WO2018126470A1 - 重组dna聚合酶 - Google Patents

重组dna聚合酶 Download PDF

Info

Publication number
WO2018126470A1
WO2018126470A1 PCT/CN2017/070609 CN2017070609W WO2018126470A1 WO 2018126470 A1 WO2018126470 A1 WO 2018126470A1 CN 2017070609 W CN2017070609 W CN 2017070609W WO 2018126470 A1 WO2018126470 A1 WO 2018126470A1
Authority
WO
WIPO (PCT)
Prior art keywords
methionine
dna polymerase
protein
amino acid
mutated
Prior art date
Application number
PCT/CN2017/070609
Other languages
English (en)
French (fr)
Inventor
王林
刘芬
董宇亮
章文蔚
徐崇钧
斯内扎娜.德马纳克
Original Assignee
深圳华大智造科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳华大智造科技有限公司 filed Critical 深圳华大智造科技有限公司
Priority to EP17889883.9A priority Critical patent/EP3613851A4/en
Priority to PCT/CN2017/070609 priority patent/WO2018126470A1/zh
Priority to CN201780069442.3A priority patent/CN109937252B/zh
Publication of WO2018126470A1 publication Critical patent/WO2018126470A1/zh
Priority to US16/505,121 priority patent/US10883091B2/en

Links

Images

Classifications

    • 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)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • 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)
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/22—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a Strep-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag

