CN112813088A - Preparation method of recombinant DpnI restriction endonuclease - Google Patents
Preparation method of recombinant DpnI restriction endonuclease Download PDFInfo
- Publication number
- CN112813088A CN112813088A CN202110021206.2A CN202110021206A CN112813088A CN 112813088 A CN112813088 A CN 112813088A CN 202110021206 A CN202110021206 A CN 202110021206A CN 112813088 A CN112813088 A CN 112813088A
- Authority
- CN
- China
- Prior art keywords
- dpni
- recombinant
- vector
- restriction enzyme
- dpni restriction
- 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
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
- 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
- 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)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
- C12Y301/21004—Type II site-specific deoxyribonuclease (3.1.21.4)
-
- 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
- C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-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
- C07K2319/24—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a MBP (maltose binding protein)-tag
-
- 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
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a preparation method of recombinant DpnI restriction endonuclease, which comprises the following steps: step one, synthesizing a coding gene of DpnI, constructing the coding gene to a prokaryotic expression vector, and obtaining a recombinant vector: the amino acid sequence of DpnI is shown in SEQ ID NO. 1, and the nucleotide sequence of the coding gene of DpnI is obtained by adding a stop codon and optimizing the codon; step two, transferring the recombinant vector into methylase-deleted escherichia coli competent cells to obtain recombinant bacteria; thirdly, identifying the sequence correctness of the target fragment in the recombinant bacteria through PCR and sequencing; step four, carrying out DpnI restriction endonuclease expression on the monoclonal with the correct target fragment; and step five, purifying the DpnI restriction enzyme. Compared with the DE3 lysogenic expression method of special strains in the prior literature, the strain material used in the method of the invention does not need special treatment, the operation flow is simplified, and the success rate is high.
Description
Technical Field
The invention belongs to the fields of molecular biology and cell engineering, and particularly relates to a preparation method of recombinant DpnI restriction endonuclease.
Background
DpnI is a restriction enzyme in Diplococcus pneumoniae (Diplococcus pneumoconiae) and is characterized in that DpnI is cleaved only when its recognition site is methylated (see FIG. 1). The GATC sequence is widely existed in genomes of various species, and DpnI is a restriction enzyme type with high practicability in heredity and genetic engineering. Because commonly used cloning and expression strains including DH5a, Top10, BL21(DE3), BL21(DE3 plyS) and the like all contain endogenous methylases, an expression vector and a host bacterium genome are methylated, and a DpnI recognition site is widely present, so that DpnI cuts the host genome and an expression plasmid, the DpnI construction using a conventional T7 promoter as a vector can normally clone in strains such as DH5a or Top10, but the substrate protein expression in an expression strain such as BL21(DE3) can seriously inhibit the normal proliferation of the host bacterium and the replication of the expression vector, so that the protein cannot be normally expressed.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of DpnI restriction endonuclease. Firstly, synthesizing DpnI protein coding sequence by gene, then constructing the gene on a carrier which takes a promoter (tac, lacUV, trc and the like) which can be recognized by the RNA polymerase of the escherichia coli as a heterologous gene expression promoter by a recombination or connection mode. Directly transforming the amplified product of the connection or recombination molecular cloning reaction system into JM110 (methylase deleted) strains (DH 5a or Top10 is not used as an intermediate host), identifying the grown monoclones, and picking the monoclones with correct sequencing to directly express DpnI. Obtaining the high-purity protein by affinity and gel chromatography.
The specific technical scheme is as follows: a preparation method of recombinant DpnI restriction endonuclease is characterized by comprising the following steps: step one, synthesizing a coding gene of DpnI, constructing the coding gene to a prokaryotic expression vector, and obtaining a recombinant vector: the amino acid sequence of DpnI is shown as SEQ ID NO 1 or the similar coding amino acid sequence with the similarity of 95 percent or more, and the nucleotide sequence of the coding gene of DpnI is obtained by adding a stop codon and optimizing the codon; step two, transferring the recombinant vector into methylase-deleted escherichia coli competent cells to obtain recombinant bacteria; thirdly, identifying the sequence correctness of the target fragment in the recombinant bacteria through PCR and sequencing; step four, carrying out DpnI restriction endonuclease expression on the monoclonal with the correct target fragment; and step five, purifying the DpnI restriction enzyme.
