CN110438104A - A kind of restriction enzyme MnlI and its expression and purification method - Google Patents

A kind of restriction enzyme MnlI and its expression and purification method Download PDF

Info

Publication number
CN110438104A
CN110438104A CN201910804225.5A CN201910804225A CN110438104A CN 110438104 A CN110438104 A CN 110438104A CN 201910804225 A CN201910804225 A CN 201910804225A CN 110438104 A CN110438104 A CN 110438104A
Authority
CN
China
Prior art keywords
mnli
restriction enzyme
expression
purification method
thallus
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
Application number
CN201910804225.5A
Other languages
Chinese (zh)
Inventor
程槐旭
胡梦竹
燕东平
聂尚海
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mona (wuhan) Biotechnology Co Ltd
Original Assignee
Mona (wuhan) Biotechnology Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Mona (wuhan) Biotechnology Co Ltd filed Critical Mona (wuhan) Biotechnology Co Ltd
Priority to CN201910804225.5A priority Critical patent/CN110438104A/en
Publication of CN110438104A publication Critical patent/CN110438104A/en
Pending legal-status Critical Current

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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The present invention provides a kind of restriction enzyme MnlI and its expression and purification methods, it the described method comprises the following steps: building restriction enzyme MnlI recombinant expression carrier, conversion methylation competent cell, obtain restriction enzyme MnlI recombinant bacterial strain, Fiber differentiation, thallus is cracked, purification and recovery product is to get restriction enzyme MnlI.Expression and purification mode is easy to operate, quickly, high income, the restriction enzyme MnlI purity is high being purified into.

