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WO2024145824A1 - Mutant d'adn polymérase, sa préparation et son utilisation - Google Patents

Mutant d'adn polymérase, sa préparation et son utilisation Download PDF

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Publication number
WO2024145824A1
WO2024145824A1 PCT/CN2023/070450 CN2023070450W WO2024145824A1 WO 2024145824 A1 WO2024145824 A1 WO 2024145824A1 CN 2023070450 W CN2023070450 W CN 2023070450W WO 2024145824 A1 WO2024145824 A1 WO 2024145824A1
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Prior art keywords
dna polymerase
residue
mutant
terminus
replacing
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PCT/CN2023/070450
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English (en)
Chinese (zh)
Inventor
兰山
王佑富
徐玉群
周娇娇
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Zybio Inc
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Zybio Inc
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Priority to PCT/CN2023/070450 priority Critical patent/WO2024145824A1/fr
Priority to CN202380016585.3A priority patent/CN118556118A/zh
Publication of WO2024145824A1 publication Critical patent/WO2024145824A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)

Definitions

  • the present invention relates to the fields of biotechnology, nucleic acid amplification and enzyme engineering, and in particular to a DNA polymerase mutant, preparation and application thereof.
  • DNA polymerase is an important enzyme that plays an important role in cell DNA replication. It uses DNA as a replication template and replicates DNA from the 5' end to the 3' end. The main activity of DNA polymerase is to catalyze the synthesis of DNA (in the presence of templates, primers, dNTPs, etc.) and its complementary activities.
  • DNA polymerase is an important tool for ensuring nucleotide incorporation, SNPs detection or more extensive sequencing, such as sequencing by synthesis.
  • the substrate is a non-natural nucleotide, and most natural polymerases have low ability to process or incorporate non-natural nucleotides and cannot be used in sequencing.
  • TaqDNA polymerase also has template-independent activity, which can add a single nucleotide tail to the 3' end of each strand of the PCR double-stranded product, so that the PCR product can have a 3' protruding single A nucleotide tail; on the other hand, when only dTTP exists, it can add a single T nucleotide tail to the 3' end of the blunt-ended plasmid, generating a 3' protruding single T nucleotide tail.
  • the T-A cloning method of PCR products can be realized.
  • a DNA polymerase mutant obtained by replacing the aspartic acid residue at position 540 from the N-terminus of 9°N DNA polymerase with a serine residue;
  • a DNA polymerase mutant (ZYC24) was obtained by replacing the threonine residue at position 667 from the N-terminus of 9°N DNA polymerase with a glutamine residue.
  • the present invention provides a biological material related to the above-mentioned DNA polymerase mutant, wherein the biological material is any one of the following C1) to C5):
  • the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.
  • the expression cassette containing the nucleic acid molecule encoding the above DNA polymerase mutant refers to a DNA capable of expressing the above DNA polymerase mutant in a host cell, and the DNA may include not only a promoter for initiating transcription of the above DNA polymerase mutant gene, but also a terminator for terminating transcription of the above DNA polymerase mutant gene. Furthermore, the expression cassette may also include an enhancer sequence.
  • Existing expression vectors can be used to construct a recombinant vector containing the above-mentioned DNA polymerase mutant gene expression cassette.
  • the vector may be a plasmid, a cosmid, a phage or a virus vector.
  • the plasmid may be pET-22(b).
  • the recombinant vector may be a recombinant vector obtained by inserting the nucleic acid molecule encoding the above-mentioned DNA polymerase mutant into the multiple cloning site of the vector.
  • the microorganism can be yeast, bacteria, algae or fungi.
  • the bacteria can be Escherichia coli, such as Escherichia coli BL21 (DE3).
  • the recombinant microorganism is specifically a vector obtained by introducing the recombinant vector into Escherichia coli BL21 (DE3).
  • the transgenic cell line may or may not include propagation materials.
  • the encoding gene of the above-mentioned DNA polymerase mutant can specifically be the nucleic acid molecule described in C1) above.
  • the biological cell can be a microorganism, an animal cell or a plant cell.
  • the microorganism can be specifically Escherichia coli, such as Escherichia coli BL21 (DE3).
  • the expression of the gene encoding the DNA polymerase mutant may specifically be achieved by culturing the recombinant cell to obtain a culture, and expressing the gene encoding the DNA polymerase mutant in the recombinant cell.
  • the method may further include purifying the DNA polymerase mutant from the culture.
  • Purification of the DNA polymerase mutant from the culture may be carried out by affinity chromatography and ion exchange chromatography.
