CN103602643B - Recombinant Taq DNA polymerase and preparation method thereof - Google Patents
Recombinant Taq DNA polymerase and preparation method thereof Download PDFInfo
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- CN103602643B CN103602643B CN201310576003.5A CN201310576003A CN103602643B CN 103602643 B CN103602643 B CN 103602643B CN 201310576003 A CN201310576003 A CN 201310576003A CN 103602643 B CN103602643 B CN 103602643B
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- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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Abstract
The invention relates to the technical field of genetic engineering, and particularly relates to a method for preparing a gene recombinant expression enzyme of Thermusaquaticus TaqDNA polymerase. The nucleotide sequence of a gene and the amino acid sequence of an encoded protein are respectively shown as SEQIDNO:1 and SEQIDNO:2. According to the invention, the method comprises the following steps: cloning a gene sequence by using a gene cloning technique, and building a prokaryotic expression vector; by using a recombinant strain of a built TaqDNA polymerase gene, carrying out suspension by using an LB culture solution; after carrying out culturing for 4.5 hours at a temperature of 37 DEG C, adding IPTG (isopropyl-beta-d-thiogalactoside) with a final concentration of 0.5 mM into the obtained substance to induce for 16-20 hours; after thalli are collected in a centrifugal mode, dissociating the thalli by using 2-4 mg/ml lysozyme, so that protein fluid is obtained; and filtering the protein fluid by using a chromatographic column and a dialysis bag so as to obtain high-purity active protein TaqDNA polymerase. Compared with the prior art, stability of a production process is good, activity of obtained TaqDNA polymerase is high, the yield and purity of products are effectively ensured, and amplification effect when being applied to a gene PCR (polymerase chain reaction) is good.
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to a kind of method preparing restructuring Taq DNA polysaccharase.
Background technology
1985, polymerase chain reaction (the polymerase chain reaction be with historically new significance has invented in Cetus company of the U.S., PCR), it is a kind of method of specific DNA fragment being carried out in vitro to rapid amplifying, namely strand is unwind in double-stranded DNA at high temperature sex change, in the presence of DNA polysaccharase, dNTP, special primer, be copied into two same molecule copies according to base pair complementarity principle.
Original bacteria YT-1 (Thermus aquatjcus) has n DNA polysaccharase, but its Taq DNA polysaccharase content is low, need to carry out selective precipitation and ion exchange mechansim from its culture purifying enzyme, be difficult to meet the demand to Taq DNA polysaccharase.Therefore, commercially available Taq DNA polysaccharase is mostly gene engineering product, can effectively improve its productive rate and separation efficiency.
Although Taq DNA polysaccharase is own through merchandized handling for many years, in order to improve the expression amount of this albumen in intestinal bacteria, the trial simplifying purifying process never stops.Pluthme Fred utilizes the thermostability of Taq DNA polysaccharase, and propose freeze-thaw method and remove the macromole such as foreign protein, prepared enzyme can be used for PCR and sequencing reaction; Sun Liang first etc. utilizes the thermostability of this enzyme, through two-wheeled-70 DEG C of deep refrigerations and 75 DEG C of water-baths, bacteria lysis is made to discharge content, the cell debris of high speed centrifugation removing freeze thawing sex change and the mixture of nucleic acid-protein are to reach the object of fast purifying Taq DNA polysaccharase, and PCR amplified reaction shows that the vigor of prepared Taq DNA polysaccharase, susceptibility, specificity all reach test requirements document.Bag Yongming etc. are after inducing target protein to express with IPTG, and adopt polymine precipitation Taq DNA polysaccharase, ion exchange column Bio-Rex-70 separation and purification, completes the overexpression of Taq DNA pol gene; Zhao Meirong etc. are by the E.coli containing Taq DNA pol gene through fermentation, and cell culture fluid is through breaking walls and cracking, and crude protein extracting, through purification steps such as Sephaeryl-s-100, CM-Sephadex C50 column chromatographies, obtains restructuring Taq DNA polysaccharase; Superfine thermally denature, affinity chromatography, the cation-exchange chromatography of utilizing of Wang Jing obtains electrophoretically pure restructuring Taq DNA polysaccharase; He Gang etc. attempt carrying out global optimization to the technology of preparing of Taq archaeal dna polymerase, to insert the Pet-24a expression plasmid transform Escherichia coli strain of restructuring Taq DNA pol gene, induce 8 hours with the IPTG of 2mmol/L, higher Taq DNA polysaccharase expression efficiency can be obtained.
Summary of the invention
For the technical problem existed in currently available technology, the invention provides a kind of restructuring Taq DNA polysaccharase and preparation method thereof, this preparation method can obtain high purity Taq DNA polysaccharase.Utilize the method that the present invention sets up, can be purified to about 3.5ml Taq archaeal dna polymerase liquid from 500ml bacterium liquid, enzyme is lived and is reached 16.7U/ul.
