CN106755041A - A kind of preparation method and applications of low form bacterial DNA ligase - Google Patents

A kind of preparation method and applications of low form bacterial DNA ligase Download PDF

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Publication number
CN106755041A
CN106755041A CN201611236983.4A CN201611236983A CN106755041A CN 106755041 A CN106755041 A CN 106755041A CN 201611236983 A CN201611236983 A CN 201611236983A CN 106755041 A CN106755041 A CN 106755041A
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dna ligase
dna
temperature
low form
low
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刘喜朋
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Suzhou Kuangshi Junchi Biological Science & Technology Co Ltd
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Suzhou Kuangshi Junchi Biological Science & Technology Co Ltd
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    • 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
    • C12N9/93—Ligases (6)
    • 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
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Abstract

The invention discloses a kind of preparation method and applications of low form bacterial DNA ligase.The enzymatic activity Optimal Temperature scope of the low form DNA ligase prepared in the present invention is 4 20 degree；The connection product for being reacted in the temperature range 5 minutes is more than 90%, is very suitable for the low temperature DNA coupled reactions in gene cloning.The beneficial effects of the present invention are：Because the DNA coupled reaction temperature of gene cloning is relatively low, and the optimum temperature of traditional T4 phage DNA ligases is at 30 40 degree, DNA coupled reactions temperature with gene cloning is mismatched, cause DNA joint efficiencies low, need to react for a long time, Connection Time is up to 12 hours, greatly limits the speed of gene cloning.DNA ligase of the invention substantially reduces the DNA coupled reaction times in low-temperature catalytic activity highest, accelerates gene cloning speed.

Description

A kind of preparation method and applications of low form bacterial DNA ligase
Technical field
The invention belongs to gene engineering technology field, more particularly, to a kind of preparation side of low form bacterial DNA ligase Method and its application.
Background technology
The appearance growth-promoting of restriction endonuclease and the DNA ligase extracorporeal recombination of DNA, i.e. gene cloning gram Grand technology.Gene clone technology is the basis of modern molecular biology and genetic engineering, has greatly facilitated protein restructuring table Up to applications such as, engineered strain transformations.DNA ligase in current gene cloning is T4 phage DNA ligases.Gene cloning DNA coupled reactions temperature carried out in the range of 4-16 degree.And the optimum temperature of T4 phage DNA ligases is in 30-40 degree, with The DNA coupled reactions temperature of gene cloning is mismatched, and causes DNA joint efficiencies low, it is necessary to react for a long time, and the Connection Time is up to 12 hours, greatly limit the speed of gene cloning.
In order to improve the law temperature joining efficiency in gene cloning, the time needed for shortening coupled reaction, it is contemplated that grinding Send out DNA ligase a kind of, its active Optimal Temperature scope is 4-20 degree；For the DNA coupled reactions of gene cloning.
The beneficial effects of the present invention are：DNA ligase of the invention catalysis activity highest at low temperature, for gene The time needed for DNA coupled reactions was shortened to 5 minutes from 16 hours during clone, has significantly speeded up gene cloning speed.
The content of the invention
In order to solve low this technical bottleneck of joint efficiency run into gene cloning, it is an object of the invention to provide one Plant low form DNA ligase, preparation method and its application in gene cloning.Low form bacterial DNA ligase of the invention The enzymatic activity highest within the temperature range of 4-20 degree；The connection product for being reacted in the temperature range 5 minutes is more than 90%, very It is suitable for the low temperature DNA coupled reactions in gene cloning.Low form DNA ligase of the invention is used for gene cloning, and DNA is connected Reversed time that should be required shortened to 5 minutes from 16 hours, had significantly speeded up gene cloning speed.
Technical scheme is specific as follows.
