CN107058260A - A kind of method of high efficiency recombinant expressed restriction enzyme - Google Patents
A kind of method of high efficiency recombinant expressed restriction enzyme Download PDFInfo
- Publication number
- CN107058260A CN107058260A CN201710035737.0A CN201710035737A CN107058260A CN 107058260 A CN107058260 A CN 107058260A CN 201710035737 A CN201710035737 A CN 201710035737A CN 107058260 A CN107058260 A CN 107058260A
- Authority
- CN
- China
- Prior art keywords
- restriction enzyme
- recombinant
- high efficiency
- culture
- kanamycins
- 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.)
- Pending
Links
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)
- C12N9/16—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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
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)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A kind of method of high efficiency recombinant expressed restriction enzyme, comprises the following steps：（1）First restriction endonuclease gene is inserted, carrier conversion is imported into Escherichia coli again, screening and culturing, sequencing obtain the recombinant expression plasmid of restriction enzyme, plasmid vector is to utilize lactose operon and T7 promoter regulations, the serial expression vectors of pET of IPTG inductions；（2）The recombinant expression plasmid of restriction enzyme is converted to BL21（DE3）In pLysS competent cells, screening obtains the recombinant strains of restriction enzyme；（3）Expand the recombinant strains and induced expression restriction enzyme of culture restriction enzyme.The inventive method utilizes the inhibitory action that glucose is expressed lactose operon expression system background, and BL21（DE3）The harsh control action that pLysS bacterial strains itself are expressed lactose operon expression system background, eliminates the protection step that methylates to host DNA, not only enormously simplify recombination expression process, can apply to the recombination expression of a variety of restriction enzymes.
Description
Technical field
The invention belongs to bioengineering field, particularly a kind of method of high efficiency recombinant expressed restriction enzyme.
Background technology
Restriction enzyme is the indispensable class important tool enzyme of contemporary genetic engineering research, is modern molecular biology
Basis.From late 1960s after first restriction enzyme is found, increasing restriction enzyme
It is found, purifies and obtains application.
Limitation-modifier is mainly cloned into plasmid by the method for restriction of production restriction endonuclease in the prior art, is used
Specific methylases corresponding with restriction enzyme prepare restriction enzyme, and purpose egg is realized by the high copy number of plasmid
White high expression, the preparation procedure of this method is cumbersome, restriction enzyme yield is low, production cost is high, the guarantor that methylates of acquisition
The bacterial strain of shield can only specificity protection host DNA from the cutting of a certain restriction enzyme, application is narrow.
The content of the invention
The technical problem to be solved in the present invention is in view of the shortcomings of the prior art, it is proposed that a kind of easy to operate, cost is low,
The method of the good high efficiency recombinant expressed restriction enzyme of enzyme activity.
The technical problem to be solved in the present invention is achieved through the following technical solutions, a kind of high efficiency recombinant expressed limitation
The method of property restriction endonuclease, is characterized in, comprises the following steps：
（1）First restriction endonuclease gene is inserted, then carrier conversion is imported into Escherichia coli, screening and culturing, sequencing are limited
The recombinant expression plasmid of property restriction endonuclease processed；
The plasmid vector is to utilize lactose operon and T7 promoter regulations, the serial expression vectors of pET of IPTG inductions；
（2）The recombinant expression plasmid of restriction enzyme is converted to BL21（DE3）In pLysS competent cells, screening is obtained
The recombinant strains of restriction enzyme；
（3）Expand the recombinant strains and induced expression restriction enzyme of culture restriction enzyme.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above
Method in, described restriction enzyme is selected from：BclI, DpnI, DpnII, EcoRI, HindIII, KpnI, MluI, MseI,
NcoI, NdeI, NheI, NotI, NsiI, NspV, PstI, SalI, SbfI, SgeI, SspI, StuI, TaqI, XbaI, XhoI.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above
Method the step of（1）In, described plasmid vector is pET-28b.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above
Method the step of（1）In, after restriction endonuclease gene fragment is connected on plasmid vector, connection product is converted to big
In enterobacteria DH5 α competent cells, it is coated in screening and cloning on the LB flat boards containing glucose and kanamycins, sequencing is obtained
The recombinant expression plasmid of restriction enzyme.It is further preferred that the concentration of described glucose is 0.5-2g/100mL, block that
The concentration of mycin is 37.5-150 μ g/mL.It is further preferred that the concentration of described glucose is 1-1.5g/100mL, block that
The concentration of mycin is 50-120 μ g/mL.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above
Method the step of（2）In, the recombinant expression plasmid of restriction enzyme is converted to BL21（DE3）PLysS competent cells
In, screening and culturing on the LB flat boards containing glucose, kanamycins and chloramphenicol is coated on, the restructuring table of restriction enzyme is obtained
Up to bacterial strain.It is further preferred that the concentration of described glucose is 0.5-2g/100mL, the concentration of kanamycins is 37.5-150
μ g/mL, the concentration of chloramphenicol is 17.5-70 μ g/mL.It is further preferred that the concentration of described glucose is 1-1.5g/
100mL, the concentration of kanamycins is 50-120 μ g/mL, and the concentration of chloramphenicol is 20-50 μ g/mL.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above
Method the step of（3）In, recombinant strains line culture and picking single bacterium colony by restriction enzyme are inoculated in containing Portugal
In the LB nutrient solutions of grape sugar, kanamycins and chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in into glucose, card
In the LB nutrient solutions of that mycin and chloramphenicol, culture a period of time is shaken, IPTG induced expression restriction enzymes are added.
