CN115044571A - Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof - Google Patents

Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof Download PDF

Info

Publication number
CN115044571A
CN115044571A CN202210709994.9A CN202210709994A CN115044571A CN 115044571 A CN115044571 A CN 115044571A CN 202210709994 A CN202210709994 A CN 202210709994A CN 115044571 A CN115044571 A CN 115044571A
Authority
CN
China
Prior art keywords
hhh
protein
gpd
sis
dna
Prior art date
Legal status (The legal status 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 status listed.)
Granted
Application number
CN202210709994.9A
Other languages
Chinese (zh)
Other versions
CN115044571B (en
Inventor
张立奎
殷有成
李铮
姜董豪
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangling College Of Yangzhou University
Original Assignee
Guangling College Of Yangzhou University
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 Guangling College Of Yangzhou University filed Critical Guangling College Of Yangzhou University
Priority to CN202210709994.9A priority Critical patent/CN115044571B/en
Publication of CN115044571A publication Critical patent/CN115044571A/en
Application granted granted Critical
Publication of CN115044571B publication Critical patent/CN115044571B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

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
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2497—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing N- glycosyl compounds (3.2.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention discloses extreme thermophilic archaeaSulfolobus islandicusThe amino acid sequence of the REY15A recombinant HhH-GPD (Sis-HhH-GPD) protein is shown as SEQ ID NO.2, and the nucleotide sequence is shown as SEQ ID NO. 1. The invention constructs a genetic engineering bacterium for expressing the Sis-HhH-GPD protein by utilizing a genetic engineering technology, the genetic engineering bacterium can efficiently express the Sis-HhH-GPD protein, the subsequent purification steps are simple and easy to implement, and a large number of recombinase eggs can be easily obtainedWhite. The recombinant Sis-HhH-GPD protein can specifically excise 1-meA in DNA, and the binding capacity of the recombinant Sis-HhH-GPD protein to DNA containing 1-meA is obviously higher than that to normal DNA. The recombinant Sis-HhH-GPD protein provided by the invention is used as a detection reagent for DNA methylation, and has wide application prospects in the fields of medical treatment and molecular biology.