The present invention provides a kind of preparation method of low form DNA ligase and its application in gene cloning, specific step It is rapid as follows：
（1）The recombinant expression plasmid of constructed dna ligase
Based on cryophile genomic information, the DNA ligase gene of its coding is designed, on this basis, modification optimization DNA The rare codon of enzyme coding gene is connected, the DNA ligase base of psychrophile strain is obtained using full genome synthesis or round pcr Cause, and by gene cloning to prokaryotic expression carrier pET28 etc., constructed dna ligase recombinant expression plasmid；
（2）Recombination expression DNA ligase
The DNA ligase recombinant expression plasmid conversion Bacillus coli expression host BL21 that will be built（DE3）Deng, obtain DNA connection Enzyme recombinant strains；It is carried out into DNA ligase induced expression with derivant IPTG again；
（3）The DNA ligase of affinity purification expression
The Escherichia coli after induced expression DNA ligase are collected by centrifugation, thalline is resuspended in non-denatured protein lysates, ultrasonic wave Broken thalline, is collected by centrifugation the Escherichia coli cracking supernatant containing DNA ligase；Recycle immobilization nickel ion affinity purification Resin purifies DNA ligase from supernatant；
（4）The enzymatic activity of the DNA ligase of purifying is detected, especially the enzymatic activity under low temperature, obtain low form DNA ligase.
Enzyme assay will be carried out by DNA ligase after purification, the DNA that therefrom filter out has excellent activity under low temperature is connected Enzyme；The technical parameter of low form DNA ligase is：In the range of 4-20 degree, DNA ligase can connect 90% in 5 minutes DNA molecular.
（5）Low form DNA ligase is used for gene cloning.
The present invention furthermore provides the gene cloning application based on low form DNA ligase.Specific method is as follows：In base Because T4 phage DNA ligases are substituted for into DNA ligase of the invention in the DNA attended operations of clone, react 5 minutes, enter Row gene cloning.
In the present invention, step（1）In, the cryophile includes Colwellia psychrerythraea, Photobacterium profundum etc..
In the present invention, step（2）It is middle Fiber differentiation is carried out with derivant IPTG actual conditions be：First by DNA ligase To OD600=0.4-1.0, adding 0.5mM derivants IPTG, that 3 are cultivated at a temperature of 30-37 DEG C is small for recombinant strains culture When or 10-30 DEG C at a temperature of culture carry out induced expression within 12 hours.
In the present invention, step（3）In, the composition of the non-denatured protein lysates is as follows：20 mM pH value are 8.0 Tris-HCl, 300 mM NaCl, 0.5mM DTT, 10vol% glycerine.
In the present invention, psychrophilic bacteria the Colwellia psychrerythraea, Photobacterium The amino acid sequence of the low form DNA ligase of profundum is respectively such as SEQ ID NO:Shown in 1-2.
Compared with prior art, the present invention has marked improvement, can greatly shorten the time of gene cloning, specifically such as Under：
（1）The gene cloning time is extremely short.The DNA coupled reaction times were foreshortened to 5 minutes by 16 hours, and required time compares is bitten based on T4 200 times of the shortening of the gene cloning of thallus DNA ligase.
（2）Gene cloning efficiency high.Because low form DNA ligase can link together 90% DNA molecular, because This cloning efficiency increases 20 times than the gene cloning based on T4 phage DNA ligases.
Brief description of the drawings
Fig. 1 is the temperature-independent of the DNA ligase catalysis activity for being psychrophilic bacteria Colwellia psychrerythraea Property.
Fig. 2 is to be based on psychrophilic bacteria Colwellia psychrerythraea, Photobacterium profundum With the gene cloning efficiency comparison of T4 phage DNA ligases.
Fig. 3 is the temperature-independent of the DNA ligase catalysis activity for being psychrophilic bacteria Photobacterium profundum Property.
Specific embodiment
With reference to the accompanying drawings and examples, specific embodiment of the invention is further described.Following examples are only For clearly illustrating technical scheme, and can not be limited the scope of the invention with this.
The preparation of the psychrophilic bacteria Colwellia psychrerythraea DNA ligases of embodiment 1
The first step, design synthesis psychrophilic bacteria Colwellia psychrerythraea DNA ligase genes, and insert PET28 expression vectors, the recombinant expression plasmid of constructed dna ligase.Restructuring psychrophilic bacteria Colwellia Psychrerythraea DNA ligases N-terminal is used with 6 continuous histidine affinity purification labels from pET28 carriers In the purifying of immobilization nickel ion affinity chromatograph.