The technical problems to be solved by the invention can also further be realized by following technical scheme.It is described above
Method the step of（3）In, recombinant strains line culture and picking single bacterium colony by restriction enzyme are inoculated in and contained
In the LB nutrient solutions of 0.5-2g/100mL glucose, 37.5-150 μ g/mL kanamycins and 17.5-70 μ g/mL chloramphenicol, shake
Overnight incubation is swung, obtained culture is inoculated in glucose containing 0.5-2g/100mL, 37.5-150 μ g/ by inoculum concentration 2-5%
In the LB nutrient solutions of mL kanamycins and 17.5-70 μ g/mL chloramphenicol, culture is shaken in 37 DEG C, 3 are reached to strain density ×
108-4×108During/mL, IPTG is added to final concentration of 0.2-0.5mM, and culture is shaken with 200-250 rpm at 15-18 DEG C
12-18 h, induced expression restriction enzyme.Trained it is further preferred that the recombinant strains of restriction enzyme are rule
It is mould that foster and picking single bacterium colony is inoculated in glucose containing 1-1.5g/100mL, 50-120 μ g/mL kanamycins and 20-50 μ g/mL chlorine
In the LB nutrient solutions of element, shaken cultivation is stayed overnight, and obtained culture is inoculated in into Portugal containing 1-1.5g/100mL by inoculum concentration 2-3%
In the LB nutrient solutions of grape sugar, 50-90 μ g/mL kanamycins and 20-50 μ g/mL chloramphenicol, culture is shaken in 35-37 DEG C, to bacterium
Density reaches 3.2 × 108-3.8×108During/mL, IPTG is added to final concentration of 0.3-0.4mM, and with 210- at 16-17 DEG C
240 rpm shake culture 14-16h, induced expression restriction enzyme.
Restriction enzyme allele is cloned into what is induced using lactose operon and T7 promoter regulations, IPTG by the inventive method
On pET series expression vectors, pass through the expression of lactose operon and T7 promoter regulation recombinant proteins.Due to recombinant protein gene
In lactose or lactose analog（Such as IPTG）In the presence of great expression, and feelings existed in lactose-free or lactose analog
Do not expressed then under condition, therefore can be with the expression of manual control recombinant protein using IPTG, and may contain in bacteria culture media
A small amount of lactose, the gene of recombinant protein trace expression, i.e. background before lactose or lactose analog are added can be caused to express, such as
Fruit recombinant protein toxicity is stronger, will kill host cell, makes cell irreproducible to the concentration for being adapted to induction.The inventive method
With reference to the characteristics of Escherichia coli itself, glucose is with the addition of in the medium, host cell is not utilized using glucose preferentially
Lactose, so that the background expression for adding restriction enzyme before derivant IPTG is suppressed, protects host DNA not limited
Property inscribe cleavage, it is ensured that host cell can breed the cell concentration of suitable induction；When the concentration of host cell reaches
When being adapted to induction, the glucose in culture medium has been consumed, and IPTG addition relative to the glucose or breast in culture medium
Sugar is all huge amount, and it is that induction restriction enzyme starts great expression to add IPTG.
Compared with prior art, the inventive method utilizes the suppression that glucose is expressed lactose operon expression system background
Effect, and BL21（DE3）The harsh control action that pLysS bacterial strains itself are expressed lactose operon expression system background, is saved
The protection step that methylates to host DNA is omited, without carrying out cumbersome methylases gene for a certain restriction enzyme
Screening, not only enormously simplify recombination expression process, can also be applied to the recombination expression of a variety of restriction enzymes, should with higher
Use promotional value.
Brief description of the drawings
Fig. 1 is Kpn I recombinant expression plasmid collection of illustrative plates in the embodiment of the present invention 22；
Fig. 2 is the detection electrophoretogram of Kpn I recombination expressions in the embodiment of the present invention 22；Wherein：1：Albumen crude enzyme liquid enzyme-specific
Cut band；2：λ DNA（Hind III digest）（Non- digestion）；M：DNA Marker.
Fig. 3 is electrophoretograms of the Kpn I through nickel affinity column after purification in the embodiment of the present invention 22；Wherein：1：5mM imidazoles；2-
3：20mM imidazoles；4：400mM imidazoles；M：Albumen Marker.
Fig. 4 is that Kpn I live electrophoretogram after purification through anion-exchange chromatography in the embodiment of the present invention 22；Wherein：1：
100mM KCl；2：300mM KCl；3：500mM KCl；4：700mM KCl；5：1M KCl；M：Albumen Marker.
Fig. 5 is Kpn I enzyme activities detection electrophoretogram in the embodiment of the present invention 22；Wherein：1：Kpn I stostes dilute 16 times；
2：Kpn I stostes dilute 32 times；3：Kpn I stostes dilute 64 times；4：Kpn I stostes dilute 128 times；5：Kpn I stostes dilute
256 times；6：Kpn I stostes dilute 512 times；7：λ DNA（HindIII digest）；M：DNA Marker.
