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WO2018107521A1 - T4 polynucléotide kinase recombinase et procédé de préparation, gène d'expression, vecteur d'expression et cellule hôte associés - Google Patents

T4 polynucléotide kinase recombinase et procédé de préparation, gène d'expression, vecteur d'expression et cellule hôte associés Download PDF

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WO2018107521A1
WO2018107521A1 PCT/CN2016/112427 CN2016112427W WO2018107521A1 WO 2018107521 A1 WO2018107521 A1 WO 2018107521A1 CN 2016112427 W CN2016112427 W CN 2016112427W WO 2018107521 A1 WO2018107521 A1 WO 2018107521A1
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polynucleotide
expression
seq
polypeptide
host cell
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Chinese (zh)
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章瑞程
史萍
蔡统聪
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Fapon Biotech Inc
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Fapon Biotech Inc
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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
    • 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)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01078—Polynucleotide 5'-hydroxyl-kinase (2.7.1.78)
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

Definitions

  • the invention relates to the field of biotechnology, in particular to a T4 polynucleotide kinase recombinase, a preparation method thereof, an expression gene, an expression vector and a host cell.
  • T4 Polynucleotide Kinase (T4 PNK), a polynucleotide 5' hydroxy kinase that catalyzes the gamma phosphate group of ATP to single or double stranded DNA, RNA, oligo 5' hydroxyl transfer of a nucleotide or a single nucleotide with a 3' phosphate group.
  • Other NTPs can also produce the same reaction: 5'-OH+NTP ⁇ 5'-P+NDP.
  • T4 polynucleotide kinase can exhibit 5' phosphatase activity, catalyzing single- or double-stranded DNA, RNA, oligonucleotides or with 3' phosphate groups Transfer of the 5' phosphate group of a single nucleotide to ADP forms ATP.
  • Other NTPs can also produce the same reaction: 5'-P+NDP ⁇ 5'-OH+NTP (optimal pH is around 6.4).
  • T4 polynucleotide kinase can catalyze single- or double-stranded DNA, RNA, oligonucleotides or 5' phosphate groups of single nucleotides with a 3' phosphate group. Exchange reaction with the gamma phosphate group of ATP. Other NTPs can also produce the same reaction: 5'-P+NTP+NDP ⁇ 5'-P+NDP+NTP.
  • T4 PNK also has 3' phosphatase activity, which can catalyze the dephosphorylation of 3' phosphorylated polynucleotide: 3'-P ⁇ 3'-OH+Pi (optimum pH is about 5.9).
  • the kinase activity of T4 PNK is near the C-terminus, while the phosphatase activity is near the N-terminus.
  • T4 PNK is widely used, such as oligonucleotides, DNA or RNA 5' end labeling, as probes for Southern, Northern, EMSA, etc., gel electrophoresis markers, DNA sequencing primers, PCR primers, etc.; Phosphorylation of the 5' end of acid, DNA or RNA ensures that the subsequent ligation reaction proceeds smoothly; catalyzing the 5' phosphorylation of the 3' phosphorylated single nucleotide, allowing the single nucleotide to interact with the 3' end of the DNA or RNA Even The 3' terminal phosphate group is removed.
  • T4 polynucleotide kinase cloned by genetic engineering technology is highly expressed in the pET system, but the following problems exist: easy expression and formation in the absence of soluble label In vivo, a small amount of active protein can be obtained by renaturation of inclusion bodies, but during storage, proteins tend to aggregate to form aggregates, forming precipitates with poor stability; increasing soluble label can increase the ratio of soluble protein, but the protein activity after purification is better. Low, if the label removal cost is high and the resulting active protein is less stable.
  • T4 polynucleotide kinase recombinase with better stability and higher activity.
  • T4 polynucleotide kinase recombinase it is also necessary to provide a method for producing the above T4 polynucleotide kinase recombinase, an expression gene, an expression vector, and a host cell.
  • a T4 polynucleotide kinase recombinase comprising:
  • An expression gene including:
  • polypeptide (c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1, wherein one or more amino acids are deleted, replaced or increased, and The polypeptide has beta-glucanase activity; or
  • polypeptide (d) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide being SEQ ID
  • the polypeptide consisting of the amino acid sequence shown in No. 1 has at least 98% homology.
  • An expression gene including:
  • polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polynucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID No. 2, wherein one or more codons are deleted , substitute or increase.
  • An expression vector comprising the above expressed gene.
  • the expression vector is pET-28a.