Second step, recombinantly expresses psychrophilic bacteria Colwellia psychrerythraea DNA ligases.By psychrophilic bacteria Colwellia psychrerythraea DNA ligases recombinant expression plasmid conversion Bacillus coli expression host BL21（DE3）, Obtain psychrophilic bacteria Colwellia psychrerythraea DNA ligase recombinant strains.Strain culturing will be expressed extremely OD600=0.6, adds 0.5mM derivant IPTG, is cultivated 12 hours at a temperature of 16 DEG C, induction psychrophilic bacteria Colwellia PsychrerythraeaDNA connects expression of enzymes.
The amino acid sequence of psychrophilic bacteria Colwellia psychrerythraea DNA ligase recombinant proteins（Nitrogen end → carbon teminal）Such as SEQ IDNO:Shown in 1.
3rd step, psychrophilic bacteria Colwellia psychrerythraea DNA ligase affinity purifications.Step 2 is lured After Escherichia coli after leading are collected by centrifugation, thalline is resuspended in protein lysate（20 mM Tris-HCl, pH 8.0, 300 MM NaCl, 0.5mM DTT, 10vol% glycerine）.Ultrasonic disruption thalline, is collected by centrifugation and contains psychrophilic bacteria Colwellia The cellular lysate supernatant of psychrerythraea DNA ligases.It is thermophilic using immobilization nickel ion affinity purification purifying resin Cold bacterium Colwellia psychrerythraea DNA ligases.
4th step, detects the enzymatic activity and enzyme activity of psychrophilic bacteria Colwellia psychrerythraea DNA ligases Temperature dependency.It is 4 notched 750bp double-stranded DNAs of base-pair as substrate by the use of collochore, adds psychrophilic bacteria Colwellia psychrerythraea DNA ligases, determine the enzymatic activity and temperature dependency of DNA ligase.Specific enzyme Active temperature dependence result is shown in Fig. 1.Result shows that psychrophilic bacteria Colwellia psychrerythraea DNA ligases exist There is highest enzymatic activity in the range of 5-20 degree, and activity is very low under 30 degree.
5th step, the gene cloning efficiency of psychrophilic bacteria Colwellia psychrerythraea DNA ligases.Herein On the basis of, entered instead of T4 phage DNA ligases using psychrophilic bacteria Colwellia psychrerythraea DNA ligases Row gene cloning, by carrier segments be inserted into genetic fragment 4,8,12,16,20 degree of insulations are attached reaction in 5 minutes, most Connection product is converted into bacillus coli DH 5 alpha competent cell afterwards, positive colony number is identified, compared to T4 phage DNA ligases, Clone's number of Colwellia psychrerythraea DNA ligases improves 20 times to the greatest extent（Fig. 2, CpsDNA ligase）.
The preparation of the psychrophilic bacteria Photobacterium profundum DNA ligases of embodiment 2
The first step, design synthesis psychrophilic bacteria Photobacterium profundum DNA ligase genes, and insert pET28 Expression vector, the recombinant expression plasmid of constructed dna ligase.Restructuring psychrophilic bacteria Photobacterium profundum DNA Ligase N-terminal is affine for immobilization nickel ion with 6 continuous histidine affinity purification labels from pET28 carriers Chromatographic purifying.
Second step, recombinantly expresses psychrophilic bacteria Photobacterium profundum DNA ligases.By psychrophilic bacteria Photobacterium profundum DNA ligases recombinant expression plasmid conversion Bacillus coli expression host BL21（DE3）, Obtain psychrophilic bacteria Photobacterium profundum DNA ligase recombinant strains.Strain culturing will be expressed extremely OD600=0.6, adds 0.5mM derivant IPTG, is cultivated 12 hours at a temperature of 16 DEG C, induces psychrophilic bacteria Photobacterium profundum DNA ligases are expressed.
The amino acid sequence of psychrophilic bacteria Photobacterium profundum DNA ligase recombinant proteins（Nitrogen end → Carbon teminal）Such as SEQ IDNO:Shown in 2.
3rd step, psychrophilic bacteria Photobacterium profundum DNA ligase affinity purifications.Step 2 is lured After Escherichia coli after leading are collected by centrifugation, thalline is resuspended in protein lysate（20 mM Tris-HCl, pH 8.0, 300 MM NaCl, 0.5mM DTT, 10vol% glycerine）.Ultrasonic disruption thalline, is collected by centrifugation and contains psychrophilic bacteria The cellular lysate supernatant of Photobacterium profundum DNA ligases.Using immobilization nickel ion affinity purification tree Fat purifies psychrophilic bacteria Photobacterium profundum DNA ligases.