An object of the present invention is to provide a kind of restructuring Taq archaeal dna polymerase, its protein amino acid sequence is as shown in SEQ ID NO:2.
Another object of the present invention is to provide a kind of gene of described Taq archaeal dna polymerase of encoding, and its nucleotide sequence is as shown in SEQ ID NO:1.
Another object of the present invention is to provide a kind of method preparing restructuring Taq archaeal dna polymerase, and concrete steps are as follows:
(1) nucleotide sequence shown in SEQ ID NO:1 is cloned;
(2) nucleotide fragments of step (1) gained is connected with plasmid pET32a, construction recombination plasmid pET32a-Taq;
(3) recombinant plasmid pET32a-Taq is transformed into competent escherichia coli cell
e.coliin BL21, obtain expression strain;
(4) expression strain utilizes LB nutrient solution to carry out suspension culture, cultivates after 4.5 hours for 37 DEG C, and adding final concentration is that the IPTG induction of 0.5 mM is after 16-20 hour; Dissociate thalline, filters; Obtain described restructuring Taq archaeal dna polymerase, its aminoacid sequence is as shown in SEQ ID NO:2.
In step (1), described nucleotide sequence be from thermus aquaticus (
thermusaquaticus) in the Taq DNA pol gene separated, described gene coded protein can be used for preparation restructuring Taq archaeal dna polymerase.The nucleotide sequence of described gene is as shown in SEQ ID NO:1, and the protein amino acid sequence of described genes encoding is as shown in SEQ ID NO:2.
Further, in step (4), described in the thalline that dissociates to dissociate thalline for adopting N,O-Diacetylmuramidase; Describedly to be filtered into chromatography column and dialysis tubing continuous filtration.
In the present invention further embodiment, the concentration of described N,O-Diacetylmuramidase is 4mg/ml; Described chromatography column is the ion exchange column resin of Bio-Rex 70;
The present invention is on the basis of the restructuring Taq DNA pol gene expression vector built, and carries out the optimization of expression system, improves its Taq archaeal dna polymerase expression and purification efficiency, to obtain the Taq DNA polysaccharase of stable, high vigor.The present invention by the method for gene clone, from thermus aquaticus (
thermusaquaticus) in be cloned into a Taq DNA pol gene.By preparing the method for restructuring Taq archaeal dna polymerase, recombinant bacterial strain abduction delivering prepares high vigor Taq DNA polysaccharase, and enzyme is lived and reached 16.7U/ul.
Accompanying drawing explanation
Fig. 1 be purifying of the present invention Taq DNA polymerase protein matter electrophorogram (1-10 be collect Taq DNA polysaccharase; 11 is commercially available Taq DNA polysaccharase, as positive control; M is standard protein molecular weight).
Fig. 2 is that Taq DNA polysaccharase prepared by the present invention carries out PCR and detects enzymic activity electrophorogram (1-4 swimming lane is that the Taq archaeal dna polymerase extracting preparation of the present invention react the PCR of template DNA, dilution 40 times respectively, 30 times, 20 times, 10 times; 5-7 swimming lane is that commercially available Taq DNA polysaccharase (5U/ul) is reacted the PCR of template DNA, dilutes 8 times respectively, 7 times, 6 times, 5 times; M is standard DNA molecular weight).
Fig. 3 be the Taq archaeal dna polymerase prepared of the present invention and commercially Taq archaeal dna polymerase carry out PCR and detect and compare enzymic activity electrophorogram (1-4 swimming lane is that commercially Taq archaeal dna polymerase different batches reacts the PCR of template DNA; 5-8 swimming lane is that the different batches Taq archaeal dna polymerase that the present invention extracts preparation reacts the PCR of template DNA; M is stranded DNA molecule amount).
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these examples are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted specific experiment condition in the following example, usual conveniently condition, Molecular Cloning: A Laboratory guide (Sambrook J, et al. 2008. Molecular Cloning:A Laboratory Manual, 3rd Ed.) described in condition, or according to the condition that manufacturer advises.