  • a host cell comprising the above gene expression vector, the host cell being a prokaryotic cell.
  • the host cell is E. coli.
  • the host cell is BL21.
  • a method for preparing a T4 polynucleotide kinase recombinase comprises the following steps:
  • the host cells after the expression is completed are lysed and centrifuged, and the supernatant is retained;
  • the supernatant was purified to obtain the T4 polynucleotide kinase recombinase.
  • the expression temperature is from 16 ° C to 20 ° C
  • the final concentration of the inducer is from 0.2 mM to 0.5 mM
  • the time of induction of expression is from 12 h to 24 h.
  • the T4 polynucleotide kinase recombinase was recombined by genetic engineering technology. After expression, the T4 polynucleotide kinase recombinase has good stability and high activity.
  • Figure 1 is a SDS-PAGE (12%) analysis of recombinant T4 PNK protein in Example 2, wherein lane M is protein Marker, lanes 1 and 2 are before induction, and lanes 3 and 4 are induced whole-protein Expression, protein Marker molecular weight of 116kDa, 66kDa, 45kDa, 35kDa, 25kDa, 18.4kDa and 14.4kDa;
  • Example 2 is a SDS-PAGE (12%) analysis diagram of recombinant T4 PNK in Example 2, wherein lane M is protein Marker, lane 1 is the original enzyme, lane 2 is diluted 5 times with the original enzyme, and lane 3 is diluted with the original enzyme. 10 times, Lane 4 is diluted 20 times with the original enzyme;
  • FIG. 3 is a graph comparing the activity of recombinant T4 PNK and NEB-T4 PNK in Example 2 with SDS-PAGE (12%), wherein M is Marker, lane 1 is 100 U, lane 2 is 200 U, and lane 3 is 400 U. Lane 4 is 600U.
  • the invention discloses a T4 polynucleotide kinase recombinase, comprising:
  • the T4 polynucleotide kinase recombinase was recombined by genetic engineering technology. After expression, the T4 polynucleotide kinase recombinase has good stability and high activity.
  • the invention also discloses an expression gene of the above T4 polynucleotide kinase recombinase according to an embodiment, which comprises the followings:
  • polypeptide (c) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide being SEQ ID
  • polypeptide (d) a polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polypeptide having at least 98% homology to a polypeptide consisting of the amino acid sequence set forth in SEQ ID No. 1.
  • the invention also discloses another expression gene of the above T4 polynucleotide kinase recombinase, which comprises the following:
  • polynucleotide encoding a polypeptide or a complementary strand of said polynucleotide, said polynucleotide sequence consisting of the nucleotide sequence set forth in SEQ ID No. 2, wherein one or more codons are deleted , substitute or increase.
  • the present invention also discloses an expression vector of the above T4 polynucleotide kinase recombinase according to an embodiment, which comprises the above-mentioned expression gene.
  • the expression vector is pET-28a.
  • the present invention also discloses a host cell of the above T4 polynucleotide kinase recombinase according to an embodiment, comprising the above gene expression vector, wherein the host cell is a prokaryotic cell.
  • the host cell is Escherichia coli.
  • the host cell is E. coli BL21 (DE3) (purchased from Invitrogen, USA).
  • the invention also discloses a preparation method of the above T4 polynucleotide kinase recombinase according to an embodiment, comprising the following steps:
  • the host cell can be obtained by genetic engineering technology, and the recombinant T4 PNK prokaryotic expression vector was constructed, and the codon-optimized T4 PNK gene was successfully cloned into the His-tagged pET vector to construct the above T4.
  • the pET vector expressing the gene of the polynucleotide kinase recombinase is correctly transformed, and the pET vector containing the expression gene of the T4 polynucleotide kinase recombinase is transformed into a host cell and stored in glycerol.
  • the expression temperature is 16 ° C to 20 ° C (preferably 18 ° C)
  • the final concentration of the inducer is 0.2 mM to 0.5 mM (preferably 0.25 mM)
  • the time for inducing expression is 12 h to 24 h (preferably 16h).
  • the host cells obtained after the expression of S10 are lysed and then centrifuged, and the supernatant is retained.
  • the S20 is obtained by suspending and mixing the host cells after the expression is completed in an ice bath, and then lysing the cells by ice bath, centrifuging and retaining the supernatant.
  • the lysis buffer included 50 mM Tris, 1.5 M NaCl, 10 wt% glycerol, 0.1 wt% Triton X-100, 1 mM PMSF (phenylmethylsulfonyl fluoride), and 20 mM Imidazole (imidazole), and the pH of the lysis buffer was 7.5.
  • S30 is specifically: the supernatant is equilibrated by a lysis buffer, and the Ni column is fully equilibrated with the lysis buffer after the sample is completed, and then washed thoroughly with the pre-wash buffer; further eluting the protein with the elution buffer, the Ni affinity layer