Definitions

  • the present invention relates to the field of biotechnology, and in particular to a recombinant DNA polymerase.
  • DNA polymerase The rapid and accurate replication of DNA in biological chromosomes by DNA polymerase is important for maintaining the stability of genetic material in living organisms.
  • DNA polymerases are divided into three families, called A, B and C, respectively.
  • a and B polymerases are similar in structure, the motifs of different families of polymerases are significantly different, and the recognition mechanism of nucleotides and their analogues is correspondingly represented.
  • DNA polymerase is at the core of molecular biology technology, especially in molecular cloning and PCR, site-directed mutagenesis and DNA sequencing. In these molecular biology techniques, DNA polymerase recognizes different nucleotides or nucleotide analogs and their complementarity with the template plays a key role.
  • DNA polymerases that are capable of accurately adding nucleotides or nucleotide analogs.
  • SBS sequencing-by-synthesis
  • the addition of fluorescently labeled nucleotides can help identify template DNA bases, giving DNA sequence information.
  • SBS sequencing methods have been favored for their high throughput and low price.
  • the addition and detection of artificially modified nucleotides or nucleotide analogs, such as DNA polymerases with fluorescently labeled reversible terminators plays a key role.
  • the protein provided by the present invention is any one of the following A)-C):
  • the protein shown in A) is characterized in that amino acid residues 408, 409 and 485 in the amino acid sequence of the wild-type KOD-type DNA polymerase are modified, and in the amino acid sequence of the wild-type KOD-type DNA polymerase, 53, 59, Amino acid residues of at least one of the 13 positions of 199, 243, 526, 558, 613, 641, 671, 673, 674, 692 and 709 are modified to obtain DNA polymerization Enzymatically active protein;
  • the protein shown in B) is the removal of 1-29 amino acids from the terminal (including the 29th amino acid) at the C-terminus of the amino acid sequence of the protein represented by A), retaining the remaining amino acid residues and having DNA polymerase activity by A) Derived protein;
  • the protein shown by C) is a protein derived from A) or B) having a tag sequence at the end of the amino acid sequence of the protein represented by A) or B) and having DNA polymerase activity.
  • the protein shown in A) is characterized in that amino acid residues 408, 409 and 485 in the amino acid sequence of the wild-type KOD-type DNA polymerase are modified, and in the amino acid sequence of the wild-type KOD-type DNA polymerase, 53, 59, The amino acid residues of at least 2 of the 13 positions of 199, 243, 526, 558, 613, 641, 671, 673, 674, 692 and 709 were modified to obtain a protein having DNA polymerase activity.
  • the protein shown in A) is characterized in that amino acid residues 408, 409 and 485 in the amino acid sequence of the wild-type KOD-type DNA polymerase are modified, and in the amino acid sequence of the wild-type KOD-type DNA polymerase, 53, 59, The amino acid residues of at least 3 of the 13 positions of 199, 243, 526, 558, 613, 641, 671, 673, 674, 692, and 709 are modified to obtain a protein having DNA polymerase activity.
  • the modification is an amino acid substitution.
  • the leucine at position 408 is replaced by isoleucine, tyrosine, valine, valine, cysteine, serine, alanine or methionine;
  • amino acid residues of at least one of the 13 positions of positions 53, 59, 199, 243, 526, 558, 613, 641, 671, 673, 674, 692, and 709 are replaced with methionine or phenylalanine or Alanine.
  • the replacement of the amino acid residue of at least one of the 13 positions of 692 and 709 with methionine or phenylalanine or alanine is as follows:
  • the lysine at position 53 is replaced with methionine
  • the histidine at position 59 is substituted with phenylalanine
  • the lysine at position 199 is replaced by methionine
  • the lysine at position 558 is replaced by methionine
  • the lysine at position 671 is replaced by methionine
  • the lysine at position 692 is replaced by methionine
  • the arginine at position 709 was replaced with methionine.
  • the wild type KOD type DNA polymerase amino acid sequence is sequence 1.
  • the protein has a lower adsorption capacity for the biochip than the wild-type KOD-type DNA polymerase.
  • the protein has a lower adsorption capacity to the biochip than the wild-type KOD-type DNA polymerase, and the protein is less likely to adsorb on the surface of the biochip and/or is easier to compare with the wild-type KOD-type DNA polymerase.
  • the surface of the biochip is eluted.
  • DNA molecules encoding the above proteins are also within the scope of the invention.
  • an expression cassette, recombinant vector, recombinant or transgenic cell line containing the DNA molecule is also within the scope of the invention.
  • the DNA polymerase has a lower adsorption capacity to the biochip than the wild Type KOD-type DNA polymerase;
  • the DNA polymerase has a lower adsorption capacity for the biochip than the wild-type KOD-type DNA polymerase, and the DNA polymerase is not easily adsorbed to the biochip compared to the wild-type KOD-type DNA polymerase. Surface and/or easy to elute from the surface of the biochip.
  • the product is a kit.
  • the substrate of the protein as a DNA polymerase is a nucleotide or a nucleotide analog
  • the protein is a single-stranded DNA as a template for a DNA polymerase
  • the substrate of the DNA polymerase is a nucleotide or nucleotide analog.
  • the template of the DNA polymerase is single-stranded DNA.
  • the DNA polymerase is characterized in that the polymerization activity is high when the single-stranded DNA is used as a template and/or the adsorption force to the biochip is low.
  • the polymerization activity is high when the single-stranded DNA is used as a template, and the polymerization activity of the protein is higher than that of the KOD-type DNA polymerase;
  • the polymerization reaction is carried out on a biochip.
  • biochips are all biochips commonly used in the art.
  • the product is a kit.
  • the above protein is a KOD type DNA polymerase mutant protein, specifically as follows:
  • KOD-type DNA polymerase point mutants Ue1A, Ue1B, Ue1C, Ue1D, Ue1E, Ue2A, Ue2B, Ue2C, Ue2D, Ue2E, Ue3A, Ue3B, Ue3C, Ue3D, Ue3E, Ue4A, Ue4B, Ue4C, Ue1A5, Ue1A6, Ue1B5 , Ue1B6, Ue1C5, Ue1C6, Ue1D5, Ue1D6, Ue1E5, Ue1E6, Ue2A5, Ue2A6, Ue2B5, Ue2B6, Ue2C5, Ue2C6, Ue2D5, Ue2D6, Ue2E5, Ue2E6, Ue3A5, Ue3A6, Ue3B5, Ue3B6, Ue3C5, Ue3C6, Ue3D5, Ue3D6 , Ue3E5, Ue3E
  • a C-terminally truncated KOD-type DNA polymerase mutant which is a segment in which the above KOD-type DNA polymerase point mutant is obtained by removing 29 amino acids from the C-terminus and remaining amino acid residues.
  • the invention is based on the dynamic simulation and statistical inference of the three-dimensional structure and sequence information of the existing and artificially modified DNA polymerase in nature, and improves the polymerization through semi-rational design, enzyme variant library construction and high-throughput screening.
  • the catalytic and physicochemical properties of the enzyme found that the wild-type partial site of the mutant KOD-type DNA polymerase can achieve DNA polymerase mutants which are not easily adsorbed on the surface of the chip and are easily eluted from the surface of the chip. These mutants are suitable for attachment to the surface. DNA polymerase working on the surface of the chip for DNA sequencing.
  • Fig. 1 shows the results of electrophoresis detection of purified wild-type KOD-type DNA polymerase.
  • Figure 2 is a graph showing the Michaelis kinetics of single-stranded DNA as a substrate for KOD-type DNA polymerase mutants Ue1E, Ue1E5, Ue1E6 and wild-type KOD polymerase KOD-WT.
  • Figure 3 is a graph showing the chromatogram of the chip reaction of the KOD-type DNA polymerase mutant Ue1E5 and the wild-type KOD polymerase chip activity test.
  • Figure 4 is a graph of the chip reaction highlights of the KOD-type DNA polymerase mutant Ue1E5 and the control Therminator chip elution test.
  • the present invention is a wild type KOD DNA polymerase and mutants of using DNA 2.0 Electra TM Cloning Reagents Kit Kit expression vector was constructed, using the His-tag affinity purification by column Ni.
  • amino acid sequence of the wild type KOD type DNA polymerase is the sequence 1 in the sequence listing, and the nucleotide sequence encoding the gene phaP is the sequence 2 in the sequence listing.
  • the nucleotide sequence of the wild-type KOD-type DNA polymerase encoding gene fused with the His tag is a sequence obtained by ligating 6 His tag codons at the 3' end of the sequence 2.
  • the amino acid sequence of the wild type KOD-type DNA polymerase fusion protein is obtained by linking the C-terminus of the amino acid shown in SEQ ID NO: 1 to 6 His tags.
  • the recombinant expression vector pD441-WT was introduced into E. coli BL21 competent cells (purchased from Quanjin Biotechnology Co., Ltd.), and the resistant plates (containing Kana 50 ⁇ g/ml) were applied to screen positive colonies. 3-5 positive colonies were selected, and positive colonies were identified by PCR using primers SQF (sequence 3 in the sequence listing) and primer SQR (sequence 4 in the sequence listing). A 2800 bp fragment which was substantially identical to the predicted theoretical value was obtained as a positive clone, and the positive clone was named BL21/pD441-WT.
  • BL21/pD441-WT single colonies were picked and cultured in 50 ml LB liquid culture concentrate (containing Kana 50 ⁇ g/ml) at 37 ° C, 220 rpm / min overnight. The next day was diluted 1:100, transferred to 1000 ml of LB liquid medium (containing Kana 50 ⁇ g/ml), shaken at 37 ° C, 220 rpm / min to an OD600 of 0.5-0.8, and added to a final concentration of 0.5 mM IPTG. The culture was induced overnight at 25 ° C, and the BL21/pD441-WT bacterial solution was collected after induction. At the same time, no IPTG was added as a blank control, and the uninducible BL21/pD441-WT bacterial solution was collected.
  • the BL21/pD441-WT bacterial solution was centrifuged at 8000 rpm/min for 10 min, the supernatant was discarded, the precipitated cells were collected, and the cells were resuspended in buffer 1 (50 mM KPO 4 , 500 mM NaCl, 10 mM imidazole, 5% Glycerol).
  • the crude fusion protein extract was loaded at the appropriate flow rate for Ni column affinity chromatography (affinity chromatography prepacked column HisTrap FF, 5ml, 17-5255-01, GE healthcare), and the buffer was used to balance 5CV; %buffer 2 (50 mM KPO 4 , 1 M NaCl, 5% Glycerol, pH 7.0) was eluted with 5 CV; 50% buffer 2 was eluted with 5 CV, and a Ni column affinity chromatography eluate corresponding to a peak value of 100 mAU was collected.
  • Ni column affinity chromatography affinity chromatography prepacked column HisTrap FF, 5ml, 17-5255-01, GE healthcare
  • the eluate corresponding to the peak value of 100 mAU or more was loaded at a certain flow rate for ion exchange chromatography (ion exchange prepacked column HiTrap Q HP, 5 ml, 17-1154-01, GE healthcare), and 5CV was balanced by buffer 2 after loading. , 0% buffer 2 ⁇ 60% buffer 2 linear elution, collecting ion exchange chromatography eluate corresponding to a peak value of 100 mAU or more.
  • the ion exchange chromatography eluate corresponding to the peak value of 100 mAU or more was subjected to gel chromatography (HiPrep Sephacryl S-100HR, 26 mm, 17-1194-01, GE healthcare), first using 20% ethanol. Wash 3CV, wash 3CV, use 35% buffer 3 (20mM Tris, 200mM KCl, 0.2mM EDTA, 10% Glycerol, pH 7.4) to balance 3CV, then load 1.5CV with buffer 3, collect the eluate. This is a wild-type KOD-type DNA polymerase fusion protein after purification.
  • the final yield of the wild-type KOD-type DNA polymerase fusion protein after detection and purification was 40 mg/L.
  • the purified wild-type KOD-type DNA polymerase fusion protein was subjected to SDS-PAGE (concentrated gel was 5% separation gel for 12%), and the results are shown in Fig. 1.
  • 1 is a protein Marker (PageRuler Prestained Protein Ladder, 26616, Thermo Scientific) 2
  • 3 is 10 ⁇ l diluted 20-fold after 0.05 mg/ml purified wild-type KOD-type DNA polymerase fusion protein + 10 ⁇ l of 2x loading buffer; it can be seen that the protein size in lanes 2 and 3 is about 91.5 KDa, which is consistent with the molecular weight reported in the literature.
  • the protein gel after electrophoresis was analyzed for protein purity using Quantity one software, and the purity of the wild-type KOD-type DNA polymerase fusion protein was 95% or more after purification.
  • the target protein of about 91.5 KDa was not obtained without inducing the BL21/pD441-WT bacterial solution.
  • the empty vector pD441-pelB was introduced into E. coli BL21 to obtain BL21/pD441-pelB.
  • the protein was expressed and purified by the above method, and the target protein of about 91.5 KDa was not obtained.
  • the KOD-type DNA polymerase point mutant is the 53, 59, 199, 243, 408, 409, 485, 526, 558, 613, 641, 671, 673, and the amino acid sequence of the KOD type DNA polymerase (sequence 1). At least three amino acids in positions 674, 692 and 709 are subjected to amino acid substitution, and the obtained proteins, the specific mutation positions and modes of each mutant are as follows:
  • the amino acid sequence of Ue1A is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to a histidine;
  • the amino acid sequence of Ue1B is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to an asparagine;
  • the amino acid sequence of Ue1C is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to lysine;
  • the amino acid sequence of Ue1D is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to an aspartic acid;
  • the amino acid sequence of Ue1E is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to glutamic acid;
  • the amino acid sequence of Ue2A is to mutate the 408th leucine of the sequence 1 to alanine, the tyrosine at position 409 to alanine, and the alanine at position 485 to a histidine;
  • the amino acid sequence of Ue2B is to mutate the 408th leucine of the sequence 1 to alanine, the tyrosine at position 409 to alanine, and the alanine at position 485 to an asparagine;
  • the amino acid sequence of Ue2C is to mutate the 408th leucine of the sequence 1 to alanine, the tyrosine at position 409 to alanine, and the alanine at position 485 to lysine;
  • the amino acid sequence of Ue2D is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, and the alanine at position 485 to an aspartic acid;
  • the amino acid sequence of Ue2E is to mutate the 408th leucine of the sequence 1 to alanine, the tyrosine at position 409 to alanine, and the alanine at position 485 to glutamic acid;
  • the amino acid sequence of Ue3A is to mutate the 408th leucine of the sequence 1 to valine, the tyrosine at position 409 to alanine, and the alanine at position 485 to a histidine;