embodiment 1: the cloning and analysis of full length gene coding region
Adopt commercially available bacterial genomes DNA extraction kit method extract thermus aquaticus (
thermusaquaticus) STb gene, adopt Touchdown round pcr and regular-PCR technology to carry out the full length sequence amplification of Taq DNA pol gene, amplimer comprises 2 groups (5 '-tatatgagggggatgctgccc-3 ' and 5 '-tcactccttggcggagagc-3 ').PCR primer is after 1% agarose gel electrophoresis detects, and the blob of viscose of cutting containing object band under ultraviolet lamp, uses sepharose to reclaim test kit and reclaim object fragment, in-20 DEG C of preservations.The object fragment reclaimed, spends the night in 16 DEG C of metal baths and is connected to the precious biotechnology company limited of cloning vector pMD19-T(, TaKaRa), and be transformed into competent escherichia coli cell
e.colitop10(Beijing Tian Gen biochemical technology company limited) in, coat on the LB solid medium containing 100mg/mL Amp, 37 DEG C of incubated overnight, after the blue hickie screening of IPTG/X-gal, a picking 4-10 positive colony checks order.By sequencing result by sequence alignment, be separated to a Taq DNA pol gene.Taq DNA polymerase gene sequence total length is 2499bp, and its nucleotide sequence is as shown in SEQ ID NO:1, and 832 amino acid of encoding, take ATG as initiator codon, TGA is terminator codon.Taq DNA pol gene PCR reclaims product and pET32a plasmid (precious biotechnology company limited, TaKaRa) EcoRI and NotI double digestion is carried out, after 37 DEG C of metal bath 3-4h, detect with 1% agarose gel electrophoresis, use sepharose to reclaim test kit and reclaim.Be connected with plasmid pET32a by object fragment Taq DNA pol gene, 16 DEG C are spent the night, the recombinant plasmid called after pET32a-Taq built.Be transformed into competent escherichia coli cell
e.colibL21(Beijing Tian Gen biochemical technology company limited) in, obtain expression strain, called after Taq archaeal dna polymerase bacterial strain.
embodiment 2: the cultivation of restructuring Taq archaeal dna polymerase bacterial strain and induction
Preparation LB liquid shaking bottle (the LB liquid nutrient medium of 500 ml, is divided in the triangular flask of 2 500 ml, i.e. every bottled 250 ml, stand-by after sterilizing).Take out Taq DNA polysaccharase bacterial classification, be seeded in the LB+Amp(100 μ g/ml of 4 ml) in liquid nutrient medium, cultivate 16-20 hour for 37 DEG C.The LB liquid nutrient medium of bottled 250 ml of each triangle, adding the antibiotic final concentration of Amp(is 100 μ g/ml), and the strain inoculation of cultivation with 1.25 ml; Cultivate 4.5 hours for 37 DEG C.
Add the IPTG induction that final concentration is 0.5mM, continue to cultivate 16-20 hour at 37 DEG C.
embodiment 3: restructuring Taq DNA polysaccharase purifying utensil prepares
Chromatography column selects the ion exchange column resin of Bio-Rex 70, and (size of dry powder is mesh size 100-200.Binding ability after swelling is 5-10 mg protein/ml).Take the dried resin of the Bio-Rex 70 of 7g, be placed in small beaker, wash with dd H2O, remove floating small-particle, swelling 2 hours of room temperature, period changes dd H several times
2o; With the solution C pre-equilibration containing 50 mM KCl, stand-by; With the resin that the solution C containing 50 mM KCl balances.The solution C containing 50 mM KCl again with 6-10 times of column volume after installing resin balances pillar.Check that the pH value of the solution C entering pillar and flow out pillar is constant.
embodiment 4: proteins extraction
4 DEG C, 4000 rpm, collect thalline in centrifugal 20 minutes; Be resuspended in the solution A of 30 ml, rinse the thalline collected; 4 DEG C, 8000 rpm, collect thalline in centrifugal 10 minutes; Be resuspended in 20 ml solution A, mixing.Add the N,O-Diacetylmuramidase of 80 mg, make final concentration reach 4 mg/ml; Keep 15 minutes under room temperature (25 DEG C); Add the solution B of 20 ml, 75 DEG C of water bath heat preservations 1 hour, period rocks several times; 4 DEG C, 10000 rpm, centrifugal 20 minutes, abandon precipitation, collect supernatant liquor; Magnetic stirrer, dropwise adds 5% PEI in supernatant liquor, makes final concentration reach 0.15%(volume); Under room temperature, (25 DEG C) continue stirring 10 minutes; 4 DEG C, 10000 rpm, centrifugal 15 minutes, collecting precipitation; Resuspended by the solution C containing 25 mM KCl of 12 ml, and washing precipitation; 4 DEG C, 8000 rpm, centrifugal 15 minutes, collecting precipitation; Resuspended by the solution C containing 25 mM KCl of 12 ml, and washing precipitation; 4 DEG C, 5000 rpm, centrifugal 10 minutes, collect supernatant liquor, the volume of measurement collection supernatant liquor; Add the solution C not containing KCl of 2 times of volumes, make the concentration of the final KCl of collection supernatant liquor reach 50mM.
embodiment 5: protein purification and detection
Supernatant liquor is contained on the chromatography column balanced; The solution C containing 50 mM KCl more than chromatography column balance use 6 times of volumes (about 60 ml), balances again; The solution C wash-out of sample containing 200 mM KCl, general preparation 50ml elutriant.