4th step, detects the enzymatic activity and enzyme activity temperature of psychrophilic bacteria Photobacterium profundum DNA ligases Degree dependence.It is 10 notched double-stranded DNAs of base-pair as substrate by the use of collochore, adds psychrophilic bacteria Photobacterium profundum DNA ligases, determine the enzymatic activity and temperature dependency of DNA ligase.Specific enzyme activity Property temperature dependency result is shown in Fig. 3.Result shows psychrophilic bacteria Photobacterium profundum DNA ligases in 4- There is highest enzymatic activity in the range of 28 degree, and 30,36 degree of activity are very low.
5th step, the gene cloning efficiency of psychrophilic bacteria Photobacterium profundum DNA ligases.Utilize Psychrophilic bacteria Photobacterium profundum DNA ligases carry out gene cloning instead of T4 phage DNAs ligase, By carrier segments be inserted into genetic fragment 4,8,12,16,20 degree of insulations are attached reaction in 5 minutes, finally will connection produce Thing converts bacillus coli DH 5 alpha competent cell, identifies positive colony number, compared to T4 phage DNA ligases, Clone's number of Photobacterium profundum DNA ligases improves 20 times to the greatest extent（Fig. 2, Pfr DNA ligase）.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, on the premise of the technology of the present invention principle is not departed from, some improvements and modifications can also be made, these improvements and modifications Also should be regarded as protection scope of the present invention.
Nucleotides and amino acid sequence table
SEQ ID NO:1
1 MSNVEKKISQ LQQQLNQYNH EYYVLDQPSV PDAEYDRLMT ALIDLEKTNP ELKTIDSPSQ
61 KVGGQALKSF TQVTHQLPML SLDNVFSLDD FHAFVKRVKD RLNDNQAIVF CAEPKLDGLA
121 VSLRYEHGQL IQAATRGDGS VGENITTNIR TIKSIPLKLM GTPGKDFPDI VEVRGEVFMP
181 KASFDALNTL AKKRGEKGFA NPRNAAAGSL RQLDSKITAK RNLAFYAYSL GFVGKLSDGG
241 AESTDLTNDF FANSHHERLC QLKRLGLPMC PEVRLLESEQ ACDAFYQDIL AKRSALSYEI
301 DGTVLKVDEI SLQKRLGFVA RAPRWAIAYK FPAEEELTCV EDVEFQVGRT GAITPVARLK
361 PVFVGGVTVS NATLHNQDEI TRLGLKVNDF VVIRRAGDVI PQIVSVVLDK RPDNAVDIVF
421 PTSCPVCDSA VAKPEGEAVL RCTAGLFCAA QRKEAIKHFA SRKAHDVDGL GDKLVEQLVD
481 EKLINTPADL FKLTEIQVST IDRMGKKSAT NLINGLEQAK STTLAKFIYG LGIREVGEAT
541 AANLANHFYT LAAIESASLE DLQNVSDVGE VVAKNIINFF KEEHNLAIVS GLSEVMHWPT
601 IEIKSAEELP LAEQIFVLTG TLTQMGRTEA KTALQSLGAK VSGSVSKNTH FVVAGDKAGS
661 KLTKAQDLGI SVLTEDGLVA LLAEHGITI
SEQ ID NO:2
1 MIGSEMSTDI QQQLDTLREQ LAYHGHRYYV EDNPEIPDAE YDRMMQQLLA LEAEHPELMS
61 VDSPSQRVGG TPLDSFTQVK HELPMLSLDN AFNDEELNAF EKRLLDRLIT AGHVSYCCEP
121 KLDGLAVSLM YENGVLVQAA SRGDGATGEN ITTNVRTIRS IPLKLQGEGW PVRLEVRGEV
181 FMPKKGFDEL NARALKKGDK AFANPRNAAA GSLRQLDSKI AATRPLSFYA YSVGVVEGMD
241 LAESQYERLV QLKGWGIPMC PEIRQLKTIN DVIAYYQDIG ERRQSLAYEI DGVVIKVDDV
301 ETQEQLGFVA RAPRWAIAYK FPAQEEMTLL NNVEFQVGRT GAITPVAKLE PIFVGGVTVS
361 NATLHNADEV ARLGVMIGDT VIIRRAGDVI PQIVSVVESR RPDNAQAITF PITCPVCESN
421 VERVEGEAVA RCTGGLFCQA QRKEALKHFV SRKALDVDGC GEKVIEQLVD REMVTTPAGL