Fig. 6 is Kpn I star activities and non-specific DNA enzymatic pollution detection electrophoretogram in the embodiment of the present invention 22；Its
In：1-6 is Kpn I digestion substrates 1h electrophoretogram, and 7-12 is Kpn I digestion substrates 16h electrophoretogram；1、7：Kpn I stostes
16 times of dilution；2、8：Kpn I stostes dilute 32 times；3、9：Kpn I stostes dilute 64 times；4、10：Kpn I stostes dilute 128 times；
5、11：Kpn I stostes dilute 256 times；6、12：Kpn I stostes dilute 512 times；13：λ DNA（Hind III digest）；M：
DNA Marker。
Fig. 7 is the digestion of digestion-connection-again detection electrophoretogram in the embodiment of the present invention 22；Wherein：1：Kpn I digestions
Common fragment；2：T4 DNA ligases connect post-fragment；4：Kpn I digestion junction fragments again；M：DNA
Marker。
Embodiment
Referring to the drawings, the concrete technical scheme of the present invention is further described, in order to which those skilled in the art enters
One step the present invention is understood, without constituting the limitation to its right.
A kind of embodiment 1, method of high efficiency recombinant expressed restriction enzyme, comprises the following steps：
（1）First restriction endonuclease gene is inserted, then carrier conversion is imported into Escherichia coli, screening and culturing, sequencing are limited
The recombinant expression plasmid of property restriction endonuclease processed；
The plasmid vector is to utilize lactose operon and T7 promoter regulations, the serial expression vectors of pET of IPTG inductions；
（2）The recombinant expression plasmid of restriction enzyme is converted to BL21（DE3）In pLysS competent cells, screening is obtained
The recombinant strains of restriction enzyme；
（3）Expand the recombinant strains and induced expression restriction enzyme of culture restriction enzyme.
Embodiment 2, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 1, described restriction enzyme
Enzyme is selected from：BclI, DpnI, DpnII, EcoRI, HindIII, KpnI, MluI, MseI, NcoI, NdeI, NheI, NotI, NsiI,
NspV, PstI, SalI, SbfI, SgeI, SspI, StuI, TaqI, XbaI, XhoI.
Embodiment 3, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 1, described plasmid vector is
pET-28b。
The step of embodiment 4, method of the high efficiency recombinant expressed restriction enzyme described in embodiment 1（1）In, it will limit
After property incision enzyme gene fragment is connected on plasmid vector, connection product is converted into bacillus coli DH 5 alpha competent cell,
It is coated in screening and cloning on the LB flat boards containing glucose and kanamycins, sequencing obtains the recombination expression matter of restriction enzyme
Grain.
In embodiment 5, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 4, described glucose
Concentration is 0.5g/100mL, and the concentration of kanamycins is 37.5 μ g/mL.
In embodiment 6, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 4, described glucose
Concentration is 2g/100mL, and the concentration of kanamycins is 150 μ g/mL.
In embodiment 7, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 4, described glucose
Concentration is 1g/100mL, and the concentration of kanamycins is 75 μ g/mL.
In embodiment 8, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 4, described glucose
Concentration is 1.5g/100mL, and the concentration of kanamycins is 120 μ g/mL.
In embodiment 9, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 4, described glucose
Concentration is 1g/100mL, and the concentration of kanamycins is 50 μ g/mL.
The step of embodiment 10, method of the high efficiency recombinant expressed restriction enzyme described in embodiment 1（2）In, it will limit
The recombinant expression plasmid of property restriction endonuclease processed is converted to BL21（DE3）In pLysS competent cells, it is coated on containing glucose, blocks that
Screening and culturing on the LB flat boards of mycin and chloramphenicol, obtains the recombinant strains of restriction enzyme.
In embodiment 11, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 10, described glucose
Concentration be 0.5g/100mL, the concentration of kanamycins is 37.5 μ g/mL, and the concentration of chloramphenicol is 17.5 μ g/mL.
In embodiment 12, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 10, described glucose
Concentration be 2g/100mL, the concentration of kanamycins is 150 μ g/mL, and the concentration of chloramphenicol is 70 μ g/mL.
In embodiment 13, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 10, described glucose
Concentration be 1g/100mL, the concentration of kanamycins is 75 μ g/mL, and the concentration of chloramphenicol is 35 μ g/mL.
In embodiment 14, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 10, described glucose
Concentration be 1g/100mL, the concentration of kanamycins is 50 μ g/mL, and the concentration of chloramphenicol is 20 μ g/mL.
In embodiment 15, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 10, described glucose
Concentration be 1.5g/100mL, the concentration of kanamycins is 120 μ g/mL, and the concentration of chloramphenicol is 50 μ g/mL.
The step of embodiment 16, method of the high efficiency recombinant expressed restriction enzyme described in embodiment 1（3）In, it will limit
Simultaneously picking single bacterium colony is inoculated in containing glucose, kanamycins and chloramphenicol for the recombinant strains line culture of property restriction endonuclease processed
In LB nutrient solutions, shaken cultivation is stayed overnight, and obtained culture is inoculated in the LB nutrient solutions of glucose, kanamycins and chloramphenicol
In, culture a period of time is shaken, IPTG induced expression restriction enzymes are added.