By each part electrophoresis on SDS-PAGE collected, contrast (referring to Fig. 1) with commercially available Taq archaeal dna polymerase, carry out protein size and purity detecting; The brighter swimming lane of electrophoresis represents Taq DNA polysaccharase high-content, these parts (3,4,5,6) is collected together; Be placed in dialysis tubing and use 500 ml solution D dialysis, 4 DEG C, 20 hours, period changes a dialyzate; Finally collect and obtain high purity Taq archaeal dna polymerase, the aminoacid sequence of gained high purity Taq archaeal dna polymerase is as shown in SEQ ID NO:2 after testing.
the PCR checking of embodiment 6:Taq DNA polysaccharase function and agarose electrophoresis detect
The DNA profiling reacted using plasmid pBI121 as PCR, using Kana Tag primer as PCR primer.
PCR reaction system cumulative volume is 25 μ l, wherein: 10 × PCR damping fluid (100 mmol/L Tris-HCl, pH 9.0; 500 mmol/L KCl; 20 mmol/L MgCl
20.1% gelatin, 1% Triton X-100) 2.5 μ l, each 2.5 mM of dNTP Mixture() 4ul, primer 1.0 uM, Taq archaeal dna polymerase comprises the Taq archaeal dna polymerase of embodiment 5 preparation and commercially available Taq archaeal dna polymerase, (the Taq archaeal dna polymerase of preparation dilutes 40 times respectively, 30 times, 20 times, 10 times; Commercially available Taq archaeal dna polymerase dilutes 8 times respectively, 7 times, 6 times, 5 times); DNA profiling 1 μ l(20 ng); Add sterilizing distilled water to 25 μ l.Application ABI 9700 PCR instrument carries out PCR reaction, response procedures is as follows: first 94 DEG C of denaturation 5 min, carry out 30 circulations afterwards, each circulation comprises 94 DEG C of sex change 30s, annealing temperature (annealing temperature is depending on primer) 30s, 72 DEG C of extension 1 min, and last 72 DEG C extend 10min.
PCR product, at 1% sepharose, carries out electrophoresis in 1 × TAE damping fluid; DNA is labeled as DL2000.Voltage 90 V, time 40 min; Gel imaging system carries out observation and takes pictures, and result is as shown in Fig. 2.Use SensiAnsys1.0.3 analysis software to carry out PCR product quantitative statistics the PCR imaging results of acquisition, the statistic data result of acquisition is as shown in table 1.The enzyme work calculating homemade Taq DNA polysaccharase liquid according to statistic data reaches 16.7U/ul.
Taq DNA polysaccharase prepared by table 1 the present invention carries out PCR and detects the PCR product fragment quantitative statistics data (in sample 1-8 corresponding diagram 2 1-8 swimming lane product) obtained
embodiment 7:Taq DNA polysaccharase PCR verifies and compares different batches stability in commercially Taq DNA polysaccharase PCR
The DNA profiling reacted using sea-tangle genomic dna as PCR, using mannose-phosphate mutase (Phosphomannomutase) Tag primer as PCR primer.
PCR reaction system cumulative volume is 25 μ l, wherein: 10 × PCR damping fluid (100 mmol/L Tris-HCl, pH 9.0; 500 mmol/L KCl; 20 mmol/L MgCl
20.1% gelatin, 1% Triton X-100) each 2.5 mM of 2.5 μ l, dNTP Mixture() 4ul, primer 1.0 uM, Taq DNA polysaccharase comprises Taq DNA polysaccharase 0.25U and each four different batches of commercially available Taq archaeal dna polymerase 1U of embodiment 5 preparation; DNA profiling 1 μ l(20 ng); Add sterilizing distilled water to 25 μ l.Application ABI 9700 PCR instrument carries out PCR reaction, response procedures is as follows: first 94 DEG C of denaturation 5 min, carry out 30 circulations afterwards, each circulation comprises 94 DEG C of sex change 30s, annealing temperature (annealing temperature is depending on primer) 30 s, 72 DEG C of extension 1 min, and last 72 DEG C extend 10 min.
PCR product, at 1% sepharose, carries out electrophoresis in 1 × TAE damping fluid; DNA marker is DL2000.Voltage 90 V, time 40 min; Gel imaging system carries out observation and takes pictures; Result as shown in Figure 3.
Use SensiAnsys1.0.3 analysis software to carry out PCR primer quantitative statistics the PCR imaging results of acquisition, the statistic data result of acquisition is as shown in table 2.The Taq archaeal dna polymerase of the preparation added in reaction system is each reaction 0.25U, and commercially available Taq archaeal dna polymerase is each reaction 1U.According to result display, different batches Taq archaeal dna polymerase prepared by embodiment 5 all can obtain pcr amplification, but only two batches of good amplifications of acquisition of the commercially available Taq archaeal dna polymerase of four different batches.And calculating the commercially available Taq archaeal dna polymerases of two batches of amplification according to statistic data, to obtain product amounts consistent with the product amount of homemade Taq archaeal dna polymerase liquid amplification under identical equivalent.Prove that the more commercially available polymerase activity of the polysaccharase prepared of the present invention is more stable.