  • the amino acid sequence of Ue3B is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, and the alanine at position 485 to asparagine;
  • the amino acid sequence of Ue3C is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, and the alanine at position 485 to lysine;
  • the amino acid sequence of Ue3D is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, and the alanine at position 485 to aspartic acid;
  • the amino acid sequence of Ue3E is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, and the alanine to glutamic acid at position 485.
  • the amino acid sequence of Ue4A is to mutate the 408th leucine of the sequence 1 to serine, the tyrosine at position 409 to alanine, and the alanine at position 485 to a histidine;
  • the amino acid sequence of Ue4B is to mutate the 408th leucine of the sequence 1 to a serine, the tyrosine at position 409 to alanine, and the alanine at position 485 to an asparagine;
  • the amino acid sequence of Ue4C is a serine at position 408 of leucine of sequence 1, a tyrosine at position 409, alanine at position 485, and a lysine at position 485;
  • the amino acid sequence of Ue1A5 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to histidine, and the 53rd Mutant mutation to methionine, 199th lysine mutated to methionine, 243th arginine mutated methionine, 526th lysine mutated to methionine, 674th The lysine is mutated to alanine, and the 709th arginine is mutated to methionine;
  • the amino acid sequence of Ue1A6 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to histidine, the 59th group.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue1B5 is to mutate the 408th leucine of sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to asparagine, the 53rd Mutant mutation to methionine, 199th lysine mutated to methionine, 243th arginine mutated methionine, 526th lysine mutated to methionine, 674th The lysine is mutated to alanine, and the 709th arginine is mutated to methionine;
  • the amino acid sequence of Ue1B6 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to asparagine, the 59th group Mutant mutation to phenylalanine, 558 lysine mutated to methionine, 613th arginine mutated to methionine, 641th arginine mutated to methionine, 671 The lysine is mutated to methionine, the tyrosine at position 673 is mutated to phenylalanine, and the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue1C5 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to lysine, and the 53th Mutant mutation to methionine, 199th lysine mutated to methionine, 243th arginine mutated methionine, 526th lysine mutated to methionine, 674th Lysine Change to alanine, the 709th arginine is mutated to methionine;
  • the amino acid sequence of Ue1C6 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to lysine, the 59th group Mutant mutation to phenylalanine, 558 lysine mutated to methionine, 613th arginine mutated to methionine, 641th arginine mutated to methionine, 671 The lysine is mutated to methionine, the tyrosine at position 673 is mutated to phenylalanine, and the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue1D5 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to aspartate, the 53rd position.
  • the lysine is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • 674 lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue1D6 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to aspartic acid, the 59th
  • the histidine mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine.
  • the 671 lysine is mutated to methionine
  • the 673th tyrosine is mutated to phenylalanine
  • the 692th lysine is mutated to methionine;
  • the amino acid sequence of Ue1E5 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to glutamic acid, and the 53rd Mutant mutation to methionine, 199th lysine mutated to methionine, 243th arginine mutated methionine, 526th lysine mutated to methionine, 674th The lysine is mutated to alanine, and the 709th arginine is mutated to methionine;
  • the amino acid sequence of Ue1E6 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, the alanine at position 485 to glutamic acid, the 59th group Mutant mutation to phenylalanine, 558 lysine mutated to methionine, 613th arginine mutated to methionine, 641th arginine mutated to methionine, 671 The lysine is mutated to methionine, the tyrosine at position 673 is mutated to phenylalanine, and the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue2A5 is to mutate the 408th leucine of sequence 1 to alanine
  • the tyrosine mutation at position 409 is alanine
  • the alanine at position 485 is mutated to histidine
  • the lysine at position 53 is mutated to methionine
  • the lysine at position 199 is mutated to methionine.
  • the amino acid sequence of Ue2A6 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to a histidine, and the 59th group to ammonia.
  • the acid mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine, the 671th position.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue2B5 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to an asparagine, and the 53th lysine.
  • the acid is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • Lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue2B6 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to an asparagine, and the 59th group to ammonia.
  • the acid mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine, the 671th position.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue2C5 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to lysine, and the 53th to lysine.
  • the acid is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • Lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue2C6 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to lysine, and the 59th to ammonia.
  • the acid mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine, the 671th position.
  • Lysine mutation Methionine, the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue2D5 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to an aspartic acid, the 53nd Mutant mutation to methionine, 199th lysine mutated to methionine, 243th arginine mutated methionine, 526th lysine mutated to methionine, 674th The lysine is mutated to alanine, and the 709th arginine is mutated to methionine;
  • the amino acid sequence of Ue2D6 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to an aspartic acid, the 59th group Mutant mutation to phenylalanine, 558 lysine mutated to methionine, 613th arginine mutated to methionine, 641th arginine mutated to methionine, 671 The lysine is mutated to methionine, the tyrosine at position 673 is mutated to phenylalanine, and the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue2E5 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to glutamic acid, and the 53th to lysine.
  • the acid is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • Lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue2E6 is to mutate the 408th leucine of a sequence 1 to alanine, the tyrosine at position 409 to alanine, the alanine at position 485 to glutamic acid, and the 59th group to ammonia.
  • the acid mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine, the 671th position.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue3A5 is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to histidine, and the 53th to lysine.
  • the acid is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • Lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue3A6 is to mutate the 408th leucine of SEQ ID NO: 1 to valine.
  • the tyrosine mutation at position 409 is alanine
  • the alanine at position 485 is mutated to histidine
  • the histidine at position 59 is mutated to phenylalanine
  • the lysine at position 558 is mutated to methionine.
  • the 613 arginine was mutated to methionine
  • the 641th arginine was mutated to methionine
  • the 671th lysine was mutated to methionine
  • the 673th tyrosine was mutated to phenylalanine.
  • the 692th lysine is mutated to methionine;
  • the amino acid sequence of Ue3B5 is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to asparagine, and the 53th to lysine.
  • the acid is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • Lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue3B6 is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to asparagine, and the 59th to ammonia.
  • the acid mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine, the 671th position.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue3C5 is to mutate the 408th leucine of sequence 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to lysine, and the 53th to lysine.
  • the acid is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • Lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue3C6 is to mutate the 408th leucine of SEQ ID NO: 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to lysine, and the 59th to ammonia.
  • the acid mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine, the 671th position.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue3D5 is to mutate the 408th leucine of sequence 1 to valine, the tyrosine of position 409 to alanine, the alanine of position 485 to aspartic acid, the 53rd Mutant mutation to methionine, 199th lysine mutated to methionine, 243th arginine Mutant methionine, the 526th lysine is mutated to methionine, the 674th lysine is mutated to alanine, and the 709th is arginine mutated to methionine;
  • the amino acid sequence of Ue3D6 is to mutate the 408th leucine of sequence 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to aspartic acid, the 59th group.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue3E5 is to mutate the 408th leucine of sequence 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to glutamic acid, and the 53th to lysine.
  • the acid is mutated to methionine
  • the lysine at position 199 is mutated to methionine
  • the arginine at position 243 is methionine
  • the lysine at position 526 is mutated to methionine.
  • Lysine is mutated to alanine
  • 709 is arginine mutated to methionine;
  • the amino acid sequence of Ue3E6 is to mutate the 408th leucine of sequence 1 to valine, the tyrosine at position 409 to alanine, the alanine at position 485 to glutamic acid, and the 59th group to ammonia.
  • the acid mutation is phenylalanine
  • the lysine at position 558 is mutated to methionine
  • the arginine at position 613 is mutated to methionine
  • the arginine at position 641 is mutated to methionine, the 671th position.
  • the lysine is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue4A5 is to mutate the 408th leucine of the sequence 1 to serine, the tyrosine at position 409 to alanine, the alanine at position 485 to histidine, and the lysine at position 53.
  • Is methionine the 199th lysine is mutated to methionine, the 243th arginine mutated methionine, the 526th lysine is mutated to methionine, the 674th lysine
  • the acid is mutated to alanine, and the 709th is arginine mutated to methionine;
  • the amino acid sequence of Ue4A6 is to mutate the 408th leucine of the sequence 1 to serine, the tyrosine at position 409 to alanine, the alanine at position 485 to histidine, and the histidine at position 59.
  • the acid is mutated to methionine, the tyrosine at position 673 is mutated to phenylalanine, and the lysine at position 692 is mutated to methionine;
  • the amino acid sequence of Ue4B5 is to mutate the 408th leucine of the sequence 1 to serine, the tyrosine at position 409 to alanine, the alanine at position 485 to asparagine, and the 53th lysine mutation.
  • Is methionine the 199th lysine is mutated to methionine, the 243th arginine mutated methionine, the 526th lysine is mutated to methionine, the 674th lysine
  • the acid is mutated to alanine
  • the 709th is arginine mutated to methionine;
  • the amino acid sequence of Ue4B6 is to mutate the 408th leucine of the sequence 1 to serine, the tyrosine at position 409 to alanine, the alanine at position 485 to asparagine, and the histidine at position 59.
  • the acid is mutated to methionine, the tyrosine at position 673 is mutated to phenylalanine, and the lysine at position 692 is mutated to methionine;
  • Ue4C5 has the amino acid sequence of the 408th leucine mutation serine of sequence 1, the 409th tyrosine mutation alanine, the 485th alanine mutation lysine, and the 53rd lysine mutation.
  • Is methionine the 199th lysine is mutated to methionine, the 243th arginine mutated methionine, the 526th lysine is mutated to methionine, the 674th lysine
  • the acid is mutated to alanine, and the 709th is arginine mutated to methionine;
  • the amino acid sequence of Ue4C6 is the leucine-mutated serine at position 408 of sequence 1, the tyrosine at position 409, the alanine at position 485, the alanine at position 485, and the histidine at position 59.
  • Is phenylalanine the 558th lysine is mutated to methionine
  • the 613th arginine is mutated to methionine
  • the 641th arginine is mutated to methionine
  • the acid is mutated to methionine
  • the tyrosine at position 673 is mutated to phenylalanine
  • the lysine at position 692 is mutated to methionine.
  • a recombinant vector expressing a different KOD-type DNA polymerase point mutant is a recombinant His-tagged different KOD-type DNA polymerase point mutant protein-encoding gene, which is ligated to the vector pD441-pelB, and the resulting vector is fused to different points of the His tag.
  • the mutant protein encodes a gene that is expressed by a signal peptide on the vector pD441-pelB.
  • each KOD-type DNA polymerase point mutant fusion protein is obtained by linking the C-terminus of the KOD-type DNA polymerase point mutant represented by the sequence A to 6 His tags; The sequence obtained by the corresponding mutation for the mutation position and mutation mode of the sequence 2 is performed.
  • the nucleotide sequence of the different KOD-type DNA polymerase point mutant protein-encoding gene fused with the His tag is to link the 3' end of the different KOD-type DNA polymerase point mutant protein-encoding gene shown in sequence B to 6 His-tag codes.
  • the sequence B is the sequence obtained by performing the corresponding amino acid codon mutation according to the mutation position and the mutation pattern of each mutant described above.
  • the N-terminus or C-terminus of the amino acid sequence can be tagged for purification, and the tag is generally the sequence shown in Table 1; with or without labeling, there is no effect on the performance of the enzyme. .
  • polymerase mutants used in the examples of the present invention are all polymerase mutant fusion proteins linked at the C-terminus to six His-tags, and can be replaced with unlabeled polymerase mutants as known to those skilled in the art.
  • the recombinant vector expressing the different KOD-type DNA polymerase point mutant prepared in the above 1 was introduced into BL21 to obtain a recombinant strain expressing a different KOD-type DNA polymerase point mutant fusion protein.