Description

Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an extreme thermophilic archaea recombinant HhH-GPD protein, and a preparation method and application thereof.
Background
It is understood that base methylation is one of the common types of base damage in DNA, and that methylation agents present in the cell endogenously and in the environment cause methylation of bases in DNA, and the current major base methylation classes include 7-methylguanine (N7-methylguanine, 7-meG), 3-methyladenine (3-methyaldine, 3-meA), 6-methylguanine (O6-methylguanine, O6-meG), 1-methyladenine (1-methyaldine, 1-meA), 3-methylguanine (3-methyytosine, 3-meC), 4-methylthymine (O4-methythymine, O4-meT), and Methyl Phosphotriesters (MPT). It was found that 7-meG, 3-meA and O6-meG are the major forms of DNA methylation, while 1-meA, 3-meC, O4-meT and MPT are the forms of DNA with relatively little methylation. It was found that O6-meG and O4-meT have high gene mutagenicity and gene toxicity, 3-meA, 1-meA, 3-meC and 3-meG can prevent DNA replication and transcription, and have cytotoxicity and relatively low gene mutagenicity. Thus, different types of base methylation can cause gene mutations and interfere with cellular DNA replication and repair processes, necessitating the detection of methylated bases in DNA. The existing method for detecting DNA methylation mainly comprises ultra-high performance liquid chromatography-tandem mass spectrometry, photocrosslinking sequencing, single cell real-time sequencing and the like. Although these methods can achieve the goal of detecting DNA methylation, they still have some limitations, such as cumbersome procedures or expensive equipment.
DNA glycosidase is an enzyme which hydrolyzes N-C glycosidic bond in DNA, and further excises damaged base in DNA, so that the DNA glycosidase plays an important role in repairing damaged base of DNA, avoiding cell mutation and detecting DNA mutation. Currently, DNA glycosidases can be divided into monofunctional DNA glycosidases and bifunctional DNA glycosidases. Monofunctional DNA glycosidases excise only the damaged bases in the DNA, thereby forming abasic sites. Bifunctional DNA glycosidases, on the other hand, are capable of cleaving not only specific bases in DNA, but also further cleaving phosphodiester bonds at the resulting base-free sites.
The HhH (Helix-hairpin-Helix) DNA glycosidase superfamily currently comprises members of six families: nth, OggI, MutY/Mig, AlkA, MpgII and OggII. HhH DNA glycosidase can cleave deaminated, oxidized, alkylated bases, etc., from DNA. HhH DNA glycosidases are widely distributed in bacteria, eukaryotes, and archaea, and HhH DNA glycosidases from bacteria and eukaryotes have been studied more and the study of archaea HhH DNA glycosidases is relatively rare. Sulfolobus glaucus (Fr.) KuntzeSulfolobus islandicusREY15A is an important model organism for researching archaea DNA replication and repair, and the genome thereof encodes a HhH-GPD (Sis-HhH-GPD) protein, belonging to the HhH DNA glycosidase superfamily.
Disclosure of Invention
The invention provides an extreme thermophilic archaea recombinant HhH-GPD protein and a preparation method thereof, aiming at solving the technical problems and overcoming the defects of the prior art.
Another object of the present invention is to provide a method for detecting DNA methylation, which does not require expensive instruments, does not require complicated steps, has high practicability, and can efficiently, specifically and sensitively recognize DNA methylation.
The invention provides an extreme thermophilic archaea recombinant HhH-GPD protein, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.
The nucleotide sequence of the nucleic acid molecule for coding the protein is shown as SEQ ID NO. 1.
The invention also provides a preparation method of the protein, the protein is produced by genetic engineering bacteria capable of expressing Sis-HhH-GPD (NCBI: ADX84373), and the used strain isE. coliBL21(DE3) pLysS cells can express a large amount of a target protein in a short time.
The invention constructs a genetic engineering bacterium capable of overexpressing heat-resistant HhH-GPD protein by utilizing a genetic engineering technology, and obtains electrophoresis-grade recombinase protein through steps of induction, expression, nickel ion affinity purification and the like. The protein can specifically excise 1-meA in DNA under high temperature, and has higher affinity to the DNA containing 1-meA and is obviously higher than the affinity to the normal DNA, thereby providing a simple and convenient method for detecting methylated DNA. Therefore, the protein has potential application in the field of molecular biology related to detection and repair of methylated bases in DNA.
The construction method of the genetic engineering bacteria capable of expressing the Sis-HhH-GPD comprises the following steps:
step 1, using a primer toS. islandicusCarrying out PCR amplification by using the REY15A genome as a template, and detecting the PCR amplification result by agarose gel electrophoresis; the forward primer sequence used was: 5'-CGCGGATCCATGGTTCGTAAAATACTTGAC-3', the reverse primer sequence is: 5'-CCGCTCGAGTCACGAGGAATTTTCTCTATA-3', respectively;