481 FKLSAGIVTV LDRMGPKSAQ KLVDSLANSK ETTLPRFIYS LGIREVGEAT AANLANHFET
541 LEAITAASKE QLLVVPDVGE VVANNLLNFF REAHNMDVVE DLIAVGIHWP AIDKPEEGVE
601 LPLDGKVVVL TGSLSQLTRS DAKAALQALG AKVTGSVSSK TDLLVAGEAA GSKLTKAQEL
661 DIEIWDEQAL VDFMNR
SEQUENCE LISTING
<110>The outstanding Jun Chi bio tech ltd in Suzhou
<120>A kind of preparation method and applications of low form bacterial DNA ligase
<210> 1
<211> 689
<212> PRT
<213>
<400> 1
1 MSNVEKKISQ LQQQLNQYNH EYYVLDQPSV PDAEYDRLMT ALIDLEKTNP ELKTIDSPSQ
61 KVGGQALKSF TQVTHQLPML SLDNVFSLDD FHAFVKRVKD RLNDNQAIVF CAEPKLDGLA
121 VSLRYEHGQL IQAATRGDGS VGENITTNIR TIKSIPLKLM GTPGKDFPDI VEVRGEVFMP
181 KASFDALNTL AKKRGEKGFA NPRNAAAGSL RQLDSKITAK RNLAFYAYSL GFVGKLSDGG
241 AESTDLTNDF FANSHHERLC QLKRLGLPMC PEVRLLESEQ ACDAFYQDIL AKRSALSYEI
301 DGTVLKVDEI SLQKRLGFVA RAPRWAIAYK FPAEEELTCV EDVEFQVGRT GAITPVARLK
361 PVFVGGVTVS NATLHNQDEI TRLGLKVNDF VVIRRAGDVI PQIVSVVLDK RPDNAVDIVF
421 PTSCPVCDSA VAKPEGEAVL RCTAGLFCAA QRKEAIKHFA SRKAHDVDGL GDKLVEQLVD
481 EKLINTPADL FKLTEIQVST IDRMGKKSAT NLINGLEQAK STTLAKFIYG LGIREVGEAT
541 AANLANHFYT LAAIESASLE DLQNVSDVGE VVAKNIINFF KEEHNLAIVS GLSEVMHWPT
601 IEIKSAEELP LAEQIFVLTG TLTQMGRTEA KTALQSLGAK VSGSVSKNTH FVVAGDKAGS
661 KLTKAQDLGI SVLTEDGLVA LLAEHGITI
<210> 2
<211> 676
<212> PRT
<213>
<400> 2
1 MIGSEMSTDI QQQLDTLREQ LAYHGHRYYV EDNPEIPDAE YDRMMQQLLA LEAEHPELMS
61 VDSPSQRVGG TPLDSFTQVK HELPMLSLDN AFNDEELNAF EKRLLDRLIT AGHVSYCCEP
121 KLDGLAVSLM YENGVLVQAA SRGDGATGEN ITTNVRTIRS IPLKLQGEGW PVRLEVRGEV
181 FMPKKGFDEL NARALKKGDK AFANPRNAAA GSLRQLDSKI AATRPLSFYA YSVGVVEGMD
241 LAESQYERLV QLKGWGIPMC PEIRQLKTIN DVIAYYQDIG ERRQSLAYEI DGVVIKVDDV
301 ETQEQLGFVA RAPRWAIAYK FPAQEEMTLL NNVEFQVGRT GAITPVAKLE PIFVGGVTVS
361 NATLHNADEV ARLGVMIGDT VIIRRAGDVI PQIVSVVESR RPDNAQAITF PITCPVCESN
421 VERVEGEAVA RCTGGLFCQA QRKEALKHFV SRKALDVDGC GEKVIEQLVD REMVTTPAGL
481 FKLSAGIVTV LDRMGPKSAQ KLVDSLANSK ETTLPRFIYS LGIREVGEAT AANLANHFET
541 LEAITAASKE QLLVVPDVGE VVANNLLNFF REAHNMDVVE DLIAVGIHWP AIDKPEEGVE
601 LPLDGKVVVL TGSLSQLTRS DAKAALQALG AKVTGSVSSK TDLLVAGEAA GSKLTKAQEL
661 DIEIWDEQAL VDFMNR

Claims (6)

1. a kind of preparation method of low form bacterial DNA ligase, it is characterised in that comprise the following steps that：
（1）Build the recombinant expression plasmid of low form DNA ligase
Based on cryophile genomic information, design synthesizes the DNA ligase gene of its coding, on this basis, optimizes DNA Connect the rare codon of enzyme gene, and the DNA ligase gene cloning that will optimize is to prokaryotic expression carrier pET28 etc., structure DNA ligase recombinant expression plasmid；
（2）Recombination expression DNA ligase
The DNA ligase recombinant expression plasmid conversion Bacillus coli expression host BL21 that will be built（DE3）, obtain DNA ligase Recombinant strains；It is carried out into low form DNA ligase induced expression with derivant IPTG again；
（3）The DNA ligase of affinity purification expression
