CN106318922A - Preparation method of Pfu DNA polymerase - Google Patents
Preparation method of Pfu DNA polymerase Download PDFInfo
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- CN106318922A CN106318922A CN201610682612.2A CN201610682612A CN106318922A CN 106318922 A CN106318922 A CN 106318922A CN 201610682612 A CN201610682612 A CN 201610682612A CN 106318922 A CN106318922 A CN 106318922A
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1241—Nucleotidyltransferases (2.7.7)
- C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/07—Nucleotidyltransferases (2.7.7)
- C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a preparation method of Pfu DNA polymerase. The method includes the specific steps of firstly, cloning the nucleotide sequence shown in SEQ ID NO:1; secondly, connecting a nucleotide segment obtained in the first step with a plasmid pET32a to establish a recombinant plasmid pET32a-Pfu; thirdly, converting the recombinant plasmid pET32a-Pfu into an escherichia coli competent cell E.coli BL21 to obtain an expression strain; fourthly, conducting suspension cultivation on the expression strain through an LB culture solution, adding the IPTG with final concentration of 0.5 mM for 16-20 h after cultivation is conducted at 37 DEG C till the OD 600 value is 0.6, dissociating thallus, conducting filtering, and obtaining the recombinant Pfu DNA polymerization.
Description
Technical field
The present invention relates to gene engineering technology field, be specifically related to a kind of method preparing Pfu archaeal dna polymerase.
Background technology
1985, the polymerase chain reaction (polymerase being with historically new significance invented in Cetus company of the U.S.
Chain reaction, PCR), be a kind of method that specific DNA fragmentation is carried out in vitro rapid amplifying, i.e. double-stranded DNA exists
Under high temperature, strand is unwind in degeneration, archaeal dna polymerase, dNTP, special primer participation under, multiple according to base pair complementarity principle
Make two same molecule copies.
Archaeal dna polymerase (DNA polymerase), is the important enzyme of cell DNA duplication, is mainly used in polymerase chain
In reaction (Polymerase Chain Reaction).Archaeal dna polymerase is broadly divided into six types: A, B, C, D, X, Y, generally
Used belongs to Type B polymerase.Pfu archaeal dna polymerase is one of best polymerase of fidelity.
PfuDNA polymerase (Pfu DNA polymerase) is to find in thermophilic ancient core biology Pyrococcus, one
Class can in vivo carry out the enzyme of DNA replication dna.This enzyme contains 2 protein protomers (P45 and P50), and for polymer, molecular weight is about
90kDa.This enzyme has 5'-3' polymerase activity and 3'-5' exonuclease activity simultaneously, can correct the most in the polymerization
The base that mistake mixes, fidelity is high.In experiment in vitro, at polymerase chain reaction (polymerase chain
Reaction, PCR) in, use Pfu polymerase can be quick, the amplification of DNA fragments of high-fidelity.
Initially use Pfu archaeal dna polymerase is directly isolated and purified from thalline, but is difficult to obtain substantial amounts of enzyme, later
Its gene recombinaton is expressed in other host cell by researcher, more extracted purification can meet application requirement.According to
Described in existing document, the extraction of Pfu archaeal dna polymerase is it is generally required to two steps, and first step is breaking cellular wall, the weight that will obtain
Group the thalline methods such as ultrasonic, lysozyme or two kinds of methods combine fracturing cell walls, and this step needs ice bath low temperature, prevents
Enzyme denaturation inactivates.Second step is thermal denaturation, will broken after cell maybe will broken after cell centrifugation take supernatant and be placed in
20-30min in the water-bath of 75-80 DEG C, utilizes the thermostability of polymerase, removes remaining most of thermo-labile impurity protein, reduces
The impurity interference that subsequent purification separates, this is also the conventional extracting method of other recombinant thermostable dna polymerase.
At present Pfu archaeal dna polymerase has the sales volume of tens dollars every year in the whole world, its price the most costly, therefore
It is necessary high expressed Pfu archaeal dna polymerase system is furtherd investigate, in order to reduce production cost, and be conducive to it to push away
Extensively.
Summary of the invention
An object of the present invention is to provide the preparation method of a kind of Pfu archaeal dna polymerase, specifically comprises the following steps that
(1) clone's nucleotide sequence shown in SEQ ID NO:1；
(2) nucleotide fragments and the plasmid pET32a of step (1) gained are attached, construction recombination plasmid pET32a-
Pfu；
(3) recombiant plasmid pET32a-Pfu is transformed in competent escherichia coli cell E.coli BL21, it is thus achieved that express
Bacterial strain；
(4) expression strain utilizes LB culture fluid to carry out suspension culture, after 37 DEG C of cultivations are 0.6 to OD600 value, adds the denseest
After the IPTG that degree is 0.5mM induces 16-20 hour；Dissociate thalline, filters；Obtain restructuring Pfu archaeal dna polymerase.