  • the recombinant strains expressing the different KOD-type DNA polymerase point mutant fusion proteins prepared above are purified and purified to obtain different KOD-type DNA polymerases. Point mutant fusion protein.
  • the purity of each of the different KOD-type DNA polymerase point mutant fusion proteins was determined to be 95% or more by the above-described one.
  • the C-terminally truncated KOD-type DNA polymerase mutant is a mutant obtained by deleting 29 amino acids of the carbon terminus of the KOD-type DNA polymerase point mutant prepared by the above two; specifically, the point mutants Ue1A, Ue1B, Ue1C Mutants ⁇ Ue1A, ⁇ Ue1B, ⁇ Ue1C, ⁇ obtained by deleting 29 amino acids at the carbon end of Ue1D, Ue1E, Ue2A, Ue2B, Ue2C, Ue2D, Ue2E, Ue3A, Ue3B, Ue3C, Ue3D, Ue3E, Ue4A, Ue4B, Ue4C6 Ue1D, ⁇ Ue1E, ⁇ Ue2A, ⁇ Ue2B, ⁇ Ue2C, ⁇ Ue2D, ⁇ Ue2E, ⁇ Ue3A, ⁇ Ue3B, ⁇ Ue3C,
  • the C-terminally truncated KOD-type DNA polymerase mutant fusion protein was obtained by ligating the C-terminus of the C-terminal truncated KOD-type DNA polymerase mutant to six His tags.
  • the preparation method is the same as the above two.
  • the point mutant fusion proteins in this example were all expressed and purified according to the method in Example 1 above.
  • the polymerase activity assay was carried out according to the literature method of Nishioka, M., et al. (2001. J. Biotechnol. 88).
  • One enzyme unit was defined as: unit enzyme was polymerized in a 50 ⁇ l reaction system at 75 ° for 30 min to polymerize 10 nmol dNTPs. The amount of acid insoluble material.
  • the reaction solution was: 20 mM Tris-HCl (pH 7.5), 8 mM MgCl 2 , 50 ⁇ g/ml BSA, 0.15 mM each dNTPs (dATP, dTTP, dCTP, dGTP), [methyl-3H]-TTP (0.13 mCi/nmol) 150 mg/ml activated calf thymus DNA, 7.5 mM DTT, 1 ⁇ L of wild-type KOD-type DNA polymerase (1 mg/ml), total reaction volume was 50 ⁇ l.
  • the above reaction solution was reacted at 75 ° for 30 min, and then the polymerization activity of the wild-type KOD-type DNA polymerase was calculated to be 2 U/ ⁇ l according to the enzyme activity definition.
  • the above polymerization reaction is carried out by taking a wild type KOD type DNA polymerase fusion protein and a KOD type DNA polymerase point mutant fusion protein as an example.
  • the KOD-type DNA polymerase point mutant is Ue1E;
  • the amino acid sequence of the KOD-type DNA polymerase point mutant Ue1E is to mutate the 408th leucine of the sequence 1 to isoleucine, and mutate the tyrosine at position 409 to alanine, and will be 485
  • the alanine is mutated to glutamic acid
  • the nucleotide sequence of the coding gene is a mutation of nucleotide C of position 1222 of sequence 2 to A
  • a nucleotide of position 1224 of G is mutated to T
  • the 1225th is nucleus
  • the T-mutation of the Glycosidic acid is G
  • the nucleotide A mutation at 1226 is C
  • the nucleotide T at position 1227 is A
  • the nucleotide C at position 1454 is mutated to A.
  • the fusion protein was added with six his tags at the C-terminus of the respective mutant.
  • Table 2 shows wild type and mutant Ue1E polymerase activity
  • the relative reaction rate of the KOD-type DNA polymerase mutant was detected by a microplate reader using a Cy3 fluorescent dye-labeled dATP (dATP-Cy3) and a Cy5 fluorescent dye-labeled DNA template (template DNA-Cy5), and the mutations were approximated.
  • dATP-Cy3 Cy3 fluorescent dye-labeled dATP
  • template DNA-Cy5 Cy5 fluorescent dye-labeled DNA template
  • Single-stranded primers with 5'Cy5 fluorescent labeling S1A (sequence 5 in the sequence listing) and S2A (sequence 6 in the sequence listing) were templated with template DNA (synthesized by Kingsray, sequence 7 in the sequence listing) by 1:1 :1
  • the mixture was mixed at an equimolar concentration, and annealed at 65 ° C for 1 min, 40 ° C for 1 min, and 4 ° C for 10 min.
  • the annealed product was stored in the dark at -20 ° C to obtain a Cy5 fluorescent dye-labeled template DNA-Cy5.
  • the enzyme activity assay was performed using a BioTek microplate reader.
  • the reaction was carried out in 384 plates (Corning black, clear bottom 384 plates), and the total volume of the reaction solution was 50 ⁇ l.
  • the reaction system is: 2UKOD polymerase mutant fusion protein, 1 ⁇ M dATP-Cy3, 10 ⁇ M dTTP, 10 ⁇ M dCTP, 10 ⁇ M dGTP, template DNA-Cy5 according to 2, 4, 5, 8, 10, 20, 40, 80nmol 8 concentration gradients
  • the experiment was carried out, and the enzyme reaction buffer was 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 pH 8.8; and the reaction temperature was 25 °C.
  • the enzyme reaction is recorded in a kinetic detection mode, and the data is recorded every minute.
  • the detection conditions are
  • the data table or the enzyme activity curve can be directly derived, and the reaction rate of the relative fluorescence value can be approximated.
  • the reaction rate of the fluorescence value depends on the concentration of the template DNA-Cy5. Therefore, by detecting the activity of the KOD-type DNA polymerase mutant under different concentrations of the template DNA-Cy5, the Km value can be approximately determined, that is, the reaction rate is reached.
  • the template DNA concentration corresponding to half of the maximum reaction rate.
  • KOD-WT wild type KOD-type DNA polymerase fusion protein
  • KOD type DNA polymerase point mutants are Ue1E, Ue1E5, Ue1E6, preparation method example 1;
  • the amino acid sequence of the KOD-type DNA polymerase point mutant Ue1E is to mutate the 408th leucine of the sequence 1 to isoleucine, and mutate the tyrosine at position 409 to alanine, and the 485th position
  • the amino acid is mutated to glutamic acid;
  • the nucleotide sequence encoding the gene is a mutation of nucleotide 1222 of sequence 2 to A, the nucleotide of position 1224 is mutated to T, and the nucleotide of 1225 is
  • the T mutation is G
  • the 1226 nucleotide A mutation is C
  • the 1227 nucleotide T mutation is A
  • the 1454 nucleotide C is mutated to A.
  • the amino acid sequence of the KOD-type DNA polymerase point mutant Ue1E5 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to Glutamate, the 53rd lysine is mutated to methionine, the 199th lysine is mutated to methionine, the 243th arginine is methionine, and the 526th lysine is mutated.
  • the nucleotide sequence encoding the gene is the mutation of nucleotide 1222 of sequence 2
  • the 1224th nucleotide G is mutated to T
  • the 1225th nucleotide is mutated to G
  • the 1226th nucleotide A is mutated to C
  • the 1227th nucleotide is mutated to A.
  • nucleotide 1454 Mutated to A, and mutated nucleotide A at position 158 to T, nucleotide A at position 159 to G, and mutated T at nucleotide position 596 and nucleotide 597 to G.
  • nucleotide 726 of the 727th nucleotide was mutated to A
  • nucleotide of the 728th nucleotide was mutated to T
  • nucleotide of the 729th nucleotide was c-mutated to G
  • nucleotide of the 1577th nucleotide A was mutated to T, the 1578th.
  • the nucleotide A is mutated to G, and the nucleotide A at position 2021 is mutated to T, and the nucleotide at position 2125 is mutated to G, and the nucleotide at position 2126 is mutated to T, and the 2127th nucleus is mutated.
  • the T-glycosidic acid is mutated to G.
  • the amino acid sequence of the KOD-type DNA polymerase point mutant Ue1E6 is to mutate the 408th leucine of the sequence 1 to isoleucine, the tyrosine at position 409 to alanine, and the alanine at position 485 to Glutamate, the 59th histidine is mutated to phenylalanine, the 558th lysine is mutated to methionine, the 613th arginine is mutated to methionine, and the 641th arginine is mutated.
  • the acid sequence is to mutate the nucleotide 1222 of sequence 2 to A, the nucleotide at position 1224 to G, and to mutate T at 1225 to G, the mutation of nucleotide 1226.
  • the 1227th nucleotide T is mutated to A
  • the 1454th nucleotide C is mutated to A
  • the 175th nucleotide C is mutated to T
  • the 176th nucleotide A is mutated to T.
  • the 1673th nucleotide A was mutated to T
  • the 1674th nucleotide A was mutated to G
  • the 1837th nucleotide C was mutated to A
  • the 1838th nucleotide G was mutated to T
  • 1839 nucleotide mutation T is G
  • will be 1921 Nucleotide C mutation is A, 1922 nucleotide G mutation to T, 1923 nucleotide T mutation to G
  • the nucleotide at nucleotide A is mutated to T
  • the nucleotide at nucleotide A is mutated to G
  • mutated nucleotide A of position 2018 to T and mutated nucleotide A of position 2075 to T.
  • the fusion protein was added with six his tags at the C-terminus of the respective mutant.
  • the Michaelis curve is shown in Figure 2. It can be seen that the mutant Ue1E, Ue1E5, Ue1E6 showed better polymerization when compared with wild-type KOD polymerase (KOD-WT) when using single-stranded DNA as a template. active.
  • KOD-WT wild-type KOD polymerase
  • the results indicate that when the Km value of the enzyme is adjusted to approximate the affinity of the enzyme and the DNA, the polymerization activity of the enzyme, that is, the catalytic reaction efficiency, is not affected, and the DNA binding affinity of the mutant modified enzyme body is achieved without affecting the specific catalytic properties. .
  • Increasing the Km value facilitates the rapid dissociation of the enzyme from the DNA molecule, which can accelerate the completion of the reaction to a certain extent, and facilitate the elution of the enzyme from the surface of the chip.
  • the above-mentioned KOD-type DNA polymerase mutant Ue1E5 was selected (the amino acid mutations 408, 409, 485, 53, 199, 243, 526, 674, and 709 were changed to isoleucine, alanine relative to the wild-type amino acid sequence.
  • Test experiments were carried out with acid, glutamic acid, methionine, methionine, methionine, methionine, alanine, and methionine.
  • the template DNA (synthesized by Kingsray, sequence 7 in the sequence listing) was immobilized on a biochip gencbio amino substrate (Gencbio, GCB01003), and the KOD-type DNA polymerase point mutant fusion protein Ue1E5 was tested in a non-aqueous solution and template.
  • the specific reaction method of DNA reaction is as follows:
  • the membrane was washed with 0.2M PH7.0 PB (0.2M NaH 2 PO 4 , 0.2M Na 2 HPO 4 ) buffer, and then the template DNA immobilization reaction was carried out, and an appropriate concentration of 30 ng/ ⁇ l of template DNA was added to fill the whole sheet.
  • the chip was reacted at room temperature for 15 min to allow the negatively charged template DNA to be sufficiently immobilized on the surface of the positively charged aminated chip using an enzyme reaction buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl).
  • the chip was washed with 2 mM MgSO 4 pH 8.8 and a reaction temperature of 25 ° C to remove excess template DNA.
  • the reaction is sufficient, and on the other hand, the enzyme and the template DNA immobilized on the chip not only react but also exhibit better polymerization activity.
  • FIG. 3 The results of the chip reaction are shown in Figure 3, where A is wild-type KOD polymerase and B is the mutant Ue1E5 polymerase. It can be seen that the chip has few bright spots in Figure 3A, indicating unmodified or mutated wild-type KOD polymerase. It is less active or inactive when reacting with the template DNA on the surface of the chip; on the contrary, the bright spot of the chip in Figure 3B is more and more dense, indicating that the modified mutant Ue1E5 is more active than the wild type when reacting with the template DNA on the surface of the chip. better.
  • the KOD-type DNA polymerase point mutant fusion proteins Ue1E and Ue1E6 were detected by the same method, and the results were not significantly different from Ue1E5, and both had good polymerization activity.
  • the fusion protein was added with six his tags at the C-terminus of the respective mutant.
  • mutant KOD DNA polymerase KOD Ue1E5 example DNA polymerase eluted chip test, while commercial enzyme Therminator (Therminator TM DNA Polymerase, NEB , M0261S) the experiment as control.
  • Therminator Therminator TM DNA Polymerase, NEB , M0261S
  • the pre-chip processing and chip activity reaction are the same as the above-mentioned chip activity test experimental methods.
  • the chip was washed 3-4 times with enzyme reaction buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 pH 8.8 @ 25 ° C), and then added without enzyme but included
  • enzyme reaction buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 10 mM KCl, 2 mM MgSO 4 pH 8.8 @ 25 ° C
  • the enzymatic reaction of dNTP and reaction buffer was carried out at 65 ° C for 15 min, and then the chip was washed twice with an enzyme reaction buffer, and the chip was observed by a fluorescence microscope according to the detection condition of the fluorescent label Cy3 (530 nm excitation wavelength, 568 nm emission wavelength). And taking pictures.
  • FIG. 4A is a blank chip and thus has no reaction, and the picture has no bright spots
  • 4B is the commercial enzyme Therminator, its picture highlights are more bright than the blank control
  • Figure 4C is the mutant Ue1E5, which still has a few bright spots compared with the control, but significantly less than the Therminator, indicating that the mutant elutes
  • the effect is better than Therminator, indicating that the mutant is easy to elute, it is not easy to adsorb the surface of the chip, and does not interfere with subsequent experiments.
  • the wild type was incapable of performing elution experiments due to poor polymerization activity, so the commercial Therminator was used as a control.
  • the KOD-type DNA polymerase point mutant fusion protein Ue1E6 was detected by the same method, and the results were not significantly different from Ue1E5, and were easier to elute than Therminator.
  • Example 3 Activity test of C-terminal truncated KOD-type DNA polymerase mutant fusion protein
  • the fusion protein was added with six his tags at the C-terminus of the respective mutant.
  • the C-terminally truncated KOD-type DNA polymerase mutant fusion protein prepared in the third embodiment of Example 1 was used.
  • the KOD-type DNA polymerase point mutant fusion proteins Ue1A, Ue1B, Ue1C, Ue1D, Ue1E, Ue2A, Ue2B, Ue2C, Ue2D, Ue2E, Ue3A, Ue3B, Ue3C, Ue3D, Ue3E, Ue4A, Ue4B and Ue4C6 were used as controls.
  • the C-terminally truncated KOD-type DNA polymerase mutant fusion protein of the present Example was detected by the method of the fourth embodiment, and was also easily eluted, and it was difficult to adsorb the surface of the chip.
  • the experiments of the present invention prove that the DNA polymerase mutant prepared by the invention can be polymerized by using artificially modified nucleotides or nucleotide analogs as a substrate, exhibits good polymerization activity and is not easily adsorbed on the chip.
  • the surface which is easily eluted from the surface of the chip, enables the addition of a single nucleotide or nucleotide analog in each reaction cycle and is easy to elute without interfering with the next sequencing cycle.