Taq archaeal dna polymerase prepared by table 2 the present invention and commercially Taq archaeal dna polymerase carry out PCR and detect the PCR product fragment quantitative statistics data (in sample 1-8 corresponding diagram 3 1-8 swimming lane product) obtained
Related solution formula is as follows:
LB liquid nutrient medium: Tryphone 5g; Yeast Extract 2.5g; NaCl 5g; The ddH20 adding sterilizing is 500 ml to volume.
Solution A is filled a prescription: 5 ml 1 M Tris-HCl, pH 8.0; 0.9 g Dextrose(glucose); 0.2 ml 0.5 M EDTA; The ddH20 adding sterilizing is 100 ml to volume.
Solution B is filled a prescription: 10 mM Tris(pH 8.0); 50 mM KCl; 1 mM EDTA; 1 mM PMSF; 0.5% Tween 20; 0.5% NP-40; The ddH20 adding sterilizing is 100 ml to volume.
4 × solution C formula: 20 mM HEPES(pH 7.9); 1 mM EDTA; 0.5 mM PMSF; 0.5% Tween 20; 0.5% NP-40; The ddH20 adding sterilizing is 250 ml to volume.
4 × solution D formula: 20 mM HEPES(pH 7.9); 100 mM KCl; 0.1 mM EDTA; 0.5 mM PMSF; 1 mM DTT; The ddH20 adding sterilizing is 250 ml to volume.
10 × PCR reaction buffer is filled a prescription: 500 mM KCl, 100 mM Tris-HCl, pH 9.0(25 DEG C), 20 mM MgCl2,0.1% gelatin, 1% Triton X-100.
1 × TAE damping fluid: dilute with 50 × TAE damping fluid mother liquor.Tris alkali 242g, Glacial acetic acid 57.1ml, Na
2eDTA2H2O 37.2g, the ddH20 adding sterilizing is 1000ml to volume.
Although the invention describes concrete example, having a bit is obvious to those skilled in the art, namely can make various changes the present invention and change under the premise without departing from the spirit and scope of the present invention.Therefore, claims cover all these variations within the scope of the present invention.
<110> Qingdao Si Neng Genetic Biotechnologies company limited
<120> mono-kind restructuring Taq archaeal dna polymerase and preparation method thereof
<130> 0
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2499
<212> DNA
<213> thermus aquaticus
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atgaggggga tgctgcccct ctttgagccc aagggccggg tcctcctggt ggacggccac 60
cacctggcct accgcacctt ccacgccctg aagggcctca ccaccagccg gggggagccg 120
gtgcaggcgg tctacggctt cgccaagagc ctcctcaagg ccctcaagga ggacggggac 180
gcggtgatcg tggtctttga cgccaaggcc ccctccttcc gccacgaggc ctacgggggg 240
tacaaggcgg gccgggcccc cacgccggag gactttcccc ggcaactcgc cctcatcaag 300
gagctggtgg acctcctggg gctggcgcgc ctcgaggtcc cgggctacga ggcggacgac 360
gtcctggcca gcctggccaa gaaggcggaa aaggagggct acgaggtccg catcctcacc 420
gccgacaaag acctttacca gctcctttcc gaccgcatcc acgccctcca ccccgagggg 480
tacctcatca ccccggcctg gctttgggaa aagtacggcc tgaggcccga ccagtgggcc 540
gactaccggg ccctgaccgg ggacgagtcc gacaaccttc ccggggtcaa gggcatcggg 600
gagaagacgg cgaggaagct tctggaggag tgggggagcc tggaagccct cctcaagaac 660
ctggaccggc tgaagcccgc catccgggag aagatcctgg cccacatgga cgatctgaag 720
ctctcctggg acctggccaa ggtgcgcacc gacctgcccc tggaggtgga cttcgccaaa 780
aggcgggagc ccgaccggga gaggcttagg gcctttctgg agaggcttga gtttggcagc 840
ctcctccacg agttcggcct tctggaaagc cccaaggccc tggaggaggc cccctggccc 900
ccgccggaag gggccttcgt gggctttgtg ctttcccgca aggagcccat gtgggccgat 960
cttctggccc tggccgccgc cagggggggc cgggtccacc gggcccccga gccttataaa 1020
gccctcaggg acctgaagga ggcgcggggg cttctcgcca aagacctgag cgttctggcc 1080
ctgagggaag gccttggcct cccgcccggc gacgacccca tgctcctcgc ctacctcctg 1140
gacccttcca acaccacccc cgagggggtg gcccggcgct acggcgggga gtggacggag 1200
gaggcggggg agcgggccgc cctttccgag aggctcttcg ccaacctgtg ggggaggctt 1260