  • the present invention provides any of the following applications:
  • the DNA polymerase can use nucleotides or nucleotide analogs as substrates.
  • the nucleotide analogs are substances obtained by modifying nucleotides.
  • the nucleotide analogs can specifically be substances obtained by modifying nucleotides with fluorescent groups.
  • the coding gene of the above-mentioned DNA polymerase mutant can be obtained by mutating one or several nucleotides in the DNA sequence encoding 9°N DNA polymerase, and/or connecting the coding sequence of the tag shown in Table 1 in the middle and/or 5′ end and/or 3′ end of its sequence.
  • the present invention also provides a kit for implementing a non-natural nucleotide incorporation reaction, wherein the modified nucleotide is modified at the 3' sugar hydroxyl group so that the substituent is larger in size than the naturally occurring 3' hydroxyl group, and the modified nucleotide at the 5' phosphate group of the template so that the substituent is larger in size than the naturally occurring 5' phosphate group, and the performance of the enzyme is described by detecting the speed at which the separated protein incorporates the non-natural nucleotide by FRET signal at a specified time.
  • Preferred examples are that the 411th serine mutates to alanine, the 411th serine mutates to leucine, the 457th leucine mutates to threonine, the 461st glutamine mutates to alanine, and the 676th threonine mutates to glutamine.
  • Mutant refers to a gene that has at least one base (nucleotide) change, deletion or insertion relative to the natural or wild-type gene.
  • the mutation (change, deletion and/or insertion of one or more nucleotides) may be in the coding region of the gene or in the intron, 3'UTR, 5'UTR or promoter region.
  • a mutant gene may be a gene that has an insertion in the promoter region that can increase or decrease gene expression; it may be a gene with a deletion that results in the production of a non-functional protein, a truncated protein, a dominant negative protein or no protein; or, it may be a gene with one or more point mutations that results in a change in the amino acid of the encoded protein or results in abnormal splicing of the gene transcript.
  • Wild-type refers to the form found in nature.
  • a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence found in an organism that has not been intentionally modified by human manipulation.
  • the mutant 9°N DNA polymerase has higher polymerization activity than the wild-type polymerase.
  • the mutant catalyzes the generation of more substrates in the same amount of time.
  • Figure 1 Schematic diagram of the 9°N DNA polymerase structure and mutation site. Site-directed mutagenesis is performed based on the structural diagram.
  • Figure 2 SDS-PAGE electrophoresis of 9°N DNA polymerase after purification, the purity can reach more than 90%.
  • Figure 3 FRET detection results of ZYC4, showing that the initial reaction rate V0 (the amount of template added substrate per unit time) is 1.37 times that of the wild type.
  • Figure 4 FRET detection results of ZYC5, showing that the initial reaction rate V0 (the amount of substrate added to the template per unit time) is 1.33 times that of the wild type.
  • Figure 5 FRET detection results of ZYC11, showing that the initial reaction rate V0 (the amount of template added substrate per unit time) is 1.35 times that of the wild type.
  • the isolated protein is SEQ NO:1 and a mutant having one of the following: R406A/L, S407I/K, S411A/L, I412L, L457T/A, R460G, Q461A, K464T, Y481A, Q483L/E, R484A/L, K487R, I488A, S492D, Y494R, D540S, T541A, T667Q.
  • Example 1 Preparation of wild-type 9°N DNA polymerase SEQ NO: 1 (9N-WT) and mutant 9°N DNA polymerase proteins
  • the plasmid containing the 9N-WT gene fragment subcloned into the pET-22(b) vector was transformed into Transetta (DE3) (Beijing Quanshijin Biotechnology Co., Ltd.) Escherichia coli to obtain recombinant protein engineering bacteria, which were inoculated into LB medium containing ampicillin and cultured at 37°C and 200rpm for 3 to 4 hours for activation.
  • the activated bacterial solution was added to a new LB medium containing ampicillin at a ratio of 1:100, and cultured at 37°C with shaking until OD600nm reached 0.8-1.1. After cooling in an ice water bath, IPTG was added at a final concentration of 0.5mM and cultured overnight at 25°C with shaking. The induced bacterial solution was centrifuged at 8000rpm for 10 minutes to collect the bacteria.
  • the buffer used for protein purification is as follows:
  • Lysis buffer 50 mM MOPS, 500 mM NaCl, 5% Glycerol, pH 7.6;
  • a buffer 50mM MOPS, 500mM NaCl, 20mM Imidazole, 5% Glycerol, pH 7.6;
  • C buffer 50 mM MOPS, 50 mM NaCl, 5% Glycerol, pH 7.0;
  • D Buffer 50mM MOPS, 50mM NaCl, 500mM Imidazole, 5% Glycerol, pH 7.0;
  • E Buffer 50 mM MOPS, 5% Glycerol, pH 7.0;
  • F Buffer 50mM MOPS, 1M Nacl, 5% Glycerol, pH 7.0;
  • Dialysis buffer 20 mM Tris-HCl, 200 mM KCl, 0.2 mM EDTA, pH 7.4.