Abstract

提供一种重组DNA聚合酶，其为如下A)-C)中任一种：A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第408、409和485位氨基酸残基进行修饰，且对所述野生型KOD型DNA聚合酶氨基酸序列中第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少一位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白；B)所示的蛋白为将A)所示蛋白的氨基酸序列C端的自末端起1-29个氨基酸去除，保留其余氨基酸残基且具有DNA聚合酶活性的由A)衍生的蛋白质；C)所示的蛋白为将A)或B)所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A)或B)衍生的蛋白质。

Description

重组DNA聚合酶 技术领域
本发明涉及生物技术领域，尤其涉及一种重组DNA聚合酶。
背景技术
DNA聚合酶快速而准确地复制在生物染色体中的DNA，对于维持生命体中遗传物质稳定性具有重要意义。基于与大肠杆菌聚合酶I、II和III的氨基酸序列差异，DNA聚合酶分为三个家族，分别称为A、B和C族。尽管在结构上A、B族聚合酶的核苷酸结合位点相似，但不同族聚合酶序列模体(motif)则有显著不同，并相应表现在对核苷酸及其类似物的识别机理上的差异。与其在生命体中功能紧密相关，DNA聚合酶在分子生物学技术中居于核心地位，尤其表现在分子克隆及PCR、定点突变和DNA测序方面。在这些分子生物学技术中，DNA聚合酶识别不同的核苷酸或核苷酸类似物及其与模板的互补性起到关键作用。
许多实际应用技术依赖于能够准确加入核苷酸或核苷酸类似物的DNA聚合酶。例如，在边合成边测序(SBS)中，加入经过荧光标记的核苷酸能够帮助识别模板DNA碱基，从而给出DNA序列信息。近年来，与其它测序方法相比，SBS测序方法以其通量高、价格低而获得青睐。在这一测序方法中，加入和检测人工修饰的核苷酸或核苷酸类似物，如带有荧光标记的可逆终止子(reversible terminator)的DNA聚合酶起到关键性作用。在DNA聚合反应中，已有聚合酶对人工修饰的核苷酸或核苷酸类似物的聚合能力不足经常成为限制性因素，鉴于此，增进聚合酶对人工修饰的核苷酸或核苷酸类似物的聚合效能成为这类测序方法的重要一步。
发明公开
本发明的一个目的是提供一种蛋白质。
本发明提供的蛋白质，是如下A)-C)中任一种：
A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第408、409和485位氨基酸残基进行修饰，且对所述野生型KOD型DNA聚合酶氨基酸序列中第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少一位的氨基酸残基进行修饰，得到具有DNA聚合 酶活性的蛋白；
B)所示的蛋白为将A)所示蛋白的氨基酸序列C端的自末端起1-29个氨基酸去除(包括第29个氨基酸)，保留其余氨基酸残基且具有DNA聚合酶活性的由A)衍生的蛋白质；
C)所示的蛋白为将A)或B)所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A)或B)衍生的蛋白质。
上述蛋白质中，
A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第408、409和485位氨基酸残基进行修饰，且对所述野生型KOD型DNA聚合酶氨基酸序列中第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少2位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述蛋白质中，
A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第408、409和485位氨基酸残基进行修饰，且对所述野生型KOD型DNA聚合酶氨基酸序列中第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少3位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
上述蛋白质中，所述修饰为氨基酸置换。
上述蛋白质中，
第408位的亮氨酸置换为异亮氨酸、酪氨酸、脯氨酸、缬氨酸、半胱氨酸、丝氨酸、丙氨酸或甲硫氨酸；
第409位的酪氨酸置换为丙氨酸或甘氨酸；
第485位的丙氨酸置换为组氨酸、天冬酰胺、赖氨酸、天冬氨酸或谷氨酸；
第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少一位的氨基酸残基置换为甲硫氨酸或苯丙氨酸或丙氨酸。
上述蛋白质中，
所述第53、59、199、243、526、558、613、641、671、673、674、 692和709位这13位中至少一位的氨基酸残基置换为甲硫氨酸或苯丙氨酸或丙氨酸为如下：
第53位的赖氨酸置换为甲硫氨酸；
第59位的组氨酸置换为苯丙氨酸；
第199位的赖氨酸置换为甲硫氨酸；
第243位的精氨酸置换为甲硫氨酸；
第526位的赖氨酸置换为甲硫氨酸；
第558位的赖氨酸置换为甲硫氨酸；
第613位的精氨酸置换为甲硫氨酸；
第641位的精氨酸置换为甲硫氨酸；
第671位的赖氨酸置换为甲硫氨酸；
第673位的酪氨酸置换为苯丙氨酸；
第674位的赖氨酸置换为丙氨酸；
第692位的赖氨酸置换为甲硫氨酸；
第709位的精氨酸置换为甲硫氨酸。
上述蛋白质中，
所述野生型KOD型DNA聚合酶氨基酸序列为序列1。
上述蛋白质中，
所述蛋白质的对生物芯片的吸附能力低于所述野生型KOD型DNA聚合酶。
上述蛋白质中，
所述蛋白质的对生物芯片的吸附能力低于所述野生型KOD型DNA聚合酶体现在与所述野生型KOD型DNA聚合酶相比，所述蛋白质不易吸附于生物芯片表面和/或易于从生物芯片表面洗脱。
编码上述蛋白的DNA分子也是本发明保护的范围；
或含有所述DNA分子的表达盒、重组载体、重组菌或转基因细胞系也是本发明保护的范围。
上述蛋白或上述DNA分子或所述表达盒、重组载体、重组菌或转基因细胞系在制备DNA聚合酶中的应用也是本发明保护的范围。
上述应用中，所述DNA聚合酶的对生物芯片的吸附能力低于所述野生 型KOD型DNA聚合酶；
或，所述DNA聚合酶的对生物芯片的吸附能力低于所述野生型KOD型DNA聚合酶体现在与所述野生型KOD型DNA聚合酶相比，所述DNA聚合酶不易吸附于生物芯片表面和/或易于从生物芯片表面洗脱。
上述蛋白或上述DNA分子或所述的表达盒、重组载体、重组菌或转基因细胞在测序中的应用也是本发明保护的范围；
或上述蛋白或上述DNA分子或所述的表达盒、重组载体、重组菌或转基因细胞在制备用于测序的产品中的应用也是本发明保护的范围。
上述应用中，所述产品为试剂盒。
上述蛋白质中，所述蛋白作为DNA聚合酶的作用底物为核苷酸或核苷酸类似物；
上述蛋白质中，所述蛋白作为DNA聚合酶的模板为单链DNA；
上述应用中，所述DNA聚合酶的底物为核苷酸或核苷酸类似物。
上述应用中，所述DNA聚合酶的模板为单链DNA。
上述应用中，所述DNA聚合酶具有如下特征：以单链DNA为模板时聚合活性高和/或对生物芯片吸附力低。
上述应用中，所述以单链DNA为模板时聚合活性高体现在以单链DNA为模板时所述蛋白的聚合活性高于所述KOD型DNA聚合酶；
上述应用中，所述聚合反应在生物芯片上进行。
上述生物芯片为所有的本领域常用的生物芯片。
上述应用中，所述产品为试剂盒。
上述蛋白为KOD型DNA聚合酶突变体蛋白，具体为如下：
KOD型DNA聚合酶点突变体：Ue1A、Ue1B、Ue1C、Ue1D、Ue1E、Ue2A、Ue2B、Ue2C、Ue2D、Ue2E、Ue3A、Ue3B、Ue3C、Ue3D、Ue3E、Ue4A、Ue4B、Ue4C、Ue1A5、Ue1A6、Ue1B5、Ue1B6、Ue1C5、Ue1C6、Ue1D5、Ue1D6、Ue1E5、Ue1E6、Ue2A5、Ue2A6、Ue2B5、Ue2B6、Ue2C5、Ue2C6、Ue2D5、Ue2D6、Ue2E5、Ue2E6、Ue3A5、Ue3A6、Ue3B5、Ue3B6、Ue3C5、Ue3C6、Ue3D5、Ue3D6、Ue3E5、Ue3E6、Ue4A5、Ue4A6、Ue4B5、Ue4B6、Ue4C5、Ue4C6
或C端截短型KOD型DNA聚合酶突变体，其为上述KOD型DNA聚合酶点突变体除去自C末端起29个氨基酸，剩余氨基酸残基组成的区段。
本发明以对自然界已有及经过人工改造的DNA聚合酶的三维结构及序列信息分别进行动力学模拟与统计推断为基础，通过半理性设计、酶变体库构建和高通量筛选达到改善聚合酶的催化及理化特性，发现突变KOD型DNA聚合酶野生型部分位点，可以实现DNA聚合酶突变体不易被吸附于芯片表面、易于从芯片表面洗脱，这些突变体为适合于在附着于芯片表面的DNA测序工作的DNA聚合酶。
附图说明
图1为对纯化后的野生型KOD型DNA聚合酶纯度检测电泳结果。
图2为对KOD型DNA聚合酶突变体Ue1E、Ue1E5、Ue1E6和野生型KOD聚合酶KOD-WT以单链DNA为底物的米氏动力学曲线图。
图3为KOD型DNA聚合酶突变体Ue1E5和野生型KOD聚合酶芯片活性测试的芯片反应亮点图。
图4为KOD型DNA聚合酶突变体Ue1E5和对照组Therminator芯片洗脱测试的芯片反应亮点图。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、野生型KOD型DNA聚合酶及其突变体的制备
本发明将野生型KOD型DNA聚合酶及其突变体均采用DNA 2.0的ElectraTMCloning Reagents Kit试剂盒进行表达载体的构建，利用His标签进行Ni柱亲和纯化。
一、野生型KOD型DNA聚合酶的制备
野生型KOD型DNA聚合酶的氨基酸序列为序列表中序列1，其编码基因phaP的核苷酸序列为序列表中序列2。
1、重组表达载体pD441-WT的构建
重组表达载体pD441-WT为将融合his标签的野生型KOD型DNA聚合酶编码基因按ElectraTMCloning Reagents Kit(DNA2.0,EKT-02)试剂盒说明书操作步骤重组到载体pD441-pelB(DNA2.0,pD441‐pelB)上，得到的载体，融合His标签的野生型KOD型DNA聚合酶编码基因，通过载体 pD441-pelB上的信号肽引导表达。
融合His标签的野生型KOD型DNA聚合酶编码基因的核苷酸序列为在序列2的3’端连接有6个His标签密码子得到的序列。
野生型KOD型DNA聚合酶融合蛋白的氨基酸序列为将序列1所示的氨基酸的C端连接6个His标签得到。
2、重组菌的构建
将重组表达载体pD441-WT导入大肠杆菌BL21感受态细胞(购自全式金生物科技有限公司)中，涂抹抗性平板(含卡那50μg/ml)筛选阳性菌落。挑选3-5个阳性菌落，利用引物SQF(序列表中序列3)和引物SQR(序列表中序列4)对阳性菌落进行菌液PCR鉴定。得到与预计的理论值基本一致的2800bp大小的片段为阳性克隆，将该阳性克隆命名为BL21/pD441-WT。
3、野生型KOD型DNA聚合酶融合蛋白的表达和纯化
挑取BL21/pD441-WT单菌落，于50ml LB液体培养集中(含卡那50μg/ml)中，37℃，220rpm/min，过夜培养。次日按1:100稀释量，转接于1000mlLB液体培养基中(含卡那50μg/ml)，37℃，220rpm/min振荡培养至OD600为0.5-0.8，加入终浓度为0.5mM的IPTG，25℃过夜诱导培养，收集诱导后BL21/pD441-WT菌液。同时设不加IPTG作为空白对照，收集未诱导BL21/pD441-WT菌液。
将上述诱导后BL21/pD441-WT菌液，转速为8000rpm/min离心10min，弃上清，收集沉淀菌体，重悬菌体细胞于buffer 1(50mMKPO4，500mM NaCl，10mM imidazole，5％Glycerol，PH 7.0)中，另加入PMSF(终浓度0.5mM)、Triton X-100(终浓度0.5％)、Lysozyme(终浓度0.25％)，后室温孵育30min，12000rpm/min、4℃离心30min，冰浴，超声破碎细胞；以12000rpm/min的转速离心30min，将离心所得上清液于75℃水浴20min，期间注意定时混匀，使受热均匀；12000rpm/min，4℃离心30min，将上清液用0.22um滤膜过滤，即为融合蛋白粗提物。
将融合蛋白粗提物以适当流速上样进行Ni柱亲和层析(亲和层析预装柱HisTrap FF，5ml，17-5255-01，GE healthcare)，上样后利用buffer1平衡5CV；3％buffer 2(50mMKPO4，1M NaCl，5％Glycerol，PH 7.0)洗脱5CV；50％buffer 2洗脱5CV，收集大于等于100mAU峰值对应的Ni柱亲和层析洗脱 液。
将大于等于100mAU峰值对应的洗脱液按一定的流速上样进行离子交换层析(离子交换预装柱HiTrap Q HP，5ml，17-1154-01，GE healthcare)，上样后利用buffer2平衡5CV，0％buffer 2→60％buffer 2线性洗脱，收集大于等于100mAU峰值对应的离子交换层析洗脱液。
将大于等于100mAU峰值对应的离子交换层析洗脱液进行凝胶层析(凝胶层析预装柱HiPrep Sephacryl S-100HR，26mm，17-1194-01，GE healthcare)，先用20％乙醇洗3CV，水洗3CV，利用100％buffer 3(20mM Tris，200mM KCl，0.2mM EDTA，10％Glycerol，PH7.4)平衡3CV后上样，再用buffer 3洗脱1.5CV，收集洗脱液即为纯化后野生型KOD型DNA聚合酶融合蛋白。