Further, the prokaryotic expression vector is a vector which can recognize a promoter tac, lacUV or trc by the escherichia coli self polymerase; the nucleotide sequence of the coding gene of DpnI is shown in SEQ ID NO. 2.
Further, the prokaryotic expression vector is pGEX-6p-1 vector; the methylase deficient E.coli is JM 110.
Further, the first step is specifically as follows: using high-fidelity enzyme to amplify the DpnI coding gene, using BamHI and SalI restriction enzyme to double-enzyme cut pGEX-6p-1 vector, and inserting the vector into pGEX-6p-1 in a homologous recombination mode; ensuring correct reading frame, and using GST protein as purification label.
Further, in the step two, 200-;
40-80. mu.l of the recombinant reaction system contained: 200 ng of pGEX-6p-1 vector, 50-100ng of DpnI encoding gene, 2-4. mu.l of expase recombinase and 8-16. mu.l of 5 Xbuffer.
Further, the second step is specifically as follows: taking 200-; finally 10000g of the supernatant is centrifuged, and the thallus is evenly coated on an ampicillin resistance LB plate; the recombination reaction product is a product obtained by reacting 40-80 mul of recombination reaction system at 37 ℃ for 25 min;
the third step is specifically as follows: selecting monoclonal strains, culturing in a 2.5ml test tube at 220rpm and 37 deg.C for 10hr, performing PCR on the bacterial liquid, and performing PCR reaction by using universal sequencing primers at two ends of the multiple cloning sites of the vector; the monoclonal strains which are identified to be correct by PCR are subjected to inspection and sequencing, and the cloned strains with correct sequences are determined by comparison; the fourth step is specifically as follows: selecting a single clone, inoculating the single clone into 10ml of LB culture medium with corresponding resistance of the vector, carrying out shaking culture overnight, inoculating the single clone into a 1L of double-antibody LB shaking flask the next day, carrying out shaking culture at the speed of 220rpm and 37 ℃ until the OD is 0.6-0.8, adding IPTG with the final concentration of 0.5mM for induction, and expressing for 21 hours.
Further, the fifth step is specifically: centrifuging for 5min by 10000rcf centrifugal force to collect thalli, removing a supernatant culture medium, suspending the thalli by using 30ml of precooled buffer solution containing 100mM KCl and 25mM Tris pH7.5, then performing bacterium breaking for 5min by using high pressure 650bar, simultaneously adding protease inhibitor, centrifuging for 30min by 14000rcf to remove precipitates, adding corresponding affinity purification medium into the supernatant for combination, then performing specific site protease mediated column label cutting or direct elution without cutting, performing AKTA mediated elution separation process on eluted protein, performing gel chromatography, and determining the purity of the obtained recombinant DpnI restriction endonuclease.
Further, the method also comprises a sixth step of measuring the enzyme activity of the DpnI restriction enzyme.
Further, the sixth step is specifically: DpnI was added to the reaction system at various dilution ratios, 1. mu.g of plasmid DNA was digested at 37 ℃ for one hour, and the specific activity of the enzyme was finally confirmed by agarose electrophoresis.
Further, in the first step, the fusion expression protein constructed in the prokaryotic expression vector is one of MBP, GST and 6 HIS.
Further, in the second step, the methylase deletion-sensitive Escherichia coli used is one of JM110 and ER 2925.
DpnI recombinant protein can be obtained by dissolving strains such as JM110, ER2925 and the like, but the steps are complicated, the period is long, and the success rate is low. The invention does not need solution source, and the operation is simpler. Compared with the DE3 lysogenic expression method of special strains in the prior literature, the strain material used in the method is more easily obtained (commercially purchased) and the operation flow is simpler. The purity obtained by affinity purification using GST, MBP and the like is higher, and the purification process only needs two steps and has small loss (Siwek, W. et al, 2012Nucleic acids research 40, 7563-.