step 2, carrying out double enzyme digestion on the PCR amplification product obtained in the step 1 and the pET-28a Plus vector ((II))BamHI/XhoI) Carrying out reaction;
step 3, recovering the PCR product after enzyme digestion and pET-28a Plus vector fragment through agarose gel electrophoresis, and carrying out ligation reaction;
step 4, transforming the ligation product obtained in the step 3 into competent cellsE. coliCulturing in DH5 alpha overnight (i.e. coating on LB plate containing kanamycin, culturing overnight at 37 ℃), picking out clone, extracting plasmid, and carrying out sequencing verification to obtain clone plasmid with correct gene sequence;
step 5, transforming the cloning plasmid with correct gene sequence into competent cellsE. coliBL21(DE3) plysS cells to obtain a genetically engineered bacterium capable of expressing the Sis-HhH-GPD, and carrying out inducible expression;
and 6, purifying the enzyme through ultrasonic crushing and nickel ion affinity purification to obtain an electrophoresis-grade pure protein sample Sis-HhH-GPD.
The invention further provides application of the protein in detecting DNA methylation.
The molecular weight of the protein Sis-HhH-GPD obtained by the construction method is about 26 kDa, 1-meA in DNA can be specifically excised, and phosphodiester bonds can be further cut at the formed non-base sites.
The protein Sis-HhH-GPD can be 40 o C ～ 90 o Cutting at a temperature in the range of CDNA having 1-meA, the optimum temperature for cleavage reaction being 70 o C。
The protein Sis-HhH-GPD can cut DNA containing 1-meA within the pH range of 8.0-9.5.
The protein Sis-HhH-GPD does not need divalent metal ions for the cleavage reaction.
The cleavage reaction of the protein Sis-HhH-GPD does not need the presence of salt, and high salt can inhibit the activity of the enzyme.
The protein Sis-HhH-GPD can bind to DNA containing 1-meA and can not bind to normal DNA.
The invention has the following beneficial effects:
the invention constructs a genetic engineering bacterium for expressing the Sis-HhH-GPD protein by utilizing a genetic engineering technology, the genetic engineering bacterium can efficiently express the Sis-HhH-GPD protein, the subsequent purification steps are simple and easy to implement, and a large amount of recombinase protein (about 2 mg recombinant protein can be obtained by fermentation liquor per liter) can be easily obtained; the recombinant Sis-HhH-GPD protein has high activity and can specifically excise 1-meA in DNA; the recombinant Sis-HhH-GPD protein has a significantly higher binding capacity to 1-meA-containing DNA than to normal DNA.
Based on the research results, the recombinant Sis-HhH-GPD protein has wide application prospect in the fields of medical treatment and molecular biology as a detection reagent for DNA methylation.
In conclusion, the extreme thermophilic archaea recombinant HhH-GPD protein can cut off and combine methylated bases in DNA, has the advantages of high expression quantity, easiness in purification, high activity and the like, has market implementation possibility in the aspect of DNA methylation detection, and is expected to produce great economic benefit.
Drawings
FIG. 1 is a schematic diagram showing the induction, expression and purification results of Sis-HhH-GPD.
FIG. 2 is a schematic diagram of the analysis of Sis-HhH-GPD cleaved DNA.
FIG. 3 is a graph showing the effect of temperature on the cleavage of DNA by Sis-HhH-GPD.
FIG. 4 is a graph showing the effect of pH on the cleavage of DNA by Sis-HhH-GPD.
FIG. 5 is a schematic diagram showing the effect of divalent metal ions on the cleavage of DNA by Sis-HhH-GPD.
FIG. 6 is a graph showing the effect of salt concentration on the cleavage of DNA by Sis-HhH-GPD.
FIG. 7 is a graph showing the results of analysis of Sis-HhH-GPD binding DNA.
Detailed Description
The technical scheme of the invention is further explained in detail by combining the embodiment as follows: the present embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments.
The sources of reagents and materials involved in the examples are as follows:
the OMEGA PCR kit is purchased from Tiangen Biochemical technology (Beijing) Co., Ltd, the pET-28a Plus vector is provided by high energy physics research institute of Chinese academy of sciences, the OMEGA gel recovery kit is purchased from Tiangen Biochemical technology (Beijing) Co., Ltd,E. coliDH5 alpha competent cell was obtained from Gentiangen Biotech (Beijing) Ltd, OMEGA plasmid extraction kit was obtained from Gentiangen Biotech (Beijing)E. coli BL21(DE3) plysS cells were purchased from holo-gold biosciences.
In addition to the biological materials and reagents specifically mentioned above, the other materials and reagents mentioned in the present invention are commercially available and commercially available to the public at home and abroad, and will not be described one by one here. In the present invention, "%" is a volume percentage.