gagggggagg agaggctcct ttggctttac cgggaggtgg agaggcccct ttccgctgtc 1320
ctggcccaca tggaggccac gggggtgcgc ctggacgtgg cctatctcag ggccttgtcc 1380
ctggaggtgg ccgaggagat cgcccgcctc gaggccgagg tcttccgcct ggccggccac 1440
cccttcaacc tcaactcccg ggaccagctg gaaagggtcc tctttgacga gctagggctt 1500
cccgccatcg gcaagacgga gaagaccggc aagcgctcca ccagcgccgc cgtcctggag 1560
gccctccgcg aggcccaccc catcgtggag aagatcctgc agtaccggga gctcaccaag 1620
ctgaagagca cctacattga ccccttgccg gacctcatcc accccaggac gggccgcctc 1680
cacacccgct tcaaccagac ggccacggcc acgggcaggc taagtagctc cgatcccaac 1740
ctccagaaca tccccgtccg caccccgctt gggcagagga tccgccgggc cttcatcgcc 1800
gaggaggggt ggctattggt ggccctggac tatagccaga tagagctcag ggtgctggcc 1860
cacctctccg gcgacgagaa cctgatccgg gtcttccagg aggggcggga catccacacg 1920
gagaccgcca gctggatgtt cggcgtcccc cgggaggccg tggaccccct gatgcgccgg 1980
gcggccaaga ccatcaactt cggggtcctc tacggcatgt cggcccaccg cctctcccag 2040
gagctagcca tcccttacga ggaggcccag gccttcattg agcgctactt tcagagcttc 2100
cccaaggtgc gggcctggat tgagaagacc ctggaggagg gcaggaggcg ggggtacgtg 2160
gagaccctct tcggccgccg ccgctacgtg ccagacctag aggcccgggt gaagagcgtg 2220
cgggaggcgg ccgagcgcat ggccttcaac atgcccgtcc agggcaccgc cgccgacctc 2280
atgaagctgg ctatggtgaa gctcttcccc aggctggagg aaatgggggc caggatgctc 2340
cttcaggtcc acgacgagct ggtcctcgag gccccaaaag agagggcgga ggccgtggcc 2400
cggctggcca aggaggtcat ggagggggtg tatcccctgg ccgtgcccct ggaggtggag 2460
gtggggatag gggaggactg gctctccgcc aaggagtga 2499
<210> 2
<211> 832
<212> PRT
<213> thermus aquaticus
<400> 2
Met Arg Gly Met Leu Pro Leu Phe Glu Pro Lys Gly Arg Val Leu Leu
1 5 10 15
Val Asp Gly His His Leu Ala Tyr Arg Thr Phe His Ala Leu Lys Gly
20 25 30
Leu Thr Thr Ser Arg Gly Glu Pro Val Gln Ala Val Tyr Gly Phe Ala
35 40 45
Lys Ser Leu Leu Lys Ala Leu Lys Glu Asp Gly Asp Ala Val Ile Val
50 55 60
Val Phe Asp Ala Lys Ala Pro Ser Phe Arg His Glu Ala Tyr Gly Gly
65 70 75 80
Tyr Lys Ala Gly Arg Ala Pro Thr Pro Glu Asp Phe Pro Arg Gln Leu
85 90 95
Ala Leu Ile Lys Glu Leu Val Asp Leu Leu Gly Leu Ala Arg Leu Glu
100 105 110
Val Pro Gly Tyr Glu Ala Asp Asp Val Leu Ala Ser Leu Ala Lys Lys
115 120 125
Ala Glu Lys Glu Gly Tyr Glu Val Arg Ile Leu Thr Ala Asp Lys Asp
130 135 140
Leu Tyr Gln Leu Leu Ser Asp Arg Ile His Ala Leu His Pro Glu Gly
145 150 155 160
Tyr Leu Ile Thr Pro Ala Trp Leu Trp Glu Lys Tyr Gly Leu Arg Pro
165 170 175
Asp Gln Trp Ala Asp Tyr Arg Ala Leu Thr Gly Asp Glu Ser Asp Asn
180 185 190
Leu Pro Gly Val Lys Gly Ile Gly Glu Lys Thr Ala Arg Lys Leu Leu
195 200 205
Glu Glu Trp Gly Ser Leu Glu Ala Leu Leu Lys Asn Leu Asp Arg Leu
210 215 220
Lys Pro Ala Ile Arg Glu Lys Ile Leu Ala His Met Asp Asp Leu Lys
225 230 235 240
Leu Ser Trp Asp Leu Ala Lys Val Arg Thr Asp Leu Pro Leu Glu Val
245 250 255
Asp Phe Ala Lys Arg Arg Glu Pro Asp Arg Glu Arg Leu Arg Ala Phe
260 265 270
Leu Glu Arg Leu Glu Phe Gly Ser Leu Leu His Glu Phe Gly Leu Leu
275 280 285
Glu Ser Pro Lys Ala Leu Glu Glu Ala Pro Trp Pro Pro Pro Glu Gly
290 295 300
Ala Phe Val Gly Phe Val Leu Ser Arg Lys Glu Pro Met Trp Ala Asp
305 310 315 320
Leu Leu Ala Leu Ala Ala Ala Arg Gly Gly Arg Val His Arg Ala Pro
325 330 335
Glu Pro Tyr Lys Ala Leu Arg Asp Leu Lys Glu Ala Arg Gly Leu Leu
340 345 350
Ala Lys Asp Leu Ser Val Leu Ala Leu Arg Glu Gly Leu Gly Leu Pro
355 360 365
Pro Gly Asp Asp Pro Met Leu Leu Ala Tyr Leu Leu Asp Pro Ser Asn
370 375 380
Thr Thr Pro Glu Gly Val Ala Arg Arg Tyr Gly Gly Glu Trp Thr Glu
385 390 395 400
Glu Ala Gly Glu Arg Ala Ala Leu Ser Glu Arg Leu Phe Ala Asn Leu