In embodiment 17, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 14, by restriction enzyme
The recombinant strains line culture of enzyme and picking single bacterium colony is inoculated in glucose containing 0.5g/100mL, 37.5 μ g/mL cards that is mould
In the LB nutrient solutions of element and 17.5 μ g/mL chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in by inoculum concentration 2% and contained
In the LB nutrient solutions of 0.5g/100mL glucose, 37.5 μ g/mL kanamycins and 17.5 μ g/mL chloramphenicol, training is shaken in 35 DEG C
Support, 3 × 10 are reached to strain density8During/mL, IPTG is added to final concentration of 0.2mM, and culture is shaken with 200rpm at 15 DEG C
12h, induced expression restriction enzyme.
In embodiment 18, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 14, by restriction enzyme
Enzyme recombinant strains line culture and picking single bacterium colony be inoculated in glucose containing 2g/100mL, 150 μ g/mL kanamycins and
In the LB nutrient solutions of 70 μ g/mL chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in containing 2g/ by inoculum concentration 5%
In the LB nutrient solutions of 100mL glucose, 150 μ g/mL kanamycins and 70 μ g/mL chloramphenicol, culture is shaken in 37 DEG C, to bacterium
Density reaches 4 × 108During/mL, IPTG is added to final concentration of 0.5mM, and culture 18h is shaken with 250rpm at 18 DEG C, is lured
Lead expression restriction enzyme.
In embodiment 19, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 14, by restriction enzyme
Enzyme recombinant strains line culture and picking single bacterium colony be inoculated in glucose containing 1g/100mL, 75 μ g/mL kanamycins and
In the LB nutrient solutions of 35 μ g/mL chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in containing 1g/ by inoculum concentration 2%
In the LB nutrient solutions of 100mL glucose, 75 μ g/mL kanamycins and 35 μ g/mL chloramphenicol, culture is shaken in 37 DEG C, to nectar
Degree reaches 3.5 × 108During/mL, IPTG is added to final concentration of 0.5mM, and 16 h of culture are shaken with 230rpm at 16 DEG C, is lured
Lead expression restriction enzyme.
In embodiment 20, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 14, by restriction enzyme
Enzyme recombinant strains line culture and picking single bacterium colony be inoculated in glucose containing 1g/100mL, 50 μ g/mL kanamycins and
In the LB nutrient solutions of 20 μ g/mL chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in containing 1g/ by inoculum concentration 2%
In the LB nutrient solutions of 100mL glucose, 50 μ g/mL kanamycins and 20 μ g/mL chloramphenicol, culture is shaken in 36 DEG C, to nectar
Degree reaches 3.2 × 108During/mL, IPTG is added to final concentration of 0.3mM, and culture 16h is shaken with 210rpm at 16 DEG C, is lured
Lead expression restriction enzyme.
In embodiment 21, the method for the high efficiency recombinant expressed restriction enzyme described in embodiment 14, by restriction enzyme
Simultaneously picking single bacterium colony is inoculated in glucose containing 1.5g/100mL, 120 μ g/mL kanamycins for the recombinant strains line culture of enzyme
In the LB nutrient solutions of 50 μ g/mL chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in by inoculum concentration 3% and contained
In the LB nutrient solutions of 1.5g/100mL glucose, 120 μ g/mL kanamycins and 50 μ g/mL chloramphenicol, culture is shaken in 37 DEG C,
3.8 × 10 are reached to strain density8During/mL, IPTG is added to final concentration of 0.4mM, and shakes culture at 17 DEG C with 240 rpm
16h, induced expression restriction enzyme.
Embodiment 22, experimental example, the present embodiment describes this by taking restriction enzyme Kpn I and pET-28b carriers as an example
The method of the described high efficiency recombinant expressed restriction enzyme of invention.
1st, material
1.1 bacterial strains and plasmid
Friedlander's bacillus（Klebsiella pneumoniae 0K8）Bacterial strain：Purchased from American Type Culture Collecti（ATCC）；
BL21（DE3）PLysS bacterial strains, λ DNA（Hind III digest）：Purchased from Thermo Fisher Scientific Inc.；
PET-28b, pUC19 and common fragment（Common fragment）（Size is 894bp DNA fragmentation, nucleotide sequence
See SEQ ID NO.3）：Purchased from Jiangsu the Foolish Old Man's Life Science Co., Ltd.
1.2 instrument and reagent
Cell high pressure cracker：Purchased from Guangzhou cumulative nano biological Science and Technology Co., Ltd.；
Protein Marker and nucleic acid molecular weight standard：Purchased from Jin Sirui bio tech ltd；
Restriction enzyme, KOD high-fidelity DNA polymerases, T4 DNA ligases and 1 × CutOne Buffer（51mM potassium acetates
（Potassium Acetate）, 22mM tri-（Methylol）Aminomethane-acetic acid（Tris-Acetate）, 10mM magnesium ions
（Magnesium）、0.1mg/mL BSA、pH8.0@25℃）：Purchased from Jiangsu the Foolish Old Man's Life Science Co., Ltd；
Ni affinitive layer purification posts（Toyopearl AF-Chelate-650M）And anion exchange chromatography（Toyopearl
GigaCap Q-650M）：Purchased from Japanese Tosoh Corporation；
B-PER bacterioproteins carry reagent：Purchased from Thermo Fisher Scientific Inc.；
Remaining reagent is that domestic analysis is pure.