检测纯化后野生型KOD型DNA聚合酶融合蛋白最终得率为40mg/L。
将纯化后野生型KOD型DNA聚合酶融合蛋白进行SDS-PAGE(浓缩胶为5％分离胶为12％)，结果如图1所示，1为蛋白Marker(PageRuler Prestained Protein Ladder，26616，Thermo Scientific)，2为10μl1mg/ml纯化后野生型KOD型DNA聚合酶融合蛋白+10μl 2x上样缓冲液，3为10μl稀释20倍后的0.05mg/ml纯化后野生型KOD型DNA聚合酶融合蛋白+10μl 2x上样缓冲液；可以看出，泳道2和泳道3中蛋白大小约91.5KDa，与文献报道分子量一致。
对电泳后的蛋白胶利用Quantity one软件分析蛋白纯度，纯化后野生型KOD型DNA聚合酶融合蛋白纯度能达到95％或以上。
未诱导BL21/pD441-WT菌液未得到约91.5KDa大小的目的蛋白。
将空载体pD441-pelB导入大肠杆菌BL21中，得到BL21/pD441-pelB。采用上述方法进行表达和纯化蛋白，也未得到约91.5KDa大小的目的蛋白。
二、KOD型DNA聚合酶突变体融合蛋白的制备
KOD型DNA聚合酶点突变体为将KOD型DNA聚合酶的氨基酸序列(序列1)中第53、59、199、243、408、409、485、526、558、613、641、671、673、674、692和709位中至少三个氨基酸进行氨基酸置换，得到的蛋白，各突变体具体突变位置及方式如下：
Ue1A其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸；
Ue1B其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺；
Ue1C其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸；
Ue1D其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸；
Ue1E其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸；
Ue2A其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸；
Ue2B其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺；
Ue2C其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸；
Ue2D其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸；
Ue2E其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸；
Ue3A其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸；
Ue3B其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺；
Ue3C其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸；
Ue3D其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸；
Ue3E其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸
Ue4A其氨基酸序列为将序列1的第408位亮氨酸突变为丝氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸；
Ue4B其氨基酸序列为将序列1的第408位亮氨酸突变为丝氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺；
Ue4C其氨基酸序列为将序列1的第408位亮氨酸突变位丝氨酸，第409位酪氨酸突变位丙氨酸，第485为丙氨酸突变位赖氨酸；
Ue1A5其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue1A6其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue1B5其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue1B6其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue1C5其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突 变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue1C6其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue1D5其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue1D6其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue1E5其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue1E6其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue2A5其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第 409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue2A6其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue2B5其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue2B6其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue2C5其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue2C6其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为 甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue2D5其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue2D6其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue2E5其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue2E6其氨基酸序列为将序列1的第408位亮氨酸突变为丙氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue3A5其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue3A6其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第 409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue3B5其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue3B6其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue3C5其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue3C6其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为赖氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue3D5其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸 突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue3D6其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue3E5其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue3E6其氨基酸序列为将序列1的第408位亮氨酸突变为缬氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue4A5其氨基酸序列为将序列1的第408位亮氨酸突变为丝氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue4A6其氨基酸序列为将序列1的第408位亮氨酸突变为丝氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为组氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue4B5其氨基酸序列为将序列1的第408位亮氨酸突变为丝氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue4B6其氨基酸序列为将序列1的第408位亮氨酸突变为丝氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为天冬酰胺，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；
Ue4C5其氨基酸序列为将序列1的第408位亮氨酸突变位丝氨酸，第409位酪氨酸突变位丙氨酸，第485为丙氨酸突变位赖氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；
Ue4C6其氨基酸序列为将序列1的第408位亮氨酸突变位丝氨酸，第409位酪氨酸突变位丙氨酸，第485为丙氨酸突变位赖氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸。
1、表达KOD型DNA聚合酶点突变体的重组载体的制备
表达不同KOD型DNA聚合酶点突变体的重组载体为将融合His标签的的不同KOD型DNA聚合酶点突变体蛋白编码基因重组到载体pD441-pelB上，得到的载体，融合His标签的不同点突变体蛋白编码基因，通过载体pD441-pelB上的信号肽引导表达。
每个KOD型DNA聚合酶点突变体融合蛋白的氨基酸序列为将序列A所示的KOD型DNA聚合酶点突变体的C端连接6个His标签得到的；序列A 为序列2按的突变位置及突变方式进行相应的突变得到的序列。
融合His标签的不同KOD型DNA聚合酶点突变体蛋白编码基因的核苷酸序列为将序列B所示的不同KOD型DNA聚合酶点突变体蛋白编码基因的3‘端连接6个His标签密码子得到的；序列B为序列1按照上述各突变体的突变位置及突变方式进行相应的氨基酸密码子突变得到的序列。
如本领域人员所知，对于不同的酶，可在氨基酸序列的N端或C端加标签，以便于纯化，标签一般为表1所示的序列；加或不加标签对于酶的性能没有影响。
表1标签的序列
Figure PCTCN2017070609-appb-000001
本发明实施例所用聚合酶突变体均为C端连接6个His标签的聚合酶突变体融合蛋白，如本领域人员所知，均可以替换为不加标签的聚合酶突变体。
2、重组菌的构建
与上述一中的2方法相同，将上述1制备的表达不同KOD型DNA聚合酶点突变体重组载体导入BL21中，得到表达不同KOD型DNA聚合酶点突变体融合蛋白的重组菌。
3、突变体的表达和纯化
与上述一野生型KOD型DNA聚合酶融合蛋白表达和纯化方法相同，将上述2制备的表达不同KOD型DNA聚合酶点突变体融合蛋白的重组菌进行表达纯化，得到不同的KOD型DNA聚合酶点突变体融合蛋白。
采用上述一的2的检测不同的KOD型DNA聚合酶点突变体融合蛋白的纯度均得到能达到95％或以上。
三、C端截短型KOD型DNA聚合酶突变体融合蛋白的制备
C端截短型KOD型DNA聚合酶突变体为将上述二制备的KOD型DNA聚合酶点突变体的碳末端起29个氨基酸删除得到的突变体；具体为将点突变体Ue1A、Ue1B、Ue1C、Ue1D、Ue1E、Ue2A、Ue2B、Ue2C、Ue2D、Ue2E、Ue3A、Ue3B、Ue3C、Ue3D、Ue3E、Ue4A、Ue4B、Ue4C6碳端29个氨基酸删除得到的突变体△Ue1A、△Ue1B、△Ue1C、△Ue1D、△Ue1E、△Ue2A、△Ue2B、△Ue2C、△Ue2D、△Ue2E、△Ue3A、△Ue3B、ΔUe3C、ΔUe3D、ΔUe3E、ΔUe4A、ΔUe4B、ΔUe4C。
C端截短型KOD型DNA聚合酶突变体融合蛋白为将C端截短型KOD型DNA聚合酶突变体的C端连接6个His标签得到的。