EXAMPLE 1 Gene cloning of the Sis-HhH-GPD protein
(1) Design of primers
Downloading sulfolobus islandii with completed sequencing from GenBankS. islandicusThe gene sequence of the genes Sis-HhH-GPD (NCBI: SiRe _0278) coded by the REY15A genome is designed to be a pair of primers containing two different restriction endonuclease cleavage sites, and the forward primer sequence is as follows: 5' -CGCGGATCCATGGTTCGTAAAATACTTGAC-3', the reverse primer sequence is: 5' -CCGCTCGAGTCACGAGGAATTTTCTCTATA-3', wherein the underlined bases are eachBamHI andXhoi enzyme cutting site.
(2) PCR amplification of the enzyme Gene
a) Using the above primer pair to download from GenBankS. islandicusThe REY15A genome was used as a template for PCR amplification.
The PCR reaction system is 50 μ L:
10 μ M Forward primer 2 μ L
10 μ M reverse primer 2 μ L
S. islandicusREY15A genomic DNA (50 ng/. mu.L) 1. mu.L
ddH 2 O 20 μL
2 × Phanta Max Master Mix 25 μL
PCR cycling parameters: 95 o C, 3 min; circulate 34 times (95) o C，30 s；55 o C，30 s；72 o C，1 min）；72 o C extension for 5 min.
b) The result of PCR amplification of the enzyme gene was detected by agarose gel electrophoresis: after the reaction, 5. mu.L of the PCR product was subjected to 1.0% agarose gel electrophoresis.
c) Purification of PCR products: the OMEGA PCR kit is adopted for recovery and purification, and the specific steps are shown in the specification. The concentration of the PCR-recovered product was measured by using a Nanodrop 2000 ultramicro spectrophotometer.
(3) Enzyme gene and plasmid vector
The PCR product and the pET-28a Plus vector were subjected to double digestion respectively: (BamHI/XhoI) And (4) reacting.
The enzyme digestion reaction system is 20 mu L:
PCR product or pET-28a Plus vector 16. mu.L
10 Xenzyme buffer (Mg) 2+ plus) 2 μL
Bam HI 1 μL
Xho I 1 μL
37 o C water bath for 2 hr. After the enzyme digestion is finished, the enzyme digestion product is detected by 1.0% agarose gel electrophoresis. By usingThe OMEGA gel recovery kit is used for gel cutting, recovery and purification, and the specific steps are shown in the specification. The concentration was measured using a Nanodrop 2000 ultramicro spectrophotometer.
(4) Ligase gene and vector: the digested PCR product and pET-30a (+) vector were recovered by agarose gel electrophoresis and subjected to ligation reaction.
The ligation reaction system was 10 μ L:
10 x Ligation Buffer 1 μL
2 mu L of pET-28a Plus vector after enzyme digestion
6 mu L of PCR product after enzyme digestion
T4 DNA Ligase 1 μL
22 o C, reacting for 2 hr.
(5) Transformation of recombinant plasmid: conversion of the ligation product toE. coliDH 5. alpha. competent cells and plated on LB plates containing kanamycin, 37 o C overnight culture. Single colonies were picked and plasmids were extracted for sequencing validation.
Pipette 5. mu.L of ligation product to 50. mu.LE. coliDH 5. alpha. competent cells were mixed well and placed in ice bath for 30 min. 42 o C standing in water bath for 90 sec, quickly returning to ice and continuing ice bath for 2 min. Adding 200. mu.L LB liquid medium to 37 o Culturing for 1 hr at 150 rpm on a shaking bed. Aspirate 100. mu.L of culture and spread on LB medium plate containing kanamycin to a final concentration of 50. mu.g/mL, 37 o C, culturing for 12-16 h to obtain 100-200 single colonies.
Subsequently, positive clones were verified: 4 single colonies were selected, inoculated into 5 mL LB medium tubes containing 50. mu.g/mL kanamycin, 37 o C shaking table 150 rpm overnight culture. Extracting plasmids by adopting an OMEGA plasmid extraction kit, and sequencing. And (3) comparing the sequencing result with the NCBI annotated sequence, and verifying positive clones to obtain a recombinant plasmid Sis-HhH-GPD-pET-28a Plus (+).
Example 2 Induction expression and purification of the Sis-HhH-GPD protein
(1) Cloning plasmid with correct gene sequence is passed through 42 o C incubation for 90 sec to SenseCompetent cellE. coli BL21(DE3) plysS cells, a genetically engineered bacterium capable of expressing the HhH-GPD protein was obtained, and IPTG (isopropyl-. beta. -D-thiogalactoside) was used as an inducer to induce the expression of the enzyme.
The recombinant plasmid Sis-HhH-GPD-pET-28a Plus (+) is transformed by heat shockE. coliBL21(DE3) pLysS expression strain, and then the expression strain was singly inoculated in LB liquid medium tube containing 50. mu.g/mL kanamycin and 34. mu.g/mL chloramphenicol, 37 o C overnight culture, 1% inoculum size transfer to 500 mL LB liquid medium containing 50. mu.g/mL kanamycin and 34. mu.g/mL chloramphenicol, 37 o C shaking to OD 600 About 0.6 hours, 0.1 mM IPTG was added and the culture was continued at room temperature for 12 hours to express the recombinant protein.
(2) The recombinase is purified by the steps of cell disruption by ultrasonic waves, heat treatment, nickel ion affinity purification and the like.