2nd, experimental method
The PCR amplifications of 2.1 Kpn I genes
According to the Kpn I gene sequences of Friedlander's bacillus bacterial strain on NCBI databases（GenBank：WP_
004176755.1）Pair of primers is designed, BamH I restriction endonuclease recognition sequences and protection base are introduced at 5 ' ends of sense primer,
5 ' ends of anti-sense primer introduce Xho I restriction endonuclease recognition sequences and protection base, and sense primer is designed as the expression cassette of Kpn I genes
It is consistent with the expression cassette of N- ends 6 × His purification tags on plasmid vector：
Primers F：5’-CCGCGGATCCATGGATGTGTTTGATAAAGT-3’（Underscore part is BamH I recognition sequences）；
Primer R：5’-TATGCTCGAGTTAACGTTTCAGGCCATAGTAGT-3’（Underscore part is Xho I recognition sequences）.
After Friedlander's bacillus bacterial strain flat board culture bacterium colony is resuspended with deionized water, 95 DEG C of 10 min of incubation, as
Pcr template, with above-mentioned primer PCR expand Kpn I genes, obtain with theoretical value Kpn I genes DNA fragmentation of the same size,
Pcr amplification reaction has used KOD high-fidelity DNA polymerases.
The structure of 2.2 restriction enzyme Kpn I recombinant expression plasmid
Kpn I PCR primer uses BamH I and Xho I double digestions after purification, and digestion products are connected to by BamH with T4 ligases
On pET-28b carriers after I and Xho I double digestions, cyclic DNA connection product is obtained, wherein, Kpn I genes DNA insertion section
Under regulation and control of the position in T7 promoters；Obtained connection product is converted to bacillus coli DH 5 alpha by chemical transformation and experienced
In state cell, and screening and cloning on the LB flat boards of glucose containing 1g/100mL and 75 μ g/mL kanamycins is coated on, is obtained through sequencing
Kpn I recombinant expression plasmid is obtained, and is named as pET-Kpn I（See accompanying drawing 1）.
The structure of 2.3 restriction enzyme Kpn I recombinant strains
BL21 is converted with pET-Kpn I（DE3）PLysS competent cells, are coated on glucose containing 1g/100mL, 75 μ g/mL cards
Screening and culturing on that mycin, the LB flat boards of 35 μ g/mL chloramphenicol, obtains Kpn I recombinant strains, by the Strain Designation
For pLysS-Kpn I.
Induced expression and the agarose gel electrophoresis detection of 2.4 destination proteins
By pLysS-Kpn I line culture and picking single bacterium colony be inoculated in glucose containing 1g/100mL, 75 μ g/mL kanamycins and
In the LB nutrient solutions of 35 μ g/mL chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in containing 1g/ by inoculum concentration 2%
In the LB nutrient solutions of 100mL glucose, 75 μ g/mL kanamycins and 35 μ g/mL chloramphenicol, culture is shaken in 37 DEG C, to nectar
Degree reaches 3 × 108-4×108During/mL, IPTG is added to final concentration of 0.5mM, and training is shaken with 200-250 rpm at 16 DEG C
Support 16h, induced expression Kpn I.
In order to which verifying purpose albumen whether there is, agarose gel electrophoresis detection is carried out, detection process is as follows：
The bacterium solution after 1 mL protein expressions is taken, 10 min are centrifuged with 4000g, supernatant discarding collects thalline, then thin with B-PER
Mycoprotein extracts reagent is extracted to bacterial protein, obtains bacterioprotein coarse body fluid；1-3 μ L bacterioprotein crude extracts are taken,
Under 1 × CutOne Buffer buffer conditions, in 20 μ L reaction systems, with substrate λ DNA（Hind III digest）At 37 DEG C
15 min are incubated, with 1% agarose gel electrophoresis detection substrate λ DNA（Hind III digest）Disappeared by bacterioprotein crude extract
The situation of change（See accompanying drawing 2）.
As a result show, substrate λ DNA（Hind III digest）Formd after being digested by bacterioprotein crude extract restricted
The fragment that restriction endonuclease Kpn I enzyme-specific slitting band, i.e. size is 1700bp（With λ DNA（Hind III digest）The bottom of for
During thing, the band can only be obtained by Kpn I digestions）, it is consistent with theoretical value, show that bacterioprotein crude extract has restriction enzyme Kpn
I activity, restriction enzyme KpnI recombination expression success.
The purifying of 2.5 destination proteins
The pLysS-Kpn I bacterium solutions after appropriate protein expression are taken, centrifuging 10 min with 4000g collects thalline, and thalline is with buffer solution
（10 25 DEG C of mM Tris-HCl pH7.4@, 50 mM KCl, 5% glycerine, 5 mM imidazoles）After being fully resuspended, crushed with cell height
Broken instrument is crushed, and with 4 DEG C of 30 min of centrifugation of 43000g, collects supernatant.Supernatant passes through Ni affinity columns, anion respectively
Displacement chromatography post is purified, and whole process of purification is carried out on 4 C or ice bath.
2.5.1 Ni affinitive layer purifications
Supernatant is taken to cross Ni affinitive layer purification posts, Fraction collection eluent, the eluent that single tube is collected carries out SDS-PAGE
Electrophoresis detection（See accompanying drawing 3）.Wherein, the buffer solution used during Ni affinity chromatographys is：Level pad（10 mM Tris-
25 DEG C of HCl pH7.4@, 50 mM KCl, 5% glycerine, 5 mM imidazoles）, wash buffer（10 mM Tris-HCl pH7.4@25
DEG C, 50 mM KCl, 5% glycerine, 20 mM imidazoles）（Rinsing 2 times）And elution buffer（10 mM Tris-HCl pH7.4@25
DEG C, 50 mM KCl, 5% glycerine, 400 mM imidazoles）.