405 410 415
Trp Gly Arg Leu Glu Gly Glu Glu Arg Leu Leu Trp Leu Tyr Arg Glu
420 425 430
Val Glu Arg Pro Leu Ser Ala Val Leu Ala His Met Glu Ala Thr Gly
435 440 445
Val Arg Leu Asp Val Ala Tyr Leu Arg Ala Leu Ser Leu Glu Val Ala
450 455 460
Glu Glu Ile Ala Arg Leu Glu Ala Glu Val Phe Arg Leu Ala Gly His
465 470 475 480
Pro Phe Asn Leu Asn Ser Arg Asp Gln Leu Glu Arg Val Leu Phe Asp
485 490 495
Glu Leu Gly Leu Pro Ala Ile Gly Lys Thr Glu Lys Thr Gly Lys Arg
500 505 510
Ser Thr Ser Ala Ala Val Leu Glu Ala Leu Arg Glu Ala His Pro Ile
515 520 525
Val Glu Lys Ile Leu Gln Tyr Arg Glu Leu Thr Lys Leu Lys Ser Thr
530 535 540
Tyr Ile Asp Pro Leu Pro Asp Leu Ile His Pro Arg Thr Gly Arg Leu
545 550 555 560
His Thr Arg Phe Asn Gln Thr Ala Thr Ala Thr Gly Arg Leu Ser Ser
565 570 575
Ser Asp Pro Asn Leu Gln Asn Ile Pro Val Arg Thr Pro Leu Gly Gln
580 585 590
Arg Ile Arg Arg Ala Phe Ile Ala Glu Glu Gly Trp Leu Leu Val Ala
595 600 605
Leu Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Gly
610 615 620
Asp Glu Asn Leu Ile Arg Val Phe Gln Glu Gly Arg Asp Ile His Thr
625 630 635 640
Glu Thr Ala Ser Trp Met Phe Gly Val Pro Arg Glu Ala Val Asp Pro
645 650 655
Leu Met Arg Arg Ala Ala Lys Thr Ile Asn Phe Gly Val Leu Tyr Gly
660 665 670
Met Ser Ala His Arg Leu Ser Gln Glu Leu Ala Ile Pro Tyr Glu Glu
675 680 685
Ala Gln Ala Phe Ile Glu Arg Tyr Phe Gln Ser Phe Pro Lys Val Arg
690 695 700
Ala Trp Ile Glu Lys Thr Leu Glu Glu Gly Arg Arg Arg Gly Tyr Val
705 710 715 720
Glu Thr Leu Phe Gly Arg Arg Arg Tyr Val Pro Asp Leu Glu Ala Arg
725 730 735
Val Lys Ser Val Arg Glu Ala Ala Glu Arg Met Ala Phe Asn Met Pro
740 745 750
Val Gln Gly Thr Ala Ala Asp Leu Met Lys Leu Ala Met Val Lys Leu
755 760 765
Phe Pro Arg Leu Glu Glu Met Gly Ala Arg Met Leu Leu Gln Val His
770 775 780
Asp Glu Leu Val Leu Glu Ala Pro Lys Glu Arg Ala Glu Ala Val Ala
785 790 795 800
Arg Leu Ala Lys Glu Val Met Glu Gly Val Tyr Pro Leu Ala Val Pro
805 810 815
Leu Glu Val Glu Val Gly Ile Gly Glu Asp Trp Leu Ser Ala Lys Glu
820 825 830
Claims (1)
1. prepare a method for restructuring Taq archaeal dna polymerase, it is characterized in that, concrete steps are as follows:
(1) nucleotide sequence shown in SEQ ID NO:1 is cloned;
(2) Taq DNA pol gene PCR recovery product and pET32a plasmid carry out EcoRI and NotI double digestion, after 37 DEG C of metal bath 3-4h, detect with 1% agarose gel electrophoresis, use sepharose to reclaim test kit and reclaim; Be connected with plasmid pET32a by object fragment Taq DNA pol gene, 16 DEG C are spent the night, the recombinant plasmid called after pET32a-Taq built; Be transformed into competent escherichia coli cell
e.colibL21, obtains expression strain, called after Taq archaeal dna polymerase bacterial strain;
(3) take out Taq DNA polysaccharase bacterial classification, be seeded in the LB+Amp liquid nutrient medium of 4ml, the concentration of Amp is 100 μ g/ml, cultivates 16-20 hour for 37 DEG C; The LB liquid nutrient medium of the bottled 250ml of each triangle, adds Amp and makes final concentration be 100 μ g/ml, and the strain inoculation of cultivation with 1.25ml; Cultivate 4.5 hours for 37 DEG C; Add the IPTG induction that final concentration is 0.5mM, continue to cultivate 16-20 hour at 37 DEG C;