The cells were collected and suspended in buffer A (20 mM Tris-HCl pH 8.0, 500 mM NaCl and 10% glycerol). Cell disruption by sonication at 12000 rpm, 4 o Centrifuging for 20 min, and subjecting the supernatant to centrifugation for 70 min o C heat treatment for 20 min at 12000 rpm 4 o And C, centrifuging for 20 min. The supernatant was passed through a Ni affinity column equilibrated with buffer A, and eluted with a gradient of buffer B (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 10% glycerol and 500 mM imidazole), and fractions were collected. All fractions were subjected to SDS-PAGE gel electrophoresis, pooled and dialyzed overnight into storage buffer (20 mM Tris-HCl pH 8.0, 1 mM DTT, 50 mM NaCl and 50% glycerol) 80-10% o And C, freezing and storing. The protein concentration was determined by UV absorption. FIG. 1 is a diagram showing the results of SDS-PAGE gel electrophoresis in the steps of induction of protein expression, ultrasonic cell disruption, heat treatment and nickel ion affinity purification.
The molecular weight of the Sis-HhH-GPD obtained by the construction method is about 26 kDa, the nucleotide sequence of the Sis-HhH-GPD is shown as SEQ ID NO.1, and the amino acid sequence of the Sis-HhH-GPD is shown as SEQ ID NO. 2. The protein can specifically excise 1-meA in DNA, and can further cut phosphodiester bonds at the formed non-base sites.
Example 3 testing of Sis-HhH-GPD for cleavage of DNA and binding to DNA
The activity of Sis-HhH-GPD cleavage containing 1-meA ssDNA was analyzed, and the effect of the optimum reaction temperature, optimum reaction pH and optimum divalent metal ion and salt concentration of the enzyme on the activity of the enzyme was investigated. In addition, the invention also detects the capability of Sis-HhH-GPD to bind DNA containing damaged bases.
Test 1: analysis of Sis-HhH-GPD cleaved DNA
Based on the fact that currently, biological companies can only synthesize DNA containing 1-meA, the invention takes the DNA containing 1-meA as a representative of methylated DNA substrate, and the activity of Sis-HhH-GPD for cutting the DNA is tested. The sequence of the fluorescent Cy 3-labeled 1-meA-containing oligonucleotide chain used in the present invention is as follows: 5'-Cy3CGA ACT GCC TGG AAT CCT GAC GAC XTG TAG CGA ACG ATC ACC TCA-3', wherein X is A, Tg (thymine glycol), 8oxoG, U (uracil), Hx (hypoxanthine), or 1-meA. The complementary nucleotide sequence is 5' -TGA GGT GAT CGT TCG CTA CAY GTC GTC AGG ATT CCA GGC AGT TCG, wherein Y is A, G, C and T.
The reaction system is 10 μ L: 20 mM Tris-HCl pH 8.0, 100 nM ssDNA, 1000 nM Sis-HhH-GPD, 1 mM DTT and 8% glycerol, 70 mM o C, reacting for 30 min. After the reaction was completed, 10. mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o C treatment for 5 min to melt the duplex. The excised products were subjected to gel electrophoresis using a 15% denaturing polyacrylamide gel (0.5 × TBE) with urea concentration of 8M. The electrophoresis results are shown in FIG. 2, and under the above reaction conditions, 77% of 1-meA in 100 nM DNA can be excised with only 1000 nM of Sis-HhH-GPD. However, Sis-HhH-GPD cannot cleave DNA containing other damaged bases, nor can it cleave normal DNA.
And (3) testing 2: effect of reaction temperature on the Activity of Sis-HhH-GPD
The reaction system is 10 μ L: 20 mM Tris-HCl pH 8.0, 100 nM ssDNA containing 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT and 8% glycerol. The reaction temperatures were 30 ℃ each o C、40 o C、50 o C、60 o C、70 o C、80 o C or 90 o C the reaction time is 30 min. After the reaction was completed, 10. mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction，95 o C reaction for 5 min to melt the double strand. The excised products were subjected to gel electrophoresis using a 15% denaturing polyacrylamide gel (0.5 × TBE) with urea concentration of 8M. The electrophoresis results are shown in FIG. 3, and the size of the Sis-HhH-GPD can be 30 o C ～90 o The optimal reaction temperature is 70 ℃ for excising 1-meA in DNA at the temperature of C o C。
And (3) testing: effect of reaction pH on the Activity of Sis-HhH-GPD
The reaction system is 10 μ L: 100 nM ssDNA containing 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT, 8% glycerol and buffers of different pH (20 mmol/L). The method for preparing buffers with different pH values is as follows: sodium phosphate buffer (pH 6.0, 6.5); Tris-HCl buffer (pH 7.0, 7.5 and 8.0); glycine buffer (pH 8.5, 9.0); sodium bicarbonate buffer (pH 10.0). 70 o C, reacting for 30 min. After the reaction was completed, 10. mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o The C reaction was performed for 5 min to melt the double strand, and the cleaved product was subjected to gel electrophoresis using a 15% denaturing polyacrylamide gel (0.5 XTBE) containing 8M urea. As shown in FIG. 4, the electrophoresis results show that Sis-HhH-GPD can cleave 1-meA from DNA in the range of pH 6.0 to 9.5, and the optimum reaction pH is 8.0 to 9.5.
And (4) testing: effect of divalent Metal ions on the Activity of Sis-HhH-GPD