From the figure 3, it may be seen that the protein content of swimming lane 4 will be generally higher than other swimming lanes, therefore the corresponding buffer solution of the swimming lane is washed
The mixing of a few pipe eluents, the dialysis taken off.
2.5.2 anion-exchange chromatography is purified
Eluent after previous step is dialysed is crossed anion exchange chromatography and is further purified, gradient elution, Fraction collection, to not
SDS-PAGE electrophoresis detections are carried out with the sample liquid that concentration elution is arrived（See accompanying drawing 4）.Wherein, anion-exchange chromatography mistake
The buffer solution used in journey is：Level pad（10 25 DEG C of mM Tris-Hcl pH7.4@, 100 mM KCl, 5% glycerine, 1
MM DTT, 0.1 mM EDTA）And gradient eluent（25 DEG C of 10mM Tris-HCl pH7.4@, 5% glycerine, 1 mM DTT, 0.1
MM EDTA and KCl, wherein, KCl concentration changes in gradient from the M of the mM of the mM of the mM of 100mM → 300 → 500 → 700 → 1）.
As shown in Figure 4, swimming lane 1 and 2, i.e., the sample obtained by KCl containing 100mM and 300 mM KCl elution
Protein content and purity are higher than the sample liquid that other concentration are afforded in liquid, by the sample under the elution of both concentration
After liquid merges, dialysis to storage buffer（10 25 DEG C of mM Tris-HCl pH7.4@, 50 mM KCl, 50% glycerine, 1 mM
DTT, 0.1 mM EDTA）, and BSA is added to the mg/mL of final concentration 0.2, Kpn I stostes are obtained, -20 DEG C are stored in.
2.6 Enzyme assay
Restriction enzyme Kpn I enzyme activity is defined as：At 37 DEG C, in 20 μ L reaction systems, 1 μ L enzymes can be in 15min completely
Digest 1 μ g λ DNA（Hind III digest）.
Obtained Kpn I stostes are carried out after gradient dilution, under 1 × CutOne Buffer buffer conditions, 20 μ L it is anti-
Answer in system, with substrate λ DNA（Hind III digest）15 min are incubated at 37 DEG C, then with 1% agarose gel electrophoresis
Detection substrate λ DNA（Hind III digest）Digestion situation（See accompanying drawing 5）.
From testing result, the restriction enzyme Kpn I obtained are purified according to the inventive method has good enzyme
Vigor is cut, Rate activity is 128000 U/mg, and yield is that 128000U/L induces bacterium solution.
2.7 restriction enzyme Kpn I quality testing
2.7.1 star activity/non-specificity DNA enzymatic pollution detection
Kpn I stostes are carried out after gradient dilution, under 1 × CutOne Buffer buffer conditions, in 20 μ L reaction systems, bottom
Thing λ DNA（Hind III digest）60 min and 16 h are incubated in 37 C, bottom is then detected with 1% agarose gel electrophoresis
Thing λ DNA（HindIII digest）Digestion situation（See accompanying drawing 6）.
As a result show, high enzyme amount（8 U）1h and ultra-long time（16 h）Digestion does not observe that substrate λ DNA produce non-spy
Specific degradation band, illustrates nonspecific DNA enzymes are not present in the enzyme of test, shows that the enzyme produced according to the present embodiment is closed
Lattice.
2.7.2 digestion-connection-digestion detection again
The Kpn I of 20 times of enzyme amount are under 1 × CutOne Buffer buffer conditions, in 20 μ L reaction systems, with substrate Common
fragment（See SEQ ID NO.3）15 min are incubated at 37 DEG C and obtain endonuclease bamhi, by these fragments T4 DNA ligases
Connection, gained connection product can again be cut more than 95% by restriction enzyme Kpn I, then without miscellaneous band, nothing in digestion result
Hangover, shows that the purified Kpn I of the present embodiment fully meet the molecular biology experiment requirement of restriction enzyme（See attached
Fig. 7）,
2.7.3 blue hickie Screening and Identification
After restriction enzyme Kpn I linearisation pUC19 carriers using 8 times or 16 times enzyme amount, connect and convert the carrier into expression
In LacZ beta fragment competent escherichia coli cell, and X-Gal/IPTG/Amp flat boards are applied, the results are shown in Table 1.
In the identification of blue hickie, the hickie of the restriction enzyme generation of production of the embodiment of the present invention（White colony）Ratio
Only 0.1-0.34% is as a result qualified.