The present invention will be further described with reference to the accompanying drawings to fully illustrate the objects, technical features and technical effects of the present invention.
Drawings
FIG. 1 shows the restriction sites of the DpnI restriction enzyme.
FIG. 2 is a flow chart showing the operation of one embodiment of the method for producing a recombinant DpnI restriction enzyme according to the present invention.
FIG. 3 shows the identification of the monoclonal species obtained in step 2 by agarose gel electrophoresis.
The target amplified gene is about 850bp, and 8 single clones picked are all correct clone length.
FIG. 4 is a SDS-PAGE gel of the DpnI restriction enzyme after affinity purification in a metal ion chelating agarose gel medium and further purification by gel chromatography.
After purification by gel chromatography, different effluent liquids of protein peaks are sampled and run on a gel chart, and the purity reaches more than 95%. A is GST-DpnI; b is DpnI after GST cleavage.
FIG. 5 is an agarose gel electrophoresis image of a commercial pFastbac vector constructed with a target fragment after cleavage with DpnI obtained by the present invention. T stands for restriction enzyme purchased from ThermoFisher Scientific.
Detailed Description
The reagents and consumables of the present invention can be purchased commercially, and are purchased from Shanghai Baisai Biotechnology GmbH, unless otherwise noted.
Example 1
In this example, the operation flow of the method for preparing recombinant DpnI restriction enzyme according to the present invention is shown in fig. 2.
The amino acid sequence of DpnI is shown as SEQ ID NO. 1 or similar coding amino acid sequence with the similarity of 95 percent and above. The coding gene obtained by adding the stop codon and codon optimization is the nucleotide sequence shown in SEQ ID NO. 2, and the nucleotide sequence obtained by adding the stop codon and codon optimization can be different from the nucleotide sequence shown in SEQ ID NO. 1 in other specific embodiments. The coding gene of DpnI is synthesized according to the nucleotide sequence shown in SEQ ID NO. 2.
The construction to pGEX-6p-1(Addgene) is described below as an example:
the DpnI coding gene is amplified by using high-fidelity enzyme, and the amplification primer pair is DpnI F (shown as SEQ ID NO: 3) and DpnI R (shown as SEQ ID NO: 4). Meanwhile, the pGEX-6p-1 vector is digested with BamHI and SalI restriction enzymes, and then inserted into pGEX-6p-1 by homologous recombination (Gibson assembly). Ensuring correct reading frame, and using GST protein as purification label.
And 2, transferring the product obtained by recombination or ligation reaction into JM110 competent cells.
JM110 competent cells were purchased from exclusively organisms. The JM110 has low transformation efficiency, and the recombination reaction system needs to be expanded by 2-4 times to obtain more positive clones, taking the recombination reaction as an example (purchased from Vazyme):
the reaction conditions were 37 ℃ 25 min.
To improve the receptive efficiency, JM110 competent cells (300 μ l) of 3 times volume under conventional conditions were used for transformation, the competent cells were taken out at-80 deg.C and then placed on ice for 10min, then recombinant reaction products were added thereto, and the mixture was gently shaken and mixed, placed on ice for 15min, then heat-shocked in a 42 deg.C water bath for 45s, taken out and placed on ice for 10min, and then 1ml of LB medium was added and cultured on a 37 deg.C shaker at 220rpm for 1 hr. Finally 10000g of the supernatant was centrifuged and the cells were evenly spread on an ampicillin resistant LB plate.
Selecting corresponding monoclonal strain, culturing in 2.5ml test tube at 220rpm 37 deg.C for 10hr, performing PCR on the bacterial solution, and performing PCR reaction with universal sequencing primers at both ends of the multiple cloning site of the vector to obtain the result shown in FIG. 3, wherein the primer pair is Verf (shown as SEQ ID NO: 5) and Verf R (shown as SEQ ID NO: 6). The monoclonal bacterial liquid which is identified by PCR to be correct is sent to a detection and comparison to determine the correct clone bacterial liquid, and the used primer pairs are Seq F (shown as SEQ ID NO: 7) and Seq R (shown as SEQ ID NO: 8).