(4) 4 DEG C, 4000rpm, collects thalline in centrifugal 20 minutes; Be resuspended in the solution A of 30ml, rinse the thalline collected; 4 DEG C, 8000rpm, collects thalline in centrifugal 10 minutes; Be resuspended in 20ml solution A, mixing; Add the N,O-Diacetylmuramidase of 80 mg, make final concentration reach 4 mg/ml; Keep 15 minutes at room temperature 25 DEG C; Add the solution B of 20ml, 75 DEG C of water bath heat preservations 1 hour, period rocks several times; 4 DEG C, 10000rpm, centrifugal 20 minutes, abandons precipitation, collects supernatant liquor; Magnetic stirrer, dropwise adds 5%PEI in supernatant liquor, makes final volume concentration reach 0.15%; Lower 25 DEG C of room temperature continues stirring 10 minutes; 4 DEG C, 10000rpm, centrifugal 15 minutes, collecting precipitation; Resuspended by the solution C containing 25mM KCl of 12ml, and washing precipitation; 4 DEG C, 8000rpm, centrifugal 15 minutes, collecting precipitation; Resuspended by the solution C containing 25mM KCl of 12ml, and washing precipitation; 4 DEG C, 5000 rpm, centrifugal 10 minutes, collect supernatant liquor, the volume of measurement collection supernatant liquor; Add the solution C not containing KCl of 2 times of volumes, make the concentration of the final KCl of collection supernatant liquor reach 50mM;
(5) supernatant liquor is contained on the chromatography column balanced; The solution C containing 50mM KCl more than chromatography column balance use 6 times of volumes, balances again; The solution C wash-out of sample containing 200 mM KCl, prepares 50ml elutriant; By each part electrophoresis on SDS-PAGE collected, contrast with commercially available Taq archaeal dna polymerase, carry out protein size and purity detecting; The brighter swimming lane of electrophoresis represents Taq DNA polysaccharase high-content, by these portion collection together; Be placed in dialysis tubing and use 500 ml solution D dialysis, 4 DEG C, 20 hours, period changed a dialyzate; Finally collect and obtain high purity Taq archaeal dna polymerase;
Described solution A formula: 5ml 1 M Tris-HCl, pH 8.0; 0.9g glucose; 0.2ml 0.5M EDTA; Add the ddH of sterilizing
2o is 100ml to volume;
Described solution B formula: 10mM Tris, pH 8.0; 50mM KCl; 1mM EDTA; 1mM PMSF; 0.5% Tween 20; 0.5% NP-40; Add the ddH of sterilizing
2o is 100ml to volume;
4 × solution C formula: 20mM HEPES, pH 7.9; 1mM EDTA; 0.5mM PMSF; 0.5% Tween 20; 0.5% NP-40; Add the ddH of sterilizing
2o is 250ml to volume;
4 × solution D formula: 20mM HEPES, pH 7.9; 100mM KCl; 0.1mM EDTA; 0.5mM PMSF; 1mM DTT; Add the ddH of sterilizing
2o is 250ml to volume.
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| CN104312992B (en) * | 2014-10-24 | 2017-05-17 | 福建农林大学 | Method for simply and quickly extracting and purifying Taq-DNA polymerase |
| CN106867978A (en) * | 2015-12-10 | 2017-06-20 | 江苏众红生物工程创药研究院有限公司 | One kind restructuring high-fidelity DNA polymerase and its encoding gene and expression |
| CN106893698A (en) * | 2015-12-17 | 2017-06-27 | 江苏众红生物工程创药研究院有限公司 | One kind restructuring Taq archaeal dna polymerases and its encoding gene and expression |
| CN106318922A (en) * | 2016-08-17 | 2017-01-11 | 青岛海大蓝科生物科技有限公司 | Preparation method of Pfu DNA polymerase |
| CN108192907B (en) * | 2018-01-26 | 2020-10-16 | 遵义医学院 | Thermostable DNA amplification fusion enzyme |
| CN108070577B (en) * | 2018-02-05 | 2021-05-28 | 中国科学院武汉病毒研究所 | Antiserum interference TaqDNA polymerase and preparation and application thereof |
| CN108949719B (en) * | 2018-07-26 | 2020-12-15 | 山西医科大学 | Coding error-prone DNA polymerase and preparation method thereof |
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