The blue hickie Screening and Identification data statistic of table 1.
| Numbering | Enzyme amount | White fleck | Macula communis number | Hickie ratio | Background locus coeruleus |
| 1 | 8U | 33 | 9633 | 0.340% | 19136 |
| 2 | 16U | 27 | 25179 | 0.107% | 3904 |
SEQUENCE LISTING
<110>Huaihai Institute of Technology
<120>A kind of method of high efficiency recombinant expressed restriction enzyme
<130> 2017
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<223>Primers F
<400> 1
ccgcggatcc atggatgtgt ttgataaagt 30
<210> 2
<211> 33
<212> DNA
<213>Artificial sequence
<220>
<223>Primer R
<400> 2
tatgctcgag ttaacgtttc aggccatagt agt 33
<210> 3
<211> 894
<212> DNA
<213>Artificial sequence
<400> 3
ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt 60
atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg 120
cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 180
caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc 240
cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca ctcattaggc 300
accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg tgagcggata 360
acaatttcac acaggaaaca gctatgacca tgattacgcc aagcttgcat gcgcggccgc 420
tgcaggctag cgtcgactct agagatatcc catggggatc cccgggtacc catatggagc 480
tcctcgagga attctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 540
ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 600
aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 660
tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatttgg tgcactctca 720
gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 780
acgcgccctg accaggtcgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga 840
ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcg 894
Claims (9)
1. a kind of method of high efficiency recombinant expressed restriction enzyme, it is characterised in that comprise the following steps：
（1）First restriction endonuclease gene is inserted, then carrier conversion is imported into Escherichia coli, screening and culturing, sequencing are limited
The recombinant expression plasmid of property restriction endonuclease processed；
The plasmid vector is to utilize lactose operon and T7 promoter regulations, the serial expression vectors of pET of IPTG inductions；
（2）The recombinant expression plasmid of restriction enzyme is converted to BL21（DE3）In pLysS competent cells, screening is obtained
The recombinant strains of restriction enzyme；
（3）Expand the recombinant strains and induced expression restriction enzyme of culture restriction enzyme.
2. the method for high efficiency recombinant expressed restriction enzyme according to claim 1, it is characterised in that described limitation
Property restriction endonuclease is selected from：BclI, DpnI, DpnII, EcoRI, HindIII, KpnI, MluI, MseI, NcoI, NdeI, NheI,
NotI, NsiI, NspV, PstI, SalI, SbfI, SgeI, SspI, StuI, TaqI, XbaI, XhoI.
3. the method for high efficiency recombinant expressed restriction enzyme according to claim 1, it is characterised in that in step（1）
In, described plasmid vector is pET-28b.
4. the method for high efficiency recombinant expressed restriction enzyme according to claim 1, it is characterised in that in step（1）
In, after restriction endonuclease gene fragment is connected on plasmid vector, connection product is converted to bacillus coli DH 5 alpha and experienced
In state cell, it is coated in screening and cloning on the LB flat boards containing glucose and kanamycins, sequencing obtains restriction enzyme
Recombinant expression plasmid.
5. the method for high efficiency recombinant expressed restriction enzyme according to claim 4, it is characterised in that described grape
The concentration of sugar is 0.5-2g/100mL, and the concentration of kanamycins is 37.5-150 μ g/mL.
6. the method for high efficiency recombinant expressed restriction enzyme according to claim 1, it is characterised in that in step（2）
In, the recombinant expression plasmid of restriction enzyme is converted to BL21（DE3）In pLysS competent cells, it is coated on containing grape
Screening and culturing on the LB flat boards of sugar, kanamycins and chloramphenicol, obtains the recombinant strains of restriction enzyme.
7. the method for high efficiency recombinant expressed restriction enzyme according to claim 6, it is characterised in that described grape
The concentration of sugar is 0.5-2g/100mL, and the concentration of kanamycins is 37.5-150 μ g/mL, and the concentration of chloramphenicol is 17.5-70 μ g/
mL。
8. the method for high efficiency recombinant expressed restriction enzyme according to claim 1, it is characterised in that in step（3）
In, by the recombinant strains of restriction enzyme line culture and picking single bacterium colony be inoculated in containing glucose, kanamycins and
In the LB nutrient solutions of chloramphenicol, shaken cultivation is stayed overnight, and obtained culture is inoculated in into glucose, kanamycins and chloramphenicol
In LB nutrient solutions, culture a period of time is shaken, IPTG induced expression restriction enzymes are added.