For example, 1L expression:
selecting a single clone, inoculating the single clone into 10ml of LB culture medium with corresponding resistance of the vector, carrying out shaking culture overnight, inoculating the single clone into a 1L of double-antibody LB shaking flask the next day, carrying out shaking culture at the speed of 220rpm and 37 ℃ until the OD is 0.6-0.8, adding IPTG with the final concentration of 0.5mM for induction, and expressing for 21 hours.
centrifuging for 5min by 10000rcf centrifugal force to collect thalli, removing a supernatant culture medium, suspending the thalli by using 30ml of precooler (100mM KCl,25mM Tris pH7.5), then performing bacterium breaking for 5min by using high pressure 650bar, after adding protease inhibitor while performing bacterium breaking, centrifuging for 30min by 14000rcf to remove precipitates, adding a corresponding affinity purification medium into the supernatant for combination (Ni, Glutathione, Dextrin and the like), then performing specific site protease mediated column label cutting or direct elution without cutting, performing AKTA (GE healthcare) mediated elution separation process on eluted protein, and performing gel chromatography. The final SDS-PAGE confirmed purity, as shown in FIG. 4. And finally, protein concentration is carried out, and the final protein concentration is determined by combining an A280 light absorption value with an extinction coefficient.
one unit is defined as the amount of enzyme required to digest 1. mu.g of methylated plasmid DNA in 1 hour at 37 ℃ in a total reaction volume of 50. mu.l.
DpnI was added to the reaction system at various dilution ratios, 1. mu.g of plasmid DNA was digested at 37 ℃ for one hour, and the specific activity of the enzyme was finally confirmed by agarose electrophoresis, as shown in FIG. 5. Finally determining that the specific activity is more than 5x 10^ 6U/mg.
The reaction conditions are as follows: 50 μ l reaction, 10U DpnI,10U DpnI (T) buffer: 100mM Tris-HCl,50mM NaCl,10mM MgCl20.025% Triton X-100(pH 7.5@25 ℃ C.) 5. mu.g of plasmid was reacted for 10min at 37 ℃. T stands for DpnI restriction enzyme purchased from ThermoFisher Scientific for control reaction, and DpnI (T) as positive control. The Negative group is the original plasmid which is not recombined with DpnI restriction endonuclease gene.
The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.
Sequence listing
<110> Shanghai Yongke Biotechnology Co., Ltd
<120> preparation method of recombinant DpnI restriction endonuclease
<130> CN100-20002PICN
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 254
<212> PRT
<213> Diplococcus pneumoniae
<400> 1
Met Glu Leu His Phe Asn Leu Glu Leu Val Glu Thr Tyr Lys Ser Asn
1 5 10 15
Ser Gln Lys Ala Arg Ile Leu Thr Glu Asp Trp Val Tyr Arg Gln Ser
20 25 30
Tyr Cys Pro Asn Cys Gly Asn Asn Pro Leu Asn His Phe Glu Asn Asn
35 40 45
Arg Pro Val Ala Asp Phe Tyr Cys Asn His Cys Ser Glu Glu Phe Glu
50 55 60
Leu Lys Ser Lys Lys Gly Asn Phe Ser Ser Thr Ile Asn Asp Gly Ala
65 70 75 80
Tyr Ala Thr Met Met Lys Arg Val Gln Ala Asp Asn Asn Pro Asn Phe
85 90 95
Phe Phe Leu Thr Tyr Thr Lys Asn Phe Glu Val Asn Asn Phe Leu Val
100 105 110
Leu Pro Lys Gln Phe Val Thr Pro Lys Ser Ile Ile Gln Arg Lys Pro
115 120 125
Leu Ala Pro Thr Ala Arg Arg Ala Gly Trp Ile Gly Cys Asn Ile Asp
130 135 140
Leu Ser Gln Val Pro Ser Lys Gly Arg Ile Phe Leu Val Gln Asp Gly
145 150 155 160