The Escherichia coli after induced expression DNA ligase are collected by centrifugation, thalline is resuspended in non-denatured protein lysates, ultrasonic wave Broken thalline, is collected by centrifugation Escherichia coli cracking supernatant；Recycling immobilization nickel ion affinity purification resin is from supernatant Purifying low form DNA ligase；
（4）The enzymatic activity and peak optimization reaction temperature of DNA ligase are detected, the low form DNA ligase of high activity is obtained
The DNA ligase of purifying is carried out into enzymatic activity and peak optimization reaction temperature measuring, identify its enzymatic activity and enzymatic activity temperature according to Lai Xing；Ensure that it can at low temperature have highest enzymatic activity, i.e., the enzymatic activity highest in the range of 4-20 degree.
2. the preparation method of a kind of low form bacterial DNA ligase according to claim 1, it is characterised in that：Step（1） In, the cryophile includes Colwellia psychrerythraea, Photobacterium profundum etc..
3. the preparation method of a kind of low form bacterial DNA ligase according to claim 1, it is characterised in that：Step（2） It is middle Fiber differentiation is carried out with derivant IPTG actual conditions be：First by the culture of low form DNA ligase recombinant strains extremely OD600=0.4-1.0, adds 0.5mM derivants IPTG and is trained at a temperature of culture 3 hours or 10-25 DEG C at a temperature of 25-37 DEG C Supporting carries out induced expression in 12 hours.
4. the low form DNA ligase that the preparation method as described in one of claims 1 to 3 is obtained.
5. low form DNA ligase as claimed in claim 4, wherein：The amino acid sequence of DNA ligase is respectively such as SEQ ID NO:Shown in 1-2.
6. the DNA ligase that the preparation method as described in one of claims 1 to 3 is obtained is used for gene cloning, improves gene gram Grand DNA coupled reaction efficiency, shortens the reaction time, accelerated gene clone.
CN201611236983.4A 2016-12-28 2016-12-28 A kind of preparation method and applications of low form bacterial DNA ligase Pending CN106755041A (en)

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CN110577935A (en) * 2019-07-29 2019-12-17 中山大学 Method for enhancing nuclear expression of T4DNA ligase
CN111235171A (en) * 2019-12-27 2020-06-05 苏州博睐恒生物科技有限公司 Preparation method and application of rapid DNA ligase

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Publication number Priority date Publication date Assignee Title
CN108220255A (en) * 2017-09-24 2018-06-29 天津强微特生物科技有限公司 A kind of high activity DNA ligase mutant of Pyrococcus furiosus
CN108220255B (en) * 2017-09-24 2021-03-16 天津强微特生物科技有限公司 Thermophilic archaea high-activity DNA ligase mutant
CN110577935A (en) * 2019-07-29 2019-12-17 中山大学 Method for enhancing nuclear expression of T4DNA ligase
CN110577935B (en) * 2019-07-29 2021-01-05 中山大学 Method for enhancing nuclear expression of T4DNA ligase
CN111235171A (en) * 2019-12-27 2020-06-05 苏州博睐恒生物科技有限公司 Preparation method and application of rapid DNA ligase

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