  • Lysis buffer was added at a ratio of 1:10 for bacterial weight (g)/buffer volume (ml) to resuspend the bacteria; PMSF was added to a final concentration of 1 mM.
  • the sample was added to a high-pressure homogenizer (ATS), the pressure was raised to 700-800 MPa, and lysis was performed at 4°C for 2-3 cycles; the lysed bacterial solution was ultracentrifuged at 4°C and 18,000 rpm for 40 min; the supernatant obtained after centrifugation was heated in a 75°C water bath for 30 min, and stirred regularly during the period to heat and mix; the crude enzyme solution obtained above was centrifuged at 16,000 rpm and 4°C for 30 min, and vacuum filtered with a 0.45 ⁇ m filter membrane (Merck Millipore). The obtained sample was used for subsequent experiments.
  • ATS high-pressure homogenizer
  • Affinity column purification was performed using a purifier (Biorad NGC Quest 100). The filtrate was loaded onto a pre-equilibrated Histrap HP column (Cat. No. 17-5248-02, cytiva), and the column was equilibrated with the Lysis buffer for 10 CV, with a retention time of 2.5 min; after all samples were loaded onto the column, the column was rinsed with Lysis buffer until the baseline of UV absorption was balanced; the column was rinsed with B buffer for 20 CV, and then with C buffer for 10 CV, and finally the target protein was eluted with D buffer until the components with a peak A280nm greater than 400 mAU were collected for the next step of purification.
  • a purifier Biorad NGC Quest 100
  • the sample eluted from the affinity column purification was diluted 5 times with E Buffer and then loaded onto a Hitrap Q HP column (Cat. No. 17-1154-01, cytiva) pre-equilibrated with E Buffer + 2% F Buffer, and the flow-through was collected;
  • E Buffer + 2% F Buffer to pre-equilibrate Hitrap SP HP (17-1152-01) column, load the above flow-through sample onto the column, rinse the column with E Buffer + 2% F Buffer for 5CV, and then use 2-50% QB Buffer for gradient elution. Start collecting when A280nm is greater than 100mAU, and discard when it is lower than 100mAU. Collect 5ml in each tube;
  • the protein eluted from the SP column was placed in a 10 kDa dialysis bag (Cat. No. 132576, Spectrum) and dialyzed into Dialysis buffer overnight.
  • the dialyzed sample was first measured for concentration using an ELISA reader (CLARIOstar Plus, BMG), and then Triton X-100 and glycerol were added to make the final concentrations 0.1% and 50%, respectively, and stored at -80°C.
  • the protein purity was determined according to the SDS-PAGE electrophoresis gel image ( Figure 2).
  • the purity of the purified wild-type 9N-WT and ZYC1-ZYC24 proteins was more than 90%, and the size of the target protein was about 92 kd.
  • Table 1 shows the mutation position and mutation information of 9°N DNA polymerase single point mutant (compared with SEQ NO: 1).
  • Primer 1 (5’-3’) CCGAGTGTCGGGACGGTGACCCAAGCTGCACCAG
  • Primer 1 and Primer 2 are reverse complementary sequences, wherein cy5 is connected to the 5' end of Primer 1 and Primer 2, and after annealing, they are complementary and matched to form a template and primer mixture for enzyme activity determination.
  • reaction was carried out in a PCR instrument.
  • the reaction system and reaction procedure were as follows, wherein 5X Anneal buffer was purchased from Solebao (Beijing).
  • the protein activity detection reaction system is shown in Table 2.
  • the above reaction was carried out in a microplate reader (CLARIOstarPlus BMG) at 42° for 40 min, and the FRET Cy5 (excitation 530 nm/emission 676 nm) signal was detected.
  • the activity of 150 pmol of non-natural base dATP-Cy3-N3 added per unit time was defined as 1 U.
  • the polymerization activity of the preferred exemplified 9°N DNA polymerase mutant is higher than that of the wild type, and the results are shown in Table 3.
  • the amino acid sequence of the ZYC5 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to alanine;
  • the amino acid sequence of the ZYC6 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to leucine;
  • the amino acid sequence of the ZYC8 mutant is that the leucine at position 457 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to threonine;
  • the amino acid sequence of the ZYC24 mutant is that the threonine at position 667 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to glutamine;
  • the speed of the preferred 9°N DNA polymerase mutant polymerizing the substrate (dATP-cy3-N3) in the initial time period of the reaction is represented by V 0.
  • the initial speed is calculated using the first 4 minutes of the reaction, and the speed of adding dATP-cy3-N3 by the wild-type 9°N DNA polymerase is 1.
  • the reaction speed of the 9°N DNA polymerase mutant is represented by its multiple relationship, see Table 4 and Figures 3-5.
  • the amino acid sequence of the ZYC5 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to alanine;
  • the amino acid sequence of the ZYC6 mutant is that the serine at position 411 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to leucine;
  • the amino acid sequence of the ZYC11 mutant is that the glutamine at position 461 of the amino acid sequence shown in SEQ ID NO: 1 is mutated to alanine;