Gln Val Arg Asp Pro Glu Lys Val Thr Lys Glu Phe Lys Gln Gly Leu
165 170 175
Phe Leu Arg Lys Ser Ser Leu Ser Ser Arg Gly Trp Thr Ile Glu Ile
180 185 190
Leu Asn Cys Ile Asp Lys Ile Glu Gly Ser Glu Phe Thr Leu Glu Asp
195 200 205
Met Tyr Arg Phe Glu Ser Asp Leu Lys Asn Ile Phe Val Lys Asn Asn
210 215 220
His Ile Lys Glu Lys Ile Arg Gln Gln Leu Gln Ile Leu Arg Asp Lys
225 230 235 240
Glu Ile Ile Glu Phe Lys Gly Arg Gly Lys Tyr Arg Lys Leu
245 250
<210> 2
<211> 765
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
atggagctgc acttcaacct ggaactggtt gaaacctaca aaagcaactc ccagaaagcg 60
cgtatcctga ccgaagattg ggtttaccgt cagagctact gcccgaactg cggcaacaac 120
ccgctgaacc acttcgaaaa caaccgtccg gttgcggatt tctactgcaa ccactgcagc 180
gaagaattcg aactgaaaag caaaaaaggc aacttcagca gcaccatcaa cgacggcgcg 240
tacgcgacca tgatgaaacg tgtgcaggcg gataacaacc cgaacttctt cttcctgacc 300
tacaccaaaa acttcgaagt taacaacttc ctggtgctgc cgaaacagtt cgttaccccg 360
aaaagcatca tccagcgtaa accgctggcg ccgaccgcga accgtgctgg ctggatcggt 420
tgcaacatcg atctgagcca ggttccgagc aaaggccgta tcttcctggt tcaggatggt 480
caggttcgtg atccggaaaa agtgaccaaa gaattcaaac agggtctgtt cctgcgtaaa 540
tctagcctga gcagccgtgg ttggaccatc gaaatcctga actgcatcga taaaatcgaa 600
ggctccgaat tcaccctgga agatatgtac cgcttcgaaa gcgacctgaa aaacatcttc 660
gttaaaaaca accacatcaa agaaaaaatc cgccagcagc tgcagatcct gcgtgataaa 720
gaaatcatcg aattcaaagg ccgtggtaaa taccgtaaac tgtaa 765
<210> 3
<211> 39
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
tccaggggcc cctgggatcc atggagctgc acttcaacc 39
<210> 4
<211> 41
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
gtcagtcacg atgcggccgc ttacagttta cggtatttac c 41
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
atggagctgc acttcaacct g 21
<210> 6
<211> 27
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
ttacagttta cggtatttac cacggcc 27
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
caatgtgcct ggatgcgttc 20
<210> 8
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
ccgctatcgc tacgtgact 19
Claims (10)
1. A preparation method of recombinant DpnI restriction endonuclease is characterized by comprising the following steps:
step one, synthesizing a coding gene of DpnI, constructing the coding gene to a prokaryotic expression vector, and obtaining a recombinant vector: the amino acid sequence of the DpnI is shown as SEQ ID NO. 1 or the similar coding amino acid sequence with the similarity of 95 percent or more, and the nucleotide sequence of the coding gene of the DpnI is obtained by adding a stop codon and optimizing a codon;
step two, transferring the recombinant vector into methylase-deleted escherichia coli competent cells to obtain recombinant bacteria;
thirdly, identifying the sequence correctness of the target fragment in the recombinant bacteria through PCR and sequencing;
step four, carrying out DpnI restriction endonuclease expression on the monoclonal with the correct target fragment;
and step five, purifying the DpnI restriction enzyme.
2. The method for preparing a recombinant DpnI restriction enzyme according to claim 1, wherein the prokaryotic expression vector is a vector in which escherichia coli self-polymerase can recognize a promoter tac, lacUV or trc; the nucleotide sequence of the coding gene of DpnI is shown in SEQ ID NO. 2.
3. The method of claim 2, wherein the prokaryotic expression vector is a pGEX-6p-1 vector; the methylase deficient E.coli is JM 110.