  • the analysis has removed a large portion of the heteroprotein, and ion exchange removes trace amounts of the heteroprotein and nucleic acid.
  • the prewash buffer included 50 mM Tris, 0.5 M NaCl, 10 wt% glycerol, 0.1 wt% Triton X-100, and 100 mM Imidazole, and the pH of the prewash buffer was 7.5.
  • the elution buffer included 50 mM Tris, 500 mM NaCl, 10 wt% glycerol, 0.1 wt% Triton X-100, and 0.5 M Imidazole, and the pH of the elution buffer was 7.5.
  • T4 PNK The gene of T4 PNK was cloned into the pET vector by vector construction, and the expression was induced under low temperature conditions.
  • the soluble ratio of the expressed protein was increased by low temperature and low IPTG concentration, and the soluble ratio of the expressed protein was increased by using high salt lysis buffer.
  • the solubility of the expressed protein is greatly increased.
  • the purification of T4 PNK is simpler and more convenient due to the addition of His tag.
  • the T4 polynucleotide kinase recombinase was recombined by genetic engineering technology. After expression, the T4 polynucleotide kinase recombinase has good stability and high activity.
  • the recombinant T4 PNK gene was successfully constructed by recombinant T4 PNK prokaryotic expression vector, and the codon-optimized T4 PNK gene was successfully cloned into His-tagged pET vector to construct the expression gene containing T4 polynucleotide kinase recombinase.
  • the pET vector (sequence as shown in SEQ ID No. 2) was transformed into the BL21 (DE3) (purchased from Invitrogen, USA) after the sequencing result was correct, and the pET vector containing the expression gene of the T4 polynucleotide kinase recombinase was transformed. In the case, the strain is obtained and stored in glycerin.
  • 100 ⁇ L of the above strain was inoculated into 10 mL of LB medium containing 50 mg/mL kanamycin; cultured for 2-3 hours until the OD600 was 1.0 or so, and 5 mL of the bacterial solution was transferred to 2 bottles containing 50 mg/mL card.
  • 500 mL LB medium of nalpenicillin the culture was continued for 3-4 hours until the OD600 was about 0.6.
  • the expression of T4 PNK was induced by adding IPTG at a final concentration of 0.25 mM.
  • the expression temperature was controlled at 18 ° C, and 16 cells were induced. After a few hours, the bacterial cells were collected. 1 L of the bacterial liquid was collected by centrifugation at 7000 rpm in a 400 mL centrifuge cup at 4 ° C for 3 min.
  • 1 L of the bacterial cells were collected, suspended and mixed in a 100 mL ice bath lysis buffer containing 50 mM Tris, 1.5 M NaCl, 10% glycerol, 0.1% Triton X-100, 0.2 mM PMSF, pH 7.5, 20 mM Imidazole; ultrasonically lyse the cells in ice bath (retain 40 ⁇ L of the whole cell suspension), centrifuge to remove the supernatant (retain 40 ⁇ L of the bacterial cell disrupted supernatant), and resuspend the pellet with an equal volume of lysis buffer (retain 40 ⁇ L of bacteria) Body broken sediment).
  • ain 40 ⁇ L of bacteria body broken sediment
  • Example 2 SDS-PAGE (12%) protein electrophoresis to verify the size and purity of T4 PNK protein
  • the recombinant T4 PNK obtained in Example 1 was subjected to SDS-PAGE protein electrophoresis detection to identify the size and purity of recombinant T4 PNK.
  • T4 PNK and NEB-T4 PNK obtained in Example 1 were separately diluted by the commercial T4 PNK (NEB-T4 PNK) of NEB, and each sample was 40 ⁇ L plus 5 ⁇ Loading Buffer 10 ⁇ L.
  • the boiling water bath was used for 10 min, and each sample was loaded with 5 ⁇ L of the protein Marker, and the concentrated gel was pressed at a constant pressure of 100 V, and the gel was separated at a constant pressure of 180 V, and the test was performed by Coomassie Brilliant Blue, and FIG. 3 was obtained.
  • each electrophoresis strip is pressed into a straight line in the concentrated gel; from the strip position in Figure 1, combined with the sequencing results, the recombinant T4 PNK is correctly determined; 18 ° C, 0.25 mM IPTG induction, The expression level of recombinant T4 PNK can reach more than 10%, and the solubility can reach more than 80%.
  • Ni affinity chromatography has removed most of the heteroproteins, and ion exchange can remove trace amounts of heteroproteins and nucleic acids.
  • Example 1 (4) Referring to Fig. 3, the recombinant T4 PNK obtained in Example 1 exhibited an enzyme activity similar to that of the commercial enzyme of NEB, and the specific activity was about 10 U/ ⁇ g.
  • a 1 L fermentation broth can obtain about 1 L of a 10 U/ ⁇ L T4 PNK working solution.
  • Example 3 Determination of activity of recombinant T4 PNK enzyme
  • T4 PNK enzyme The activity of recombinant T4 PNK enzyme was determined by [ ⁇ - 32 P]ATP incorporation.
  • Activity is defined as: Micrococcal Nuclease-treated calf thymus DNA as a substrate, required to incorporate 1 nmol of [ ⁇ - 32 P]ATP into an acid-insoluble precipitate at 37 ° C, pH 7.6 for 30 minutes.
  • the amount of enzyme is defined as 1 unit of activity.