制备方法同上述二。
实施例2、KOD型DNA聚合酶点突变体融合蛋白的性能检测
本实施例中的点突变体融合蛋白均按照上述实施例1中的方法表达纯化。
一、KOD型DNA聚合酶点突变体融合蛋白的聚合活性检测
聚合酶活性检测参照Nishioka,M.,et al.(2001.J.Biotechnol.88)文献方法进行，一个酶活单位定义为:单位酶在50μl的反应体系中于75°反应30min聚合10nmol dNTP生成酸不溶性物质的量。
反应液为：20mM Tris-HCl(pH7.5)，8mM MgCl2，50μg/ml BSA，0.15mM each dNTPs(dATP、dTTP、dCTP、dGTP)，[methyl-3H]-TTP(0.13mCi/nmol)，150mg/ml激活的小牛胸腺DNA,7.5mM DTT，1μL野生型KOD型DNA聚合酶(1mg/ml),总反应体积为50μl。
上述反应液于75°反应30min，后按酶活定义计算野生型KOD型DNA聚合酶的聚合活性为2U/μl。
以野生型KOD型DNA聚合酶融合蛋白及KOD型DNA聚合酶点突变体融合蛋白为例，进行上述聚合反应。
KOD型DNA聚合酶点突变体为Ue1E；
KOD型DNA聚合酶点突变体Ue1E的氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，且将第409位酪氨酸突变为丙氨酸，且将第485 位丙氨酸突变为谷氨酸；其编码基因核苷酸序列为将序列2第1222位核苷酸C突变为A、第1224位核苷酸G为突变为T，且将第1225为核苷酸T突变为G、第1226位核苷酸A突变为C、第1227位核苷酸T突变为A，且将第1454位核苷酸C突变为A。
融合蛋白为在各自突变体的C端加入6个his标签。
KOD型DNA聚合酶点突变体融合蛋白Ue1E聚合酶活性结果如表3所示，可以看出，KOD型DNA聚合酶点突变体具有聚合酶活性。
表2为野生型及突变体Ue1E聚合酶活性
Figure PCTCN2017070609-appb-000002
其他突变体的聚合活性结果类似。
二、KOD型DNA聚合酶点突变体融合蛋白的单碱基掺入动力学
本实施例利用Cy3荧光染料标记的dATP(dATP-Cy3)和Cy5荧光染料标记的DNA模板(模板DNA-Cy5)，利用酶标仪检测KOD型DNA聚合酶突变体相对反应速率，近似描绘各突变体的米氏动力学曲线，具体实验方法如下：
将带有5’Cy5荧光标记的单链引物S1A(序列表中序列5)和S2A(序列表中序列6)与模板DNA(由金斯瑞公司合成，序列表中序列7)按1：1：1等摩尔浓度进行混合，按65℃1min、40℃1min、4℃10min退火，退火产物避光保存至-20℃，即得到Cy5荧光染料标记的模板DNA-Cy5。
使用BioTek酶标仪进行酶活检测，反应在 384板(Corning black,clear bottom 384plates)中进行，反应液总体积50μl
反应体系为：2UKOD聚合酶突变体融合蛋白，1μM dATP-Cy3，10μM dTTP,10μM dCTP,10μM dGTP,模板DNA-Cy5按2、4、5、8、10、20、 40、80nmol 8个浓度梯度进行实验，酶反应缓冲液为20mM Tris-HCl、10mM(NH4)2SO4、10mM KCl、2mM MgSO4pH 8.8；反应温度为25℃。
酶反应以动力学检测模式，每分钟记录一次数据，检测条件为
Figure PCTCN2017070609-appb-000003
反应完成后可直接导出数据表格或者酶活曲线，可近似计算其相对荧光值的反应速率。
对荧光值的反应速率的大小取决于模板DNA-Cy5的浓度，因此通过在不同浓度的模板DNA-Cy5条件下，检测KOD型DNA聚合酶突变体的活性可以近似确定其Km值即反应速率达到最大反应速率的一半时所对应的模板DNA浓度。
以野生型KOD型DNA聚合酶融合蛋白(KOD-WT)为对照。
KOD型DNA聚合酶点突变体为Ue1E、Ue1E5、Ue1E6，制备方法实施例1；
KOD型DNA聚合酶点突变体Ue1E的氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，且将第409位酪氨酸突变为丙氨酸，且将第485位丙氨酸突变为谷氨酸；其编码基因核苷酸序列为将序列2第1222位核苷酸C突变为A、第1224位核苷酸G为突变为T，且将第1225为核苷酸T突变为G、第1226位核苷酸A突变为C、第1227位核苷酸T突变为A，且将第1454位核苷酸C突变为A。
KOD型DNA聚合酶点突变体Ue1E5的氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第53位赖氨酸突变为甲硫氨酸，第199位赖氨酸突变为甲硫氨酸，第243位精氨酸突变位甲硫氨酸，第526位赖氨酸突变为甲硫氨酸，第674位赖氨酸突变为丙氨酸，第709为精氨酸突变为甲硫氨酸；其编码基因核苷酸序列为将序列2第1222位核苷酸C突变为A、第1224位核苷酸G为突变为T，且将第1225为核苷酸T突变为G、第1226位核苷酸A突变为C、第1227位核苷酸T突变为A，且将第1454位核苷酸C 突变为A，且将第158位核苷酸A突变为T、第159位核苷酸A突变为G，且将第596位核苷酸A突变T、第597位核苷酸突变为G，且将第727位核苷酸C突变为A、第728位核苷酸G突变为T、第729位核苷酸C突变为G，且将第1577位核苷酸A突变为T、第1578位核苷酸A突变为G，且将第2021位核苷酸A突变为T，且将第2125位核苷酸C突变为G、第2126位核苷酸G突变为T、第2127位核苷酸T突变为G。
KOD型DNA聚合酶点突变体Ue1E6其氨基酸序列为将序列1的第408位亮氨酸突变为异亮氨酸，第409位酪氨酸突变为丙氨酸，第485位丙氨酸突变为谷氨酸，第59位组氨酸突变为苯丙氨酸，第558位赖氨酸突变为甲硫氨酸，第613位精氨酸突变为甲硫氨酸，第641位精氨酸突变为甲硫氨酸，第671位赖氨酸突变为甲硫氨酸，第673位酪氨酸突变为苯丙氨酸，第692位赖氨酸突变为甲硫氨酸；其编码基因核苷酸序列为将序列2第1222位核苷酸C突变为A、第1224位核苷酸G为突变为T，且将第1225为核苷酸T突变为G、第1226位核苷酸A突变为C、第1227位核苷酸T突变为A，且将第1454位核苷酸C突变为A，且将第175位核苷酸C突变为T、第176位核苷酸A突变为T，且将第1673位核苷酸A突变为T、第1674位核苷酸A突变为G，且将第1837位核苷酸C突变为A、第1838位核苷酸G突变为T、第1839位核苷酸突变T为G，且将第1921位核苷酸C突变为A、1922位核苷酸G突变为T、1923位核苷酸T突变为G，且将第2012位核苷酸A突变为T、第2013位核苷酸A突变为G，且将第2018位核苷酸A突变为T，且将第2075位核苷酸A突变为T。
融合蛋白为在各自突变体的C端加入6个his标签。
Michaelis曲线如图2所示，可以看出，在以单链DNA为模板时，与野生型KOD聚合酶(KOD-WT)相比，突变体Ue1E、Ue1E5、Ue1E6相比表现出较好的聚合活性。
另外，本结果说明调整酶的Km值大小近似酶与DNA亲和力大小时，并未影响酶的聚合活性即催化反应效率，达到理想的突变修饰酶体的DNA结合亲和力而不影响其特定的催化性质。增大Km值有利于酶与DNA分子快速解离，从而可以在一定程度上加快反应完成，另一方面有利于酶从芯片表面的洗脱。
三、KOD型DNA聚合酶点突变体融合蛋白Ue1E5芯片活性测试
本实验选取上述一种的KOD型DNA聚合酶突变体Ue1E5(相对于野生型氨基酸序列408、409、485、53、199、243、526、674、709位氨基酸突变为异亮氨酸、丙氨酸、谷氨酸、甲硫氨酸、甲硫氨酸、甲硫氨酸、甲硫氨酸、丙氨酸、甲硫氨酸)进行测试实验。
将模板DNA(由金斯瑞公司合成，序列表中序列7)固定于生物芯片gencbio氨基基片(Gencbio，GCB01003)上，测试KOD型DNA聚合酶点突变体融合蛋白Ue1E5在非水溶液中与模板DNA的反应，具体实验方法如下：
利用0.2M PH7.0的PB(0.2M NaH2PO4,0.2M Na2HPO4)缓冲液，清洗芯片，后进行模板DNA固定反应，加入适量浓度为30ng/μl模板DNA使其充满整张芯片，于室温反应15min，使带负电的模板DNA能充分固定在带正电的氨基化修饰的芯片表面,利用酶反应缓冲液(20mM Tris-HCl、10mM(NH4)2SO4、10mM KCl、2mM MgSO4pH 8.8，反应温度25℃)清洗芯片，以除去多余模板DNA。再加入含2U突变体Ue5D8聚合酶、1μM dATP-Cy3、10μM dTTP,、10μM dCTP、10μM dGTP的酶反应液(酶反应缓冲液为0.2M PH7.0的PB)，使酶反应液充满整张芯片，于65℃反应15min，后以酶反应缓冲液清洗芯片两次，以除去多余未参与反应的dNTP。完成反应后，利用荧光显微镜按荧光标记物Cy3的检测条件(530nm激发波长、568nm发射波长)对芯片进行观察和拍照。对照组野生型的KOD聚合酶采用同样方法进行实验。
图中若亮点越多、越密集说明反应充分，另一方面则说明酶与固定在芯片上的模板DNA不仅发生了反应而且还表现出较好的聚合活性。
芯片反应结果如图3所示，其中A为野生型KOD聚合酶，B为突变体Ue1E5聚合酶，可以看出，图3A中芯片亮点很少，说明未经改造或突变的野生型KOD聚合酶与芯片表面的模板DNA反应时其活性较差或无活性；相反，图3B中芯片亮点多且密集，说明改造后的突变体Ue1E5与野生型相比在与芯片表面的模板DNA反应时其活性较好。
采用同样的方法检测KOD型DNA聚合酶点突变体融合蛋白Ue1E、Ue1E6，结果与Ue1E5无显著差异，均具有较好的聚合活性。
融合蛋白为在各自突变体的C端加入6个his标签。
四、KOD型DNA聚合酶突变体Ue1E5芯片洗脱测试
在芯片反应中，上一轮反应残留物会干扰下一轮反应，且直接影响整张芯片的拍照效果，在实验中完成移除上一轮反应残留物是理想状态。
本实验以KOD型DNA聚合酶突变体Ue1E5为例进行KOD型DNA聚合酶芯片洗脱测试，同时以商业酶Therminator(TherminatorTMDNA Polymerase，NEB,M0261S)为对照进行实验。具体实验方法如下：
芯片前期处理及芯片活性反应与上述芯片活性测试实验方法相同。
完成拍照后，利用酶反应缓冲液(20mM Tris-HCl、10mM(NH4)2SO4、10mM KCl、2mM MgSO4pH 8.8@25℃)清洗芯片3-4次，后加入不含酶但包括dNTP和反应缓冲液的酶反应，于65℃反应15min，后以酶反应缓冲液清洗芯片两次，利用荧光显微镜按荧光标记物Cy3的检测条件(530nm激发波长、568nm发射波长)对芯片进行观察和拍照。
洗脱实验中，若图片亮点越多，说明有反应的发生即说明酶未洗脱干净，说明此酶不易洗脱。
实验结果如图4所示，其中A为空白对照即空白芯片，B为Therminator，C为聚合酶突变体融合蛋白Ue1E5，可以看出，图4A为空白芯片因而无反应，图片未见亮点；图4B为商业酶Therminator，其图片亮点与空白对照相比亮点较多；图4C为突变体Ue1E5，其与对照相比仍有少许亮点，但与Therminator相比明显少很多，说明本突变体洗脱效果好于Therminator，说明突变体易于洗脱，不易吸附芯片表面，不干扰后续实验。
野生型由于聚合活性差，无法进行洗脱实验，故以商业的Therminator为对照。
采用同样的方法检测KOD型DNA聚合酶点突变体融合蛋白Ue1E6，结果与Ue1E5无显著差异，均比Therminator易于洗脱。
采用同样的方法其他KOD型DNA聚合酶突变体，也易于洗脱，不易吸附芯片表面。
实施例3、C端截短型KOD型DNA聚合酶突变体融合蛋白的活性测试
融合蛋白为在各自突变体的C端加入6个his标签。
将实施例1的三制备的C端截短型KOD型DNA聚合酶突变体融合蛋白△ Ue1A、ΔUe1B、ΔUe1C、ΔUe1D、ΔUe1E、ΔUe2A、ΔUe2B、ΔUe2C、ΔUe2D、ΔUe2E、ΔUe3A、ΔUe3B、ΔUe3C、ΔUe3D、ΔUe3E、ΔUe4A、ΔUe4B和△Ue4C分别按照实施例2中的二检测聚合活性的方法和三中检测芯片活性的方法进行检测。
以KOD型DNA聚合酶点突变体融合蛋白Ue1A、Ue1B、Ue1C、Ue1D、Ue1E、Ue2A、Ue2B、Ue2C、Ue2D、Ue2E、Ue3A、Ue3B、Ue3C、Ue3D、Ue3E、Ue4A、Ue4B和Ue4C6为对照。
结果C端截短型KOD型DNA聚合酶突变体融合蛋白△Ue1A、ΔUe1B、ΔUe1C、ΔUe1D、ΔUe1E、ΔUe2A、ΔUe2B、ΔUe2C、ΔUe2D、ΔUe2E、ΔUe3A、ΔUe3B、ΔUe3C、ΔUe3D、ΔUe3E、ΔUe4A、ΔUe4B和△Ue4C的结果与KOD型DNA聚合酶突变体融合蛋白Ue1A、Ue1B、Ue1C、Ue1D、Ue1E、Ue2A、Ue2B、Ue2C、Ue2D、Ue2E、Ue3A、Ue3B、Ue3C、Ue3D、Ue3E、Ue4A、Ue4B和Ue4C6无显著差异。
表明，C端29个氨基酸删除不影响聚合酶的活性。
采用实施例2的四的方法检测本实施例的C端截短型KOD型DNA聚合酶突变体融合蛋白，也易于洗脱，不易吸附芯片表面。
工业应用
本发明的实验证明，本发明制备的DNA聚合酶突变体，能够利用人工修饰的核苷酸或核苷酸类似物作为底物进行聚合反应，表现出较好的聚合活性及不易被吸附于芯片表面、易于从芯片表面洗脱，使之能够在每个反应循环中加入单个核苷酸或核苷酸类似物且易于洗脱而不对下一个测序循环造成干扰。