The reaction system is 10 μ L: 20 mM Tris-HCl pH 8.0, 100 nM ssDNA containing 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT, 8% glycerol and 5 mM divalent metal ion (the divalent metal ion is Ca) 2+ 、Mg 2+ 、Zn 2+ 、Mn 2+ 、Ni 2+ 、Co 2+ Or Cu 2+ ）。70 o C, reacting for 30 min, adding 10 mu L formamide containing 100 mmol/L EDTA to stop the reaction after the reaction is finished, and stopping the reaction at 95 DEG C o The C reaction was performed for 5 min to melt the double strand, and the cleaved product was subjected to gel electrophoresis using a 15% denaturing polyacrylamide gel (0.5 XTBE) containing 8M urea. As shown in FIG. 5, the activity of Sis-HhH-GPD for cleaving 1-meA in DNA was independent of divalent metal ions; zn 2+ 、Ni 2+ 、Co 2+ And Cu 2+ Inhibit the activity of the enzyme to varying degrees, while Ca 2+ 、Mn 2+ And Mg 2+ Does not affect the activity of the enzyme.
And (5) testing: effect of salt concentration on the Activity of Sis-HhH-GPD
The reaction system is 10 μ L: 20 mM Tris-HCl pH 8.0, 100 nM ssDNA containing 1-meA, 1000 nM Sis-HhH-GPD, 1 mM DTT, 8% glycerol and varying concentrations of NaCl (50 mM, 100 mM, 200 mM or 400 mM); 70 o C, reacting for 30 min. After the reaction was completed, 10. mu.L of formamide containing 100 mmol/L EDTA was added to terminate the reaction, 95 o The C reaction was performed for 5 min to melt the double strand, and the cleaved product was subjected to gel electrophoresis using a 15% denaturing polyacrylamide gel (0.5 XTBE) containing 8M urea. As shown in FIG. 6, 1-meA in the cleaved DNA of Sis-HhH-GPD was independent of NaCl, and high concentration of NaCl inhibited the activity of the enzyme.
And 6, testing: analysis of Sis-HhH-GPD binding DNA
The gel retardation test system is 10 μ L: 20 mM Tris-HCl pH 8.0, 1 mM DTT, 10% glycerol, 100 nM Cy3 labeled 1-meA ssDNA or normal ssDNA, and different concentrations of Sis-HhH-GPD. After binding at room temperature for 10 min, the blocking products were separated by 4% native polyacrylamide gel electrophoresis (0.1 × TBE). The DNA bands in the image were then quantitatively analyzed by scanning and photographed using a molecular imager, and the binding rate of the enzyme to the substrate was calculated. As shown in A in FIG. 7, the scanning results show that Sis-HhH-GPD can bind to 1-meA-containing DNA with high efficiency, but weakly binds to normal DNA (B in FIG. 7).
The invention discovers that the Sis-HhH-GPD protein is bifunctional DNA glycosidase, can specifically excise 1-meA in DNA, and has obviously higher capability of combining DNA containing 1-meA than normal DNA, so the invention provides an important method for detecting DNA methylation. Compared with other DNA methylation detection methods, the method provided by the invention has the advantages of high sensitivity, low cost, easiness in operation and the like.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can understand that the modifications or substitutions within the technical scope of the present invention are included in the scope of the present invention, and therefore, the scope of the present invention should be subject to the protection scope of the claims.
Sequence listing
<110> Guangling college of Yangzhou university
<120> thermophilic archaea recombinant HhH-GPD protein, preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 684
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atggttcgta aaatacttga cacattgctg gaaatatttg aaaacaataa aagcatattg 60
aaggaaaaag gttggatagt ttcgtccgaa acttcttatg aatggtggga cggactaaaa 120
agtgcagaag aaataatcat ttcagcaata ttggttcaaa tgtcaagatg ggaaattgta 180
aagggcaaag tagaggagat gaggagtaag ggtttgactg atttttataa attatacaat 240
actactgaac aagaattata tgatgtattg aaaggaatta acttctataa gactaaggtt 300
aagaggttaa ttaatttatc taaaatcata ataaatctag gtagtgttga gaaattttat 360
gacagaaatt tacttttaag cattgatggt ataggcgaag aaacagctga ctcaatcttg 420
cttttcgcag gtcacaaacc aaactttcca ccatcagagt acggtaagag agtattatct 480
agagtattag gaattagtat aaagaaaaag aatgaggtta aaagactagt agaggagaat 540
ttagagcgaa acgtctacga atacaaatta ctacacgctg gaatagtcac tgtaggtaga 600
gcattttgtt tcattgaaaa tcccaaatgt gaagactgta tcttgaagaa agtatgtaaa 660
tattatagag aaaattcctc gtga 684
<210> 2
<211> 227
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Val Arg Lys Ile Leu Asp Thr Leu Leu Glu Ile Phe Glu Asn Asn
1 5 10 15
Lys Ser Ile Leu Lys Glu Lys Gly Trp Ile Val Ser Ser Glu Thr Ser
20 25 30
Tyr Glu Trp Trp Asp Gly Leu Lys Ser Ala Glu Glu Ile Ile Ile Ser
35 40 45
Ala Ile Leu Val Gln Met Ser Arg Trp Glu Ile Val Lys Gly Lys Val
50 55 60
Glu Glu Met Arg Ser Lys Gly Leu Thr Asp Phe Tyr Lys Leu Tyr Asn
65 70 75 80
Thr Thr Glu Gln Glu Leu Tyr Asp Val Leu Lys Gly Ile Asn Phe Tyr
85 90 95
Lys Thr Lys Val Lys Arg Leu Ile Asn Leu Ser Lys Ile Ile Ile Asn
100 105 110
Leu Gly Ser Val Glu Lys Phe Tyr Asp Arg Asn Leu Leu Leu Ser Ile
115 120 125
Asp Gly Ile Gly Glu Glu Thr Ala Asp Ser Ile Leu Leu Phe Ala Gly
130 135 140
His Lys Pro Asn Phe Pro Pro Ser Glu Tyr Gly Lys Arg Val Leu Ser
145 150 155 160
Arg Val Leu Gly Ile Ser Ile Lys Lys Lys Asn Glu Val Lys Arg Leu
165 170 175
Val Glu Glu Asn Leu Glu Arg Asn Val Tyr Glu Tyr Lys Leu Leu His
180 185 190
Ala Gly Ile Val Thr Val Gly Arg Ala Phe Cys Phe Ile Glu Asn Pro
195 200 205
Lys Cys Glu Asp Cys Ile Leu Lys Lys Val Cys Lys Tyr Tyr Arg Glu
210 215 220
Asn Ser Ser
225