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Abstract

L'invention concerne une T4 polynucléotide kinase recombinase et un procédé de préparation, un gène d'expression, un vecteur d'expression et une cellule hôte associés. La T4 polynucléotide kinase recombinase comprend : (a) un polypeptide composé de la séquence d'acides aminés représentée par la SEQ ID No. 1 ; et (b) un polypeptide ayant au moins 98 % d'homologie avec le polypeptide composé de la séquence d'acides aminés représentée par la SEQ ID No. 1 ; ou (c) un polypeptide obtenu par délétion, remplacement ou addition d'un ou plusieurs acides aminés du polypeptide composé de la séquence d'acides aminés représentée par la SEQ ID No. 1. La T4 polynucléotide kinase recombinase est obtenue par recombinaison à l'aide d'une technologie de génie génétique.
PCT/CN2016/112427 2016-12-12 2016-12-27 T4 polynucléotide kinase recombinase et procédé de préparation, gène d'expression, vecteur d'expression et cellule hôte associés Ceased WO2018107521A1 (fr)

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Cited By (2)

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CN113355379A (zh) * 2021-02-04 2021-09-07 康龙化成(北京)新药技术股份有限公司 一种经济实用的核酸链5’-羟基磷酸化方法
CN114836448A (zh) * 2022-05-19 2022-08-02 安诺优达基因科技(北京)有限公司 一种密码子优化的t4多聚核苷酸激酶的核酸分子及其表达方法

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CN114480568B (zh) * 2020-12-30 2024-04-26 安诺优达基因科技(北京)有限公司 一种磷酸化反应的检测方法及试剂盒
CN113512541B (zh) * 2021-04-29 2022-07-08 温州医科大学 一种新型磷酸化腺苷酰化酶及其制备方法与应用
CN113930405B (zh) * 2021-10-21 2022-07-19 温州医科大学 一种新型热稳定磷酸化和腺苷酰化一步法催化酶及其制备方法与应用

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CN104178467A (zh) * 2014-07-24 2014-12-03 孙启明 一种重组t4噬菌体多核苷酸激酶及其制备方法

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MIDGLEY: "polynucleotide kinase; cloning of the gene (pseT) and amplification of its product", THE EMBO JOURNALEMBO JOURNAL, vol. 4, no. 10, 31 October 1985 (1985-10-31), pages 2695 - 2703, XP055606568, ISSN: 0261-4189 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113355379A (zh) * 2021-02-04 2021-09-07 康龙化成(北京)新药技术股份有限公司 一种经济实用的核酸链5’-羟基磷酸化方法
CN114836448A (zh) * 2022-05-19 2022-08-02 安诺优达基因科技(北京)有限公司 一种密码子优化的t4多聚核苷酸激酶的核酸分子及其表达方法
CN114836448B (zh) * 2022-05-19 2023-12-05 浙江安诺优达生物科技有限公司 一种密码子优化的t4多聚核苷酸激酶的核酸分子及其表达方法

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