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Abstract

La présente invention concerne un mutant d'ADN polymérase, sa préparation et son utilisation. La séquence d'acides aminés de l'ADN polymérase est telle que représentée dans SEQ ID NO : 1 ; et au moins l'un de 18 résidus d'acides aminés en position 406, position 407, position 411, position 412, position 457, position 460, position 461, position 464, position 481, position 483, position 484, position 487, position 488, position 492, position 494, position 540, position 541 et position 667 d'une séquence d'acides aminés de SEQ ID NO : 1 est muté pour obtenir une protéine mutante à activité ADN polymérase. Par comparaison avec la polymérase de type sauvage, l'ADN polymérase mutante 9°N présente une activité de polymérisation plus élevée. Le mutant catalyse la production de davantage de substrats dans le même laps de temps.
PCT/CN2023/070450 2023-01-04 2023-01-04 Mutant d'adn polymérase, sa préparation et son utilisation Ceased WO2024145824A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/CN2023/070450 WO2024145824A1 (fr) 2023-01-04 2023-01-04 Mutant d'adn polymérase, sa préparation et son utilisation
CN202380016585.3A CN118556118A (zh) 2023-01-04 2023-01-04 一种dna聚合酶突变体、制备及其应用

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070048748A1 (en) * 2004-09-24 2007-03-01 Li-Cor, Inc. Mutant polymerases for sequencing and genotyping
US20160032377A1 (en) * 2013-03-14 2016-02-04 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
CN108018270A (zh) * 2016-11-01 2018-05-11 Pgi股份有限公司 用以提升核苷酸类似物并入的重组dna聚合酶
CN108795900A (zh) * 2017-04-27 2018-11-13 深圳华大智造科技有限公司 Dna聚合酶及其制备方法
CN111349623A (zh) * 2018-12-24 2020-06-30 深圳华大生命科学研究院 9°n dna聚合酶突变体
CN112673098A (zh) * 2018-10-31 2021-04-16 亿明达股份有限公司 聚合酶、组合物及使用方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070048748A1 (en) * 2004-09-24 2007-03-01 Li-Cor, Inc. Mutant polymerases for sequencing and genotyping
US20160032377A1 (en) * 2013-03-14 2016-02-04 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues
CN108018270A (zh) * 2016-11-01 2018-05-11 Pgi股份有限公司 用以提升核苷酸类似物并入的重组dna聚合酶
CN108795900A (zh) * 2017-04-27 2018-11-13 深圳华大智造科技有限公司 Dna聚合酶及其制备方法
CN112673098A (zh) * 2018-10-31 2021-04-16 亿明达股份有限公司 聚合酶、组合物及使用方法
CN111349623A (zh) * 2018-12-24 2020-06-30 深圳华大生命科学研究院 9°n dna聚合酶突变体

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