In the present invention, the aminoacid sequence of described Pfu archaeal dna polymerase is known in the art, and particular sequence can be found in
Genebank accession number BAA02362.1.Nucleotide sequence SEQ ID NO:1 is the coded sequence of this aminoacid sequence, by one
Fixed is codon optimized, so that Pfu archaeal dna polymerase expression efficiency in expression system is higher.
In one embodiment, described LB culture fluid consist of tryptone 10g/L；Yeast extract 5g/L；
NaCl 10g/L；Vitamin C 0.3g/L；Glucose 1g/L；Lactose 0.1g/L；Magnesium sulfate 0.5g/L；Surplus is water, and pH value is
7.6。
In another embodiment, in step (4), described in dissociate thalline for using lysozyme to dissociate thalline；Described
It is filtered into and filters continuously with chromatographic column and bag filter.In further embodiment, the concentration of described lysozyme is 4mg/
ml；Described chromatographic column is the ion exchange column resin of Bio-Rex 70.
Accompanying drawing explanation
Pfu archaeal dna polymerase protein electrophoresis figure prepared by Fig. 1 present invention
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these examples be merely to illustrate the present invention and
It is not used in restriction the scope of the present invention.The experimental technique of unreceipted specific experiment condition in the following example, generally according to routine
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 proposed by manufacturer.
Embodiment 1: the clone of full length gene coding region and analysis
Pfu gene (sequence is as shown in SEQ ID NO:1) and pET32a plasmid are carried out EcoRI and NotI double digestion,
After 37 DEG C of metal bath 3-4h, with 1% agarose gel electrophoresis detection, use agarose gel to reclaim test kit and reclaim.By purpose
Fragment Pfu DNA polymerase gene and plasmid pET32a are attached, and 16 DEG C overnight, and the recombiant plasmid built is named
pET32a-Pfu.Being transformed in competent escherichia coli cell E.coli BL21, it is thus achieved that expression strain, named Pfu DNA gathers
Synthase bacterial strain
The cultivation of embodiment 2:Pfu archaeal dna polymerase bacterial strain and induction
(the LB fluid medium of 500ml, is divided in the triangular flask of 2 500ml preparation LB liquid shaking bottle, the most bottled
250ml LB culture fluid, consisting of tryptone 10g/L；Yeast extract 5g/L；NaCl 10g/L；Vitamin C 0.3g/
L；Glucose 1g/L；Lactose 0.1g/L；Magnesium sulfate 0.5g/L；Surplus is water, and pH value is 7.6, lower same；After sterilizing stand-by).Take out
Pfu archaeal dna polymerase strain, is seeded in LB+Amp (the 100 μ g/ml) fluid medium of 4ml, cultivates 16-20 hour for 37 DEG C.
The LB fluid medium of the bottled 250ml of each triangle, adds Amp (the final concentration of 100 μ g/ml of antibiotic), and uses 1.25ml
Cultivation strain inoculation；After 37 DEG C are 0.6 to OD600 value, add the IPTG induction of final concentration of 0.5mM, 37 DEG C of continuation
Cultivate 16-20 hour.
Embodiment 3:Pfu archaeal dna polymerase purification utensil prepares
(size of dry powder is mesh size 100-200 to the ion exchange column resin of chromatographic column selection Bio-Rex 70.Molten
Binding ability after swollen is 5-10mg protein/ml).Weigh the dried resin of the Bio-Rex 70 of 7g, be placed in small beaker, use dd
H2O washes, and removes floating little granule, and swelling 2 hours of room temperature, period changes dd H2O several times；Pre-flat by the solution C containing 50mM KCl
Weighing apparatus, stand-by；The resin balanced by the solution C containing 50mM KCl.Install after resin again with 6-10 times of bed volume containing 50mM
The solution C balance pillar of KCl.Check that the pH value of the solution C entering pillar and flowing out pillar is constant.