Claims (8)

1. The extreme thermophilic archaea recombinant HhH-GPD protein is characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO. 2.
2. A nucleic acid molecule encoding the protein of claim 1, wherein: the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.
3. A method for producing a protein according to claim 1 or 2, characterized in that: the protein is produced by genetic engineering bacteria capable of expressing Sis-HhH-GPD, and the used strain isE. coliBL21(DE3) pLysS cells.
4. The method for constructing a genetically engineered bacterium capable of expressing Sis-HhH-GPD according to claim 3, comprising the steps of:
step 1, using a primer toS. islandicusCarrying out PCR amplification by using the REY15A genome as a template, and detecting the PCR amplification result by agarose gel electrophoresis; the forward primer sequence used was: 5'-CGCGGATCCATGGTTCGTAAAATACTTGAC-3', the reverse primer sequence is: 5'-CCGCTCGAGTCACGAGGAATTTTCTCTATA-3', respectively;
step 2, carrying out double enzyme digestion on the PCR amplification product obtained in the step 1 and the pET-28a Plus vector ((II))BamHI/XhoI) Carrying out reaction;
step 3, recovering the PCR product after enzyme digestion and pET-28a Plus vector fragment through agarose gel electrophoresis, and carrying out ligation reaction;
step 4, transforming the ligation product obtained in the step 3 into competent cellsE. coliCulturing in DH5 alpha overnight, extracting plasmids for sequencing verification to obtain clone plasmids with correct gene sequences;
step 5, transforming the cloning plasmid with correct gene sequence into competent cellsE. coliBL21(DE3) plysS cells, a genetically engineered strain capable of expressing Sis-HhH-GPD was obtained.
5. Use of a protein according to any one of claims 1 to 4 for detecting DNA methylation.
6. Use of a protein according to claim 5 for detecting DNA methylation, wherein: the protein has a molecular weight of 26 kDa, is capable of specifically cleaving 1-meA from DNA, and is further capable of cleaving phosphodiester bonds at the formed site without base.
7. Use of a protein according to claim 6 for detecting DNA methylation, wherein: the protein can be at 40 o C ～ 90 o C temperature range for cutting DNA containing 1-meA.
8. Use of a protein according to claim 7 for detecting DNA methylation, wherein: the protein is capable of cleaving DNA containing 1-meA at a pH in the range of 8.0 to 9.5.
CN202210709994.9A 2022-06-22 2022-06-22 Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof Active CN115044571B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210709994.9A CN115044571B (en) 2022-06-22 2022-06-22 Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210709994.9A CN115044571B (en) 2022-06-22 2022-06-22 Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN115044571A true CN115044571A (en) 2022-09-13
CN115044571B CN115044571B (en) 2023-11-24