4. The method for preparing a recombinant DpnI restriction enzyme according to claim 3, wherein the first step is specifically: using high-fidelity enzyme to amplify the DpnI coding gene, using BamHI and SalI restriction enzyme to double-enzyme cut pGEX-6p-1 vector, and inserting the vector into pGEX-6p-1 in a homologous recombination mode; ensuring correct reading frame, and using GST protein as purification label.
5. The method for preparing recombinant DpnI restriction endonuclease as claimed in claim 3, wherein 200-400. mu. lJM110 competent cells and 40-80. mu.l recombinant reaction system are adopted in the second step and mixed to transfer into the recombinant vector to obtain the recombinant strain;
the 40-80 μ l recombinant reaction system comprises: 200 ng of pGEX-6p-1 vector, 50-100ng of DpnI encoding gene, 2-4. mu.l of Exnase recombinase and 8-16. mu.l of 5 Xbuffer.
6. The method for preparing a recombinant DpnI restriction enzyme according to claim 5, wherein the second step is specifically: taking 200-; finally 10000g of the supernatant is centrifuged, and the thallus is evenly coated on an ampicillin resistance LB plate; the recombination reaction product is a product obtained by reacting 40-80 μ l of the recombination reaction system at 37 ℃ for 25 min;
the third step is specifically as follows: selecting monoclonal strains, culturing in a 2.5ml test tube at 220rpm and 37 deg.C for 10hr, performing PCR on the bacterial liquid, and performing PCR reaction by using universal sequencing primers at two ends of the multiple cloning sites of the vector; the monoclonal strains which are identified to be correct by PCR are subjected to inspection and sequencing, and the cloned strains with correct sequences are determined by comparison; the fourth step is specifically as follows: selecting a single clone, inoculating the single clone into 10ml of LB culture medium with corresponding resistance of the vector, carrying out shaking culture overnight, inoculating the single clone into a 1L of double-antibody LB shaking flask the next day, carrying out shaking culture at the speed of 220rpm and 37 ℃ until the OD is 0.6-0.8, adding IPTG with the final concentration of 0.5mM for induction, and expressing for 21 hours.
7. The method for preparing a recombinant DpnI restriction enzyme according to claim 1, wherein the fifth step is specifically: centrifuging for 5min by 10000rcf centrifugal force to collect thalli, removing a supernatant culture medium, suspending the thalli by using 30ml of precooled buffer solution containing 100mM KCl and 25mM Tris pH7.5, then performing bacterium breaking for 5min by using high pressure 650bar, simultaneously adding protease inhibitor, centrifuging for 30min by 14000rcf to remove precipitates, adding corresponding affinity purification medium into the supernatant for combination, then performing specific site protease mediated column label cutting or direct elution without cutting, performing AKTA mediated elution separation process on eluted protein, performing gel chromatography, and determining the purity of the obtained recombinant DpnI restriction endonuclease.
8. The method of claim 1, further comprising a sixth step of measuring the enzymatic activity of the DpnI restriction enzyme.
9. The method of claim 1, wherein in the first step, the fusion expression protein constructed in the prokaryotic expression vector is one of MBP, GST and 6 HIS.