Embodiment 4: Protein Extraction and purification
4 DEG C, 4000rpm, collects thalline in centrifugal 20 minutes；It is resuspended in the solution A of 30ml, rinses the thalline collected；4
DEG C, 8000rpm, collects thalline in centrifugal 10 minutes；It is resuspended in 20ml solution A, mixing.Add the lysozyme of 80mg, make the denseest
Degree reaches 4mg/ml；Keep 15 minutes under room temperature (25 DEG C)；Add the solution B of 20ml, 75 DEG C of water bath heat preservations 1 hour, period
Rock several times；4 DEG C, 10000rpm, centrifugal 20 minutes, abandons precipitation, collects supernatant；Magnetic stirrer, in supernatant
It is added dropwise over 5%PEI, makes final concentration reach 0.15% (volume)；Under room temperature, (25 DEG C) continue stirring 10 minutes；4 DEG C,
10000rpm, centrifugal 15 minutes, collects precipitation；Resuspended by the solution C containing 25mM KCl of 12ml, and wash precipitation；4 DEG C,
8000rpm, centrifugal 15 minutes, collects precipitation；Resuspended by the solution C containing 25mM KCl of 12ml, and wash precipitation；4 DEG C,
5000rpm, centrifugal 10 minutes, collects supernatant, measures the volume collecting supernatant；Add the solution without KCl of 2 times of volumes
C, makes the concentration of the final KCl of collection supernatant reach 50mM.
Supernatant is contained on the chromatographic column balanced；Chromatographic column balance uses containing of more than 6 times of volumes (about 60ml)
The solution C of 50mM KCl, balances again；The sample solution C eluting containing 200mM KCl, general preparation 50ml eluent.
Embodiment 5Pfu archaeal dna polymerase concentration measures and enzyme activity determination
(1) Pfu archaeal dna polymerase concentration uses Bradford method to be measured, with bovine serum albumin as standard, and result table
Bright purified after eluent (prepared by embodiment 4) in protein concentration be 2.9mg/ml.By Gelpro32 software analysis eluting
Liquid electrophoretogram, result shows Pfu archaeal dna polymerase high purity 95%.
(2) with calf thymus DNA as template, and with commercially available commercial Pfu archaeal dna polymerase 1U as comparison, reaction system it is
2ul (200mM Tris-HCl (PH 8.8), 100mM KCl, 100mM (NH4) 2SO4,1%Triton X-100,1mg/ml
BSA, 20mM MgSO4)；The DNA profiling of 1ul, 2ul dNTP, 1ul primer, Pfu archaeal dna polymerase (prepared by embodiment 4) and water；
PCR reaction setting see table
Result shows, Pfu archaeal dna polymerase of the present invention is compared with commercially available 1U polymerase, and enzyme is than living as 22900U/mg.
Embodiment 6 coding nucleotide sequence and the impact on Pfu archaeal dna polymerase yield of the LB culture medium
Different coding nucleotide sequences or LB culture fluid is used to repeat the preparation test of embodiment 1-4, according to enforcement
The method of example 5 measures the concentration of the Pfu archaeal dna polymerase obtained, and result is as follows:
| The concentration of Pfu archaeal dna polymerase | |
| Comparative example 1 | 0.7mg/ml |
| Comparative example 2 | 1.1mg/ml |
| Comparative example 3 | 1.6mg/ml |
| Comparative example 4 | 1.3mg/ml |
| Comparative example 5 | 1.2mg/ml |
Comparative example 1 sees the 237th-2564 bit bases in Genebank accession number D12983.1 for coding nucleotide sequence
Sequence, other preparation methoies are with embodiment 1-4
Comparative example 2 sees nucleotide sequence disclosed in Genebank accession number KF836420.1 for coding nucleotide sequence,
Other preparation methoies are with embodiment 1-4
Comparative example 3 is tryptone 10g/L for LB culture medium；Yeast extract 5g/L；NaCl 10g/L；Glucose 1g/
L；Lactose 0.1g/L；Magnesium sulfate 0.5g/L；Surplus is water, and pH value is 7.6, and other preparation methoies are with embodiment 1-4
Comparative example 4 is tryptone 10g/L for LB culture medium；Yeast extract 5g/L；NaCl 10g/L；Lactose 0.1g/
L；Magnesium sulfate 0.5g/L；Surplus is water, and pH value is 7.6, and other preparation methoies are with embodiment 1-4
Comparative example 5 is tryptone 10g/L for LB culture medium；Yeast extract 5g/L；NaCl 10g/L；Vitamin C
0.3g/L；Glucose 1g/L；Lactose 0.1g/L；Magnesium sulfate 0.5g/L；Surplus is water, and pH value is 7.0, and other preparation methoies are with real
Execute example 1-4
Related solution formula is as follows:
Solution A formulation: 5ml 1 M Tris-HCl, pH 8.0；0.9g Dextrose(glucose)；0.2ml 0.5M
EDTA；The ddH20 adding sterilizing is 100ml to volume.