Family

ID=83162531

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210709994.9A Active CN115044571B (en) 2022-06-22 2022-06-22 Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN115044571B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1837233A (en) * 2005-03-21 2006-09-27 中国科学院微生物研究所 Halophilic bacteria and its immune protein and genes encoding same and use thereof
CN105087602A (en) * 2014-05-19 2015-11-25 上海市农业科学院 Application of heat shock protein gene in construction of high-heat-tolerance escherichia coli
US20170191047A1 (en) * 2015-11-13 2017-07-06 University Of Georgia Research Foundation, Inc. Adenosine-specific rnase and methods of use
US20210230577A1 (en) * 2017-03-10 2021-07-29 President And Fellows Of Harvard College Cytosine to guanine base editor

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1837233A (en) * 2005-03-21 2006-09-27 中国科学院微生物研究所 Halophilic bacteria and its immune protein and genes encoding same and use thereof
CN105087602A (en) * 2014-05-19 2015-11-25 上海市农业科学院 Application of heat shock protein gene in construction of high-heat-tolerance escherichia coli
US20170191047A1 (en) * 2015-11-13 2017-07-06 University Of Georgia Research Foundation, Inc. Adenosine-specific rnase and methods of use
US20210230577A1 (en) * 2017-03-10 2021-07-29 President And Fellows Of Harvard College Cytosine to guanine base editor

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
"endonuclease III domain-containing protein [Sulfolobus islandicus]", GENBANK, 登录号：WP_014511900.1 *
LIKUI ZHANG等: "Biochemical characterization and mutational studies of a thermostable endonuclease III from Sulfolobus islandicus REY15A", INT J BIOL MACROMOL, vol. 193 *
李玉婷等: "极端嗜热古菌DNA修复核酸内切酶的研究进展", 微生物学报, vol. 59, no. 10 *
汤承轩等: "极端嗜热古菌核酸内切酶Ⅲ的研究进展", 微生物学报, vol. 62, no. 07 *
甘琪等: "极端嗜热古菌尿嘧啶DNA糖苷酶的研究进展", 微生物学报, vol. 60, no. 02 *

Also Published As

Publication number Publication date
CN115044571B (en) 2023-11-24

Similar Documents

Publication Publication Date Title
CN113583996B (en) Bst DNA polymerase recombinant mutant, coding DNA thereof and ultra-fast magnetic bead LAMP detection method
WO2022227880A1 (en) Novel phosphorylated adenylase, and preparation method therefor and application thereof
JP4486009B2 (en) DNA ligase mutant
CN109679932A (en) A kind of archaeal dna polymerase, recombinant vector and their preparation method and application
CN111534493A (en) Purine nucleoside phosphorylase mutant, gene and application
CN111876399B (en) Arctic-pole-derived beta-glucosidase gene, and encoded protein and application thereof
CN110951803B (en) Method for preparing neoagarobiose by combined utilization of agarase, recombinant host cell and application of recombinant host cell and expression vector
KR100358532B1 (en) Adjusting and controlling factor for expression of nitrile solution deenzyme and gene thereof
CN114134205B (en) Deaminase mediated N in DNA 4 Single base resolution localization analysis method of methyl cytosine
CN109402092B (en) Chitinase derived from marine environment and gene thereof
CN114561374A (en) Novel thermophilic endonuclease mutant and preparation method and application thereof
CN113637658A (en) dCas 9-oToV-based gene transcription system and application thereof
CN115044571B (en) Extreme thermophilic archaea recombinant HhH-GPD protein and preparation method and application thereof
EP0877084B1 (en) Thermostable diaphorase gene
CN114807085B (en) Taq enzyme mutant and application thereof
CN106834252A (en) A kind of high stable type MazF mutant and its application
CN114645033B (en) Nucleoside triphosphate hydrolase and purification method and application thereof
CN105969751B (en) Beta-glucosidase gene and application thereof
CN110093362B (en) Functional gene vector pET32a-fdhD for high-efficiency auxiliary hydrogen production and construction thereof
JP2022534004A (en) Mutants of terminal deoxynucleotidyl transferase and uses thereof
CN113481186A (en) GH18 chitinase ChiA and application thereof
JPH01225483A (en) Recombinant plasmid
CN113564141B (en) Single-cell genome amplification enzyme mutant and application thereof
JP5935382B2 (en) RrhJ1II nuclease and its gene
CN117210433B (en) Overspeed high-fidelity combined reverse transcription DNA polymerase, gene amplification and reverse transcription method based on same and reagent

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant