⁇ 400ul, take 5ul cell lysate running electrophoresis to observe the extracted genome (see Figure 19), Excess samples can be stored at -80 ° C;
  • Blank control group 100 ul of template sample DNA was added, 4 ul of 5 M NaCl was added, and treated at 65 ° C for 2 h to be cross-linked, which was used as a blank control. After extracting a part of phenol/chloroform, the breaking effect of step 6 was identified by electrophoresis.
  • Negative control group 100 ul of template sample DNA, 900 ul dilution buffer (containing 4.5 ul protease inhibitor), non-specific Mouse IgG 1 ug as antibody, incubate at 2 ° C for 2 h with gentle shaking;
  • experimental group template sample DNA taken 200ul, add 900ul dilution buffer (containing 4.5ul protease inhibitor), add the target protein specific antibody, 4 ° C, gently shake for 2h;
  • the experimental group and the negative control group were added to the Transposase-Protein A/G transposome with Adaptor1 (transposable construct was carried out according to the transposase-antibody binding protein function detection part of Example 2), and incubated at 4 °C for 10 min with gentle shaking. Adding MgCl 2 to a final concentration of Mg 2+ of 5 mM, 55 ° C for 10 min;
  • the experiment was carried out using the traditional ChIP-Seq method.
  • the number of Hela cells was 1*10 6
  • the DNA extracted after cell lysis was about 10 ug
  • the DNA obtained after chromatin immunoprecipitation was about 10 ng.
  • After filling up add A at the end, connect Y-type linker, and carry out PCR amplification. 20 ng of library can be obtained for sequencing on the machine.
  • the experiment was carried out by ChT-Seq method.
  • the number of Hela cells was about 2*10 5 , and the DNA extracted after cell lysis was about 2 ug. After transposase digestion and ligation, a library of 100 ng was obtained. Sequencing. It can be seen that the ChT-Seq method uses fewer starting cells, and the obtained sequencing library has more sample size than ChIP-Seq, with less loss in the middle, and the repeated effect is consistent and the repeatability is high.

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Abstract

La présente invention se rapporte aux domaines de la biologie moléculaire, de la génomique et de la biotechnologie, et concerne une protéine de fusion d'une protéine se liant au transposase-anticorps, ainsi qu'une préparation et une utilisation associées. La protéine de fusion selon la présente invention comprend une partie transposase, une partie peptide lieur et une partie capable de se lier à un segment Fc d'un anticorps, et présente simultanément une fonction de transposition et une fonction de liaison du segment Fc de l'anticorps. La présente invention concerne en outre un nouveau procédé d'étude de l'interaction in vivo protéine-ADN de chromatine : un procédé de séquençage-transposition de chromatine (ChT-Seq).
PCT/CN2018/084711 2018-03-27 2018-04-27 Protéine de fusion d'une protéine se liant au transposase-anticorps, préparation et utilisation associées Ceased WO2019184044A1 (fr)

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