10. The method for preparing recombinant DpnI restriction enzyme according to claim 1, wherein in the second step, the methylase deletion-sensitive e.coli used is one of JM110 and ER 2925.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110021206.2A CN112813088B (en) | 2021-01-08 | 2021-01-08 | Preparation method of recombinant DpnI restriction enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110021206.2A CN112813088B (en) | 2021-01-08 | 2021-01-08 | Preparation method of recombinant DpnI restriction enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112813088A true CN112813088A (en) | 2021-05-18 |
| CN112813088B CN112813088B (en) | 2023-06-27 |
Family
ID=75868810
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110021206.2A Active CN112813088B (en) | 2021-01-08 | 2021-01-08 | Preparation method of recombinant DpnI restriction enzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112813088B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000063368A1 (en) * | 1999-04-15 | 2000-10-26 | Zetesis S.P.A. | Recombinant protein uk114 and its use in therapy and diagnostics |
| CN101215554A (en) * | 2008-01-08 | 2008-07-09 | 江南大学 | Fire resistant L-arabinose isomerase and gene sequence thereof |
| CN108949719A (en) * | 2018-07-26 | 2018-12-07 | 山西医科大学 | A kind of coding fallibility archaeal dna polymerase and preparation method thereof |
-
2021
- 2021-01-08 CN CN202110021206.2A patent/CN112813088B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000063368A1 (en) * | 1999-04-15 | 2000-10-26 | Zetesis S.P.A. | Recombinant protein uk114 and its use in therapy and diagnostics |
| CN101215554A (en) * | 2008-01-08 | 2008-07-09 | 江南大学 | Fire resistant L-arabinose isomerase and gene sequence thereof |
| CN108949719A (en) * | 2018-07-26 | 2018-12-07 | 山西医科大学 | A kind of coding fallibility archaeal dna polymerase and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 无: "type-2 restriction enzyme DpnI [Streptococcus pneumoniae]", 《GENBANK》 * |
| 李田等: "植物启动子研究进展", 《生物技术通报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112813088B (en) | 2023-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018013990A1 (en) | Scarless dna assembly and genome editing using crispr/cpf1 and dna ligase | |
| KR20190059966A (en) | S. The Piogenes CAS9 mutant gene and the polypeptide encoded thereby | |
| US10202607B2 (en) | Cleavable fusion tag for protein overexpression and purification | |
| CN114262697B (en) | Bsu DNA polymerase and Bsu DNA polymerase mutant as well as gene, plasmid and genetic engineering bacteria thereof | |
| KR101659732B1 (en) | Protein Expression System Induced by Heavy Metals And Biosensor for Detecting Heavy Metals | |
| CN115029333A (en) | Endonuclease and its purification method and use | |
| CN114645033B (en) | Nucleoside triphosphate hydrolase and purification method and application thereof | |
| EP0877084B1 (en) | Thermostable diaphorase gene | |
| US7981653B2 (en) | Highly efficient hyperthermophilic DNA ligase | |
| CN112813088A (en) | Preparation method of recombinant DpnI restriction endonuclease | |
| CN115785283A (en) | PAG-Tn5 mutant and application thereof | |
| US8137943B2 (en) | Heat-resistant DNA ligase with high reactivity | |
| CN111849939B (en) | Nicked DNA (deoxyribonucleic acid) preference high-fidelity polymerase and application thereof | |
| CN114480345B (en) | MazF mutant, recombinant vector, recombinant engineering bacterium and application thereof | |
| US20240199691A1 (en) | Expression and purification of cas enzymes | |
| KR102667373B1 (en) | A novel fusion tag system promising soluble expression and purification in Escherichia coli using CBM66 and levan, and their applications | |
| CN114958808B (en) | CRISPR/Cas system for small-sized genome editing and special CasX protein thereof | |
| CN113151213B (en) | High-fidelity DNA polymerase, preparation method and PCR application thereof | |
| CN114164223B (en) | Antarctic soil-derived esterase and encoding gene and application thereof | |
| CN113073107B (en) | Mannase gene AbMan5, recombinant expression plasmid, recombinant expression strain, mannase and application thereof | |
| RU2310688C1 (en) | RECOMBINANT DNA ENCODING FUNCTIONALLY ACTIVE HYBRID PROTEIN OF D-AMINO ACID OXYDASE WITH CHITIN-COUPLING DOMAIN (DAOcbd), pVR1 RECOMBINANT PLASMID PROVIDING SYNTHESIS THEREOF IN Escherichia coli CELLS AND RECOMBINANT Escherichia coli STRAIN C 41 (DE3)/pVR1 AS PRODUCER OF DAOcbd | |
| CN108220317B (en) | Recombinant expression plasmid and preparation method and application thereof | |
| JP4714848B2 (en) | DNA polymerase mutant | |
| US20020119506A1 (en) | Genes encoding UMP kinase, methods for purifying UMP kinase and methods of characterizing UMP kinase | |
| CN115975987A (en) | Preparation method of AscI restriction endonuclease |
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 |