Solution B formula: 10mM Tris (pH 8.0)；50mM KCl；1mM EDTA；1mM PMSF；0.5%Tween20；
0.5%NP-40；The ddH20 adding sterilizing 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；The ddH20 adding sterilizing is 250ml to volume.
4 × solution D formula: 20mM HEPES (pH 7.9)；100mM KCl；0.1mM EDTA；0.5mM PMSF；1mM
DTT；The ddH20 adding sterilizing is 250ml to volume.
1 × PCR reaction buffer formula: 500mM KCl, 100mM Tris-HCl, pH 9.0 (25 DEG C), 20mM
MgCl2,0.1% gelatin, 1%Triton X-100.
1 × TAE buffer: dilute with 50 × TAE buffer mother solution.Tris alkali 242g, glacial acetic acid 57.1ml,
Na2EDTA 2H2O 37.2g, the ddH20 adding sterilizing is 1000ml to volume.
Although the present invention describes specific example, but has any will be apparent to practitioners skilled in the art,
The present invention can be made various changes and change the most under the premise without departing from the spirit and scope of the present invention.Therefore, appended right
Require to cover all these variation within the scope of the present invention.
Claims (5)
1. a preparation method for Pfu archaeal dna polymerase, specifically comprises the following steps that
(1) clone's nucleotide sequence shown in SEQ ID NO:1；
(2) nucleotide fragments and the plasmid pET32a of step (1) gained are attached, construction recombination plasmid pET32a-Pfu；
(3) recombiant plasmid pET32a-Pfu is transformed in competent escherichia coli cell E.coli BL21, it is thus achieved that express bacterium
Strain；
(4) expression strain utilizes LB culture fluid to carry out suspension culture, after 37 DEG C of cultivations are 0.6 to OD600 value, adds final concentration of
After the IPTG of 0.5mM induces 16-20 hour；Dissociate thalline, filters；Obtain restructuring Pfu archaeal dna polymerase.
The preparation method preparing Pfu archaeal dna polymerase the most according to claim 1, it is characterised in that described LB culture fluid
Consist of tryptone 10g/L；Yeast extract 5g/L；NaCl 10g/L；Vitamin C 0.3g/L；Glucose 1g/L；Breast
Sugar 0.1g/L；Magnesium sulfate 0.5g/L；Surplus is water, and pH value is 7.6.
The preparation method preparing Pfu archaeal dna polymerase the most according to claim 1, it is characterised in that in step (4),
The described thalline that dissociates is for using lysozyme to dissociate thalline；Described being filtered into is filtered continuously with chromatographic column and bag filter.
The preparation method preparing Pfu archaeal dna polymerase the most according to claim 3, it is characterised in that described lysozyme
Concentration is 4mg/ml.
The preparation method preparing Pfu archaeal dna polymerase the most according to claim 3, it is characterised in that described chromatographic column is
The ion exchange column resin of Bio-Rex70.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662893A (en) * | 2020-07-01 | 2020-09-15 | 济南国科医工科技发展有限公司 | Preparation method of molecular diagnostic enzyme preparation |
| CN114015672A (en) * | 2021-12-06 | 2022-02-08 | 江南大学 | Pfu DNA polymerase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602643A (en) * | 2013-11-18 | 2014-02-26 | 青岛思能基因生物技术有限公司 | Recombinant Taq DNA polymerase and preparation method thereof |
-
2016
- 2016-08-17 CN CN201610682612.2A patent/CN106318922A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103602643A (en) * | 2013-11-18 | 2014-02-26 | 青岛思能基因生物技术有限公司 | Recombinant Taq DNA polymerase and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| AKBARZADEH,A., ET AL.: "Genbank accession number:KF836420.1", 《GENBANK》 * |
| UEMORI, T., ET AL.: "Genbank accession number:D12983.1", 《GENBANK》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662893A (en) * | 2020-07-01 | 2020-09-15 | 济南国科医工科技发展有限公司 | Preparation method of molecular diagnostic enzyme preparation |
| CN114015672A (en) * | 2021-12-06 | 2022-02-08 | 江南大学 | Pfu DNA polymerase |
| CN114015672B (en) * | 2021-12-06 | 2022-05-31 | 江南大学 | Pfu DNA polymerase |
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Application publication date: 20170111 |