9. the method for high efficiency recombinant expressed restriction enzyme according to claim 8, it is characterised in that in step（3）
In, by the recombinant strains line culture of restriction enzyme, simultaneously picking single bacterium colony is inoculated in grape containing 0.5-2g/100mL
In the LB nutrient solutions of sugar, 37.5-150 μ g/mL kanamycins and 17.5-70 μ g/mL chloramphenicol, shaken cultivation is stayed overnight, will
To culture by inoculum concentration 2-5% be inoculated in glucose containing 0.5-2g/100mL, 37.5-150 μ g/mL kanamycins and
In the LB nutrient solutions of 17.5-70 μ g/mL chloramphenicol, culture is shaken in 35-37 DEG C, 3 × 10 are reached to strain density8-4×108/mL
When, IPTG is added to final concentration of 0.2-0.5mM, and culture 12-18 h, induction are shaken with 200-250 rpm at 15-18 DEG C
Express restriction enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710035737.0A CN107058260A (en) | 2017-01-17 | 2017-01-17 | A kind of method of high efficiency recombinant expressed restriction enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710035737.0A CN107058260A (en) | 2017-01-17 | 2017-01-17 | A kind of method of high efficiency recombinant expressed restriction enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107058260A true CN107058260A (en) | 2017-08-18 |
Family
ID=59598705
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710035737.0A Pending CN107058260A (en) | 2017-01-17 | 2017-01-17 | A kind of method of high efficiency recombinant expressed restriction enzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107058260A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880841A (en) * | 2018-12-31 | 2019-06-14 | 吴江近岸蛋白质科技有限公司 | A kind of preparation method of HindIII restriction endonuclease |
| CN112522234A (en) * | 2020-12-15 | 2021-03-19 | 中国农业大学 | Preparation method of restriction endonuclease FseI |
| CN114686455A (en) * | 2022-05-16 | 2022-07-01 | 江苏百时美生物科技有限公司 | Mixed restriction enzyme and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101948795A (en) * | 2010-09-07 | 2011-01-19 | 东北农业大学 | Engineering bacteria of soluble expression Not I, and construction method and application thereof |
| KR101219716B1 (en) * | 2009-02-27 | 2013-01-09 | 세종대학교산학협력단 | Method of producing recombinant endonucleases |
-
2017
- 2017-01-17 CN CN201710035737.0A patent/CN107058260A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101219716B1 (en) * | 2009-02-27 | 2013-01-09 | 세종대학교산학협력단 | Method of producing recombinant endonucleases |
| CN101948795A (en) * | 2010-09-07 | 2011-01-19 | 东北农业大学 | Engineering bacteria of soluble expression Not I, and construction method and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| E. TOKSOY等: "high-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter", 《APPL MICROBIOL BIOTECHNOL》 * |
| 李冰冰等主编: "《生化与分子生物学实验指导》", 31 August 2014 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109880841A (en) * | 2018-12-31 | 2019-06-14 | 吴江近岸蛋白质科技有限公司 | A kind of preparation method of HindIII restriction endonuclease |
| CN112522234A (en) * | 2020-12-15 | 2021-03-19 | 中国农业大学 | Preparation method of restriction endonuclease FseI |
| CN114686455A (en) * | 2022-05-16 | 2022-07-01 | 江苏百时美生物科技有限公司 | Mixed restriction enzyme and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Neal et al. | Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria | |
| JP2022106883A (en) | Methods and compositions for sequences guiding cas9 targeting | |
| Binder et al. | Transcription initiation sites in mitochondria of Oenothera berteriana. | |
| CN106480076A (en) | A kind of method of protecting high efficiency recombinant expressed restriction enzyme of utilization methylase | |
| CN106755002A (en) | Using the method for the high efficiency recombinant expressed restriction enzyme of protection of methylase | |
| CN107058260A (en) | A kind of method of high efficiency recombinant expressed restriction enzyme | |
| Haselkorn et al. | Organization and transcription of genes important in Anabaena heterocyst differentiation | |
| EP4092128A1 (en) | Factor regulating protein expression efficiency of trichoderma reesei, and regulation method and use thereof | |
| DE69839426D1 (en) | NUCLEOTIDE SEQUENCES CODING FOR SECRETATED POLYPEPTIDES FROM MYCOBACTERIUM, VECTORS CONTAINING THEM, AND APPLICATIONS FOR THE DIAGNOSIS AND PREVENTION OF TUBERCULOSIS | |
| CN104946648A (en) | Plant pollen specific promoter PCHF32 and application thereof | |
| Smith et al. | An accessory protein is required for relaxosome formation by small staphylococcal plasmids | |
| Krol et al. | Expression of large plasmids in the endosymbiotic form of Rhizobium leguminosarum | |
| CN104928318A (en) | Application of rice OsLEA5c-1 gene in heavy metal resisting rice variety cultivation | |
| CN113444743A (en) | Construction method of sheep mycoplasma pneumonia bivalent nucleic acid vaccine containing adjuvant gene | |
| CN109486779B (en) | DNA methyltransferase and soluble heterologous expression and separation and purification method thereof | |
| US8735562B2 (en) | Isolated fungal promoters and gene transcription terminators and methods of protein and chemical production in a fungus | |
| CN107338241A (en) | A kind of method that evolution is oriented to gene promoter | |
| BR112012005263A2 (en) | modified promoter | |
| CN111909929B (en) | Method for obtaining Trichoderma reesei cellulase regulation gene in targeted manner | |
| JP3549210B2 (en) | Plasmid | |
| KR101261852B1 (en) | Agarase expression vector, transgenic organism transformed with the same vector and method for the production of agarase using the same transgenic organism | |
| Jiang et al. | High-efficiency transformation of the plant pathogenic fungus Marssonina brunnea. | |
| CN110791513B (en) | Codon-optimized holothuria leucospilota alpha-amylase gene, recombinant expression vector and application thereof | |
| CN106701802A (en) | Overexpressed UGPase (Uridine Diphosphoglucose Pyrophosphorylase) gene as well as construction method and purification method for recombinant escherichia coli thereof | |
| CN117737098A (en) | Wheat plasma membrane H + Application of ATPase TaHA1 gene in wheat stripe rust regulation |
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 | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20190308 Address after: 222000 Floor 13-14, Building 1, Science and Technology Pioneering City, 17 Huaguoshan Avenue, Haizhou District, Lianyungang City, Jiangsu Province Applicant after: Mona (Lianyungang) Biotechnology Co., Ltd. Address before: 222000 Gao Songzhuan, Institute of Marine Medicine, Huaihai Institute of Technology, 108 Tongguannan Road, Haizhou District, Lianyungang City, Jiangsu Province Applicant before: Huaihai Institute of Technology |
|
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170818 |