Claims (14)

  1. 一种蛋白质，是如下A)-C)中任一种：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第408、409和485位氨基酸残基进行修饰，且对所述野生型KOD型DNA聚合酶氨基酸序列中第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少一位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白；
    B)所示的蛋白为将A)所示蛋白的氨基酸序列C端的自末端起1-29个氨基酸去除，保留其余氨基酸残基且具有DNA聚合酶活性的由A)衍生的蛋白质；
    C)所示的蛋白为将A)或B)所示蛋白的氨基酸序列末端添加标签序列且具有DNA聚合酶活性的由A)或B)衍生的蛋白质。
  2. 根据权利要求1所述的蛋白质，其特征在于：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第408、409和485位氨基酸残基进行修饰，且对所述野生型KOD型DNA聚合酶氨基酸序列中第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少2位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
  3. 根据权利要求1或2所述的蛋白质，其特征在于：
    A)所示的蛋白为将野生型KOD型DNA聚合酶氨基酸序列中第408、409和485位氨基酸残基进行修饰，且对所述野生型KOD型DNA聚合酶氨基酸序列中第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少3位的氨基酸残基进行修饰，得到具有DNA聚合酶活性的蛋白。
  4. 根据权利要求1-3中任一所述的蛋白质，其特征在于：所述修饰为氨基酸置换。
  5. 根据权利要求4所述的蛋白质，其特征在于：
    第408位的亮氨酸置换为异亮氨酸、酪氨酸、脯氨酸、缬氨酸、半胱氨酸、丝氨酸、丙氨酸或甲硫氨酸；
    第409位的酪氨酸置换为丙氨酸或甘氨酸；
    第485位的丙氨酸置换为组氨酸、天冬酰胺、赖氨酸、天冬氨酸或谷氨酸；
    第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少一位的氨基酸残基置换为甲硫氨酸或苯丙氨酸或丙氨酸。
  6. 根据权利要求5所述的蛋白质，其特征在于：
    所述第53、59、199、243、526、558、613、641、671、673、674、692和709位这13位中至少一位的氨基酸残基置换为甲硫氨酸或苯丙氨酸或丙氨酸为如下：
    第53位的赖氨酸置换为甲硫氨酸；
    第59位的组氨酸置换为苯丙氨酸；
    第199位的赖氨酸置换为甲硫氨酸；
    第243位的精氨酸置换为甲硫氨酸；
    第526位的赖氨酸置换为甲硫氨酸；
    第558位的赖氨酸置换为甲硫氨酸；
    第613位的精氨酸置换为甲硫氨酸；
    第641位的精氨酸置换为甲硫氨酸；
    第671位的赖氨酸置换为甲硫氨酸；
    第673位的酪氨酸置换为苯丙氨酸；
    第674位的赖氨酸置换为丙氨酸；
    第692位的赖氨酸置换为甲硫氨酸；
    第709位的精氨酸置换为甲硫氨酸。
  7. 根据权利要求1-5中任一所述蛋白，其特征在于：所述野生型KOD型DNA聚合酶氨基酸序列为序列1。
  8. 根据权利要求1-6中任一所述蛋白，其特征在于：
    所述蛋白质的对生物芯片的吸附能力低于所述野生型KOD型DNA聚合酶。
  9. 根据权利要求7所述蛋白，其特征在于：
    所述蛋白质的对生物芯片的吸附能力低于所述野生型KOD型DNA聚合 酶体现在与所述野生型KOD型DNA聚合酶相比，所述蛋白质不易吸附于生物芯片表面和/或易于从生物芯片表面洗脱。
  10. 编码权利要求1-8中任一所述蛋白的DNA分子；
    或含有所述DNA分子的表达盒、重组载体、重组菌或转基因细胞系。
  11. 权利要求1-8中任一所述蛋白或权利要求9所述DNA分子或所述表达盒、重组载体、重组菌或转基因细胞系在制备DNA聚合酶中的应用。
  12. 根据权利要求10所述的应用，其特征在于：所述DNA聚合酶的对生物芯片的吸附能力低于所述野生型KOD型DNA聚合酶；
    或，所述DNA聚合酶的对生物芯片的吸附能力低于所述野生型KOD型DNA聚合酶体现在与所述野生型KOD型DNA聚合酶相比，所述DNA聚合酶不易吸附于生物芯片表面和/或易于从生物芯片表面洗脱。
  13. 权利要求1-8中任一所述蛋白或权利要求9所述DNA分子或所述的表达盒、重组载体、重组菌或转基因细胞在测序中的应用；
    或权利要求1-8中任一所述蛋白或权利要求9所述DNA分子或所述的表达盒、重组载体、重组菌或转基因细胞在制备用于测序的产品中的应用。
  14. 根据权利要求12所述的应用，其特征在于：所述产品为试剂盒。
PCT/CN2017/070609 2017-01-09 2017-01-09 重组dna聚合酶 WO2018126470A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP17889883.9A EP3613851A4 (en) 2017-01-09 2017-01-09 RECOMBINANT DNA POLYMERASE
PCT/CN2017/070609 WO2018126470A1 (zh) 2017-01-09 2017-01-09 重组dna聚合酶
CN201780069442.3A CN109937252B (zh) 2017-01-09 2017-01-09 重组dna聚合酶
US16/505,121 US10883091B2 (en) 2017-01-09 2019-07-08 DNA polymerase variant and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/070609 WO2018126470A1 (zh) 2017-01-09 2017-01-09 重组dna聚合酶

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US16/505,121 Continuation-In-Part US10883091B2 (en) 2017-01-09 2019-07-08 DNA polymerase variant and application thereof

Publications (1)

Publication Number Publication Date
WO2018126470A1 true WO2018126470A1 (zh) 2018-07-12

Family

ID=62789044

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/070609 WO2018126470A1 (zh) 2017-01-09 2017-01-09 重组dna聚合酶

Country Status (4)

Country Link
US (1) US10883091B2 (zh)
EP (1) EP3613851A4 (zh)
CN (1) CN109937252B (zh)
WO (1) WO2018126470A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020048329A1 (zh) * 2018-09-03 2020-03-12 深圳华大生命科学研究院 重组型kod聚合酶
US11001816B2 (en) 2018-12-05 2021-05-11 Illumina, Inc. Polymerases, compositions, and methods of use
US11104888B2 (en) 2018-10-31 2021-08-31 Illumina, Inc. Polymerases, compositions, and methods of use
WO2022247055A1 (zh) * 2021-05-26 2022-12-01 深圳华大生命科学研究院 耐热b家族dna聚合酶突变体及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108130318B (zh) * 2018-02-28 2020-07-14 深圳市艾伟迪生物科技有限公司 突变型Taq DNA聚合酶、免核酸提取直接PCR扩增的试剂盒及其应用
WO2022082482A1 (zh) * 2020-10-21 2022-04-28 深圳华大生命科学研究院 重组型kod聚合酶

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104854237A (zh) * 2012-12-13 2015-08-19 霍夫曼-拉罗奇有限公司 具有改进活性的dna聚合酶
WO2015200693A1 (en) * 2014-06-27 2015-12-30 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009131919A2 (en) * 2008-04-22 2009-10-29 New England Biolabs, Inc. Polymerases for incorporating modified nucleotides
US8993255B2 (en) * 2008-10-10 2015-03-31 Toyo Boseki Kabushiki Kaisha Protein having fructosyl valyl histidine oxidase activity, modified protein, and use of the protein or the modified protein
CN103013945B (zh) * 2013-01-11 2014-06-11 华东理工大学 一种环氧水解酶突变体及其基因和应用
WO2016054096A1 (en) * 2014-09-30 2016-04-07 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104854237A (zh) * 2012-12-13 2015-08-19 霍夫曼-拉罗奇有限公司 具有改进活性的dna聚合酶
WO2015200693A1 (en) * 2014-06-27 2015-12-30 Illumina, Inc. Modified polymerases for improved incorporation of nucleotide analogues

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HASHIMOTO, H: "Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KODI", JOURNAL OF MOLECULAR BIOLOGY, vol. 306, no. 3, 23 February 2001 (2001-02-23), pages 469 - 477, XP004469251, ISSN: 0022-2836 *
NISHIOKA, M. ET AL., J. BIOTECHNOL., vol. 88, 2001
See also references of EP3613851A4

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020048329A1 (zh) * 2018-09-03 2020-03-12 深圳华大生命科学研究院 重组型kod聚合酶
CN112585264A (zh) * 2018-09-03 2021-03-30 深圳华大生命科学研究院 重组型kod聚合酶
CN112639089A (zh) * 2018-09-03 2021-04-09 深圳华大生命科学研究院 重组型kod聚合酶
CN112639089B (zh) * 2018-09-03 2024-02-09 深圳华大生命科学研究院 重组型kod聚合酶
US11104888B2 (en) 2018-10-31 2021-08-31 Illumina, Inc. Polymerases, compositions, and methods of use
US11560552B2 (en) 2018-10-31 2023-01-24 Illumina, Inc. Polymerases, compositions, and methods of use
US11001816B2 (en) 2018-12-05 2021-05-11 Illumina, Inc. Polymerases, compositions, and methods of use
US11634697B2 (en) 2018-12-05 2023-04-25 Illumina, Inc. Polymerases, compositions, and methods of use
WO2022247055A1 (zh) * 2021-05-26 2022-12-01 深圳华大生命科学研究院 耐热b家族dna聚合酶突变体及其应用

Also Published As

Publication number Publication date
US10883091B2 (en) 2021-01-05
CN109937252A (zh) 2019-06-25
EP3613851A4 (en) 2020-07-01
US20190330602A1 (en) 2019-10-31
CN109937252B (zh) 2023-03-28
EP3613851A1 (en) 2020-02-26

Similar Documents

Publication Publication Date Title
WO2018126470A1 (zh) 重组dna聚合酶
CN107257854B (zh) 聚合酶变体
CN112639089B (zh) 重组型kod聚合酶
US10759830B2 (en) Bacterial colicin-immunity protein protein purification system
JP6963238B2 (ja) Ｄｎａポリメラーゼ変異体
CN110914415A (zh) 工程化连接酶变体
AU2020319829A1 (en) Template-free enzymatic synthesis of polynucleotides using poly(A) and poly(U) polymerases
WO2018107521A1 (zh) T4多聚核苷酸激酶重组酶及其制备方法、表达基因、表达载体以及宿主细胞
CN112175980B (zh) 通过定点突变提高聚合酶大片段活性的方法及应用
CN112899253A (zh) 具有dna聚合酶活性的多肽、重组载体及其制备方法与应用
CN109266628B (zh) 一种融合的TaqDNA聚合酶及其应用
CN108795900B (zh) Dna聚合酶及其制备方法
WO2009113718A1 (ja) 機能改善したｒｎａポリメラーゼ変異体
US8981067B2 (en) Composition, method and kit for obtaining purified recombinant proteins
US20120135472A1 (en) Hot-start pcr based on the protein trans-splicing of nanoarchaeum equitans dna polymerase
CN114829593B (zh) 嵌合dna聚合酶及其应用
WO2010035756A1 (ja) イムノグロブリン結合タンパク質
JP5324083B2 (ja) 高反応性耐熱性ｄｎａリガーゼ
WO2022082482A1 (zh) 重组型kod聚合酶
US20230374475A1 (en) Engineered thermophilic reverse transcriptase
US20230220449A1 (en) Marine dna polymerase i
WO2024083883A1 (en) Methods and products for removal of uracil containing polynucleotides
CN117866923A (zh) 一种LdCsm突变型复合物、含该突变型复合物的检测系统及其应用
CN112813088A (zh) 重组DpnI限制性内切酶的制备方法
WO2019108660A1 (en) Zinc finger moiety attached to a resin used to purify polynucleotide molecules

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17889883

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2017889883

Country of ref document: EP