Description

A kind of restriction enzyme MnlI and its expression and purification method
Technical field
The invention belongs to field of biotechnology, it is related to a kind of restriction enzyme MnlI and its expression and purification method.
Background technique
There is a kind of enzyme that external DNA can be cut off in vivo, it can limit the intrusion of allogeneic dna sequence DNA and be allowed to lose Vigor is removed, but to the harmless effect of the DNA of itself, can thus play the role of protecting the original hereditary information of cell. Since this dissection is carried out inside DNA molecular, therefore named restriction enzyme.Its discovery promotes DNA recombinant technique Birth to greatly push the development of modern molecular biology and genetic engineering, it is indispensable to be that contemporary genetic engineering is studied Master tool.
Due to there is " limitation-modification " phenomenon in bacterial body, i.e. the DNA of itself is methylated enzyme modification, to avoid institute Cutting of the restriction enzyme of generation to itself DNA fragmentation achievees the purpose that resist external Phage Infection.Therefore in protokaryon Single recombinant expression restriction enzyme in expression system can make host dead due to DNA is by cutting.And transmethylase exists Special sex modification in identification sequence can prevent the cutting of restriction enzyme.The normal close linkage of limitation-modifier, bacterium are logical The expression for crossing both regulations makes itself DNA be first subjected to methylation protection, expresses restriction enzyme again after the completion of protection, and The allogeneic dna sequence DNA of invasion is not protected thus is degraded by restriction enzyme.Relevant transmethylase pair i.e. intracellular with Its identical particular sequence for matching restriction enzyme identification carries out methylation modification, and has the DNA of modification and resist its limit The characteristic of the cutting of property restriction endonuclease processed protects host DNA and the exogenous DNA not being methylated of degrading.
As restriction modification phenomenon is found, scientist Werner Arben, Daniel Nathans and Hamilton Smith has found restriction enzyme, illustrates effect of the restriction enzyme in molecular gene problem, later, increasingly More restriction enzymes is found, purifies and obtains application.About the research of restriction enzyme, them are focused on for many years As the practical value of toolenzyme, discovery and screening, the building of high-yielding engineering bacterial strain, enzyme purification technology and program including new enzyme Optimal improvements etc..Wherein MnlI is a kind of widely used restriction enzyme, can recognize the nucleic acid sequence of four bases It arranges (5 ' CCTCNNNNNN^ '), can be used as common restriction enzyme applied to molecular cloning, in its of biological field His direction also has important application value and research potential.
CN101225396A discloses a kind of method of restriction of production restriction endonuclease, the described method comprises the following steps: will The methylases gene opposite with the restriction enzyme is cloned into the upstream of pEEB carrier；Under the protection of methylase, Restriction endonuclease gene is cloned into the multiple cloning sites in identical carrier strong promoter downstream；Conversion expression host strain；Sieve Select the bacterial strain of stable, inducible, highly expressed restriction enzyme.
CN106755002A discloses a kind of side of high efficiency recombinant expressed restriction enzyme of protection using methylase Method, the methylase are M.CviPI；The restriction enzyme C is selected from: AatI, AscI, BanII, BglI, BmtI, BspQI, BsrDI, BssHII, BtsI, EagI, HindIII, KasI, MluI, NheI, NotI, NruI, NsiI, PstI, PvuII, SacI, SapI, SbfI, SfiI, SphI.It is directly protected using wide spectrum in the building process of the inventive method recombination engineering Shield type methylase M.CviPI, without carrying out cumbersome methylases gene screening for single restriction enzyme, using model It encloses extensively, simplifies recombinant expression process.
But due to the problems such as expression quantity is low, to isolate and purify program cumbersome, albumen yield is low, presently commercially available MnlI is restricted Not only price is high for restriction endonuclease, but also also limits its extensive use and further investigation.Meanwhile above-mentioned a series of problems Also limit the further investigation of other many widely used restriction enzymes.
Therefore it provides a kind of expression quantity is high, it is easy to isolate and purify program, albumen yield is high prepares restriction enzyme The method of MnlI is of great significance.
Summary of the invention
In view of the deficiencies of the prior art and actual demand, the present invention provides a kind of restriction enzyme MnlI and its expression Purification process, expression and purification mode is easy to operate, and quickly, high income, gained purity of protein is high, SDS-PAGE electrophoresis without miscellaneous band, Its purity is 95% or more.
To achieve this purpose, the present invention adopts the following technical scheme:
In a first aspect, the present invention provides the expression and purification method of restriction enzyme MnlI a kind of, the method includes with Lower step: building restriction enzyme MnlI recombinant expression carrier, conversion methylation competent cell obtain restriction enzyme MnlI recombinant bacterial strain, Fiber differentiation crack thallus, and purification and recovery product is to get restriction enzyme MnlI.
Preferably, the construction method of the restriction enzyme MnlI recombinant expression carrier includes by restriction enzyme MnlI genetic fragment connects pBAD plasmid, obtains restriction enzyme MnlI recombinant expression carrier pBAD-R.MnlI.
Preferably, the amino acid sequence of the restriction enzyme MnlI genetic fragment is as shown in SEQ ID NO.1.
The amino acid sequence of the SEQ ID NO.1 is specific as follows:
MDFNNFLNKATLNALLVAFANAKPITKPFIGFVIESSYKNSAYSPIELTDDIYQQNNQLLSQFTNLDF QLNGKKHQFFIQSDLSCDDFFALVYKNILIKGSTIDNDEFSKLILLSFFALRGSPDFKLNFYSLDLPRQIVSKNYL DNLFKLSTNVSDLRQLNLNFRELQEQFITGENERNTQFRINLRYFYDNFSDDLAKINLFKSNILKHNANLIKTKNI AQESRTFIERLNFYRDNVLNQTKTQHEIEQLRKNLGFIYDDTIDEKTTRNQGIVQYVRAYFPDECACCKNQYNIKD RSFTYRHSDRYYLEVHHVISFASDRTLDQIDNLVKVCPTCHRALSKNRADEQYQKELISEILINAPKAKEFCLNFT DENDCIQFIYDRLR.
Preferably, the preparation method of the methylation competent cell is the following steps are included: screening MnlI transmethylase Genetic fragment connects expression vector pACYC184, obtains transmethylase recombinant expression carrier pACYC184-M.MnlI and convert Escherichia coli ER2566 obtains methylation competent cell [pACYC184-M.MnlI, ER2566].
Preferably, the inducer of the Fiber differentiation includes arabinose.
Preferably, the final concentration of 0.1-0.3% of the inducer, for example, can be 0.1%, 0.15%, 0.2%, 0.25% or 0.3%, preferably 0.2%.
Preferably, the condition of the culture be 25-30 DEG C, 180-220rpm, cultivate 15-20h, further preferably 30 DEG C, 200rpm cultivates 16h.
Preferably, the cracking thallus is the following steps are included: collect thallus, with lysis buffer resuspension bacterium after Fiber differentiation Body obtains re-suspension liquid, carries out low-temperature ultrahigh-pressure to re-suspension liquid and is crushed, obtains lysate, be centrifuged to obtain crude product.
Preferably, the lysis buffer include final concentration of 5mmol/L~15mmol/L Tris-Hcl, EDTA, 0.5mmol/L of 0.05mmol/L~0.15mmol/L~1.5mmol/L DTT, 5% glycerol and 150mmol/L~ The Nacl of 250mmol/L.
Preferably, the volume mass of the lysis buffer and thallus ratio (2-10): 1mL/g, such as can be 2:1mL/ G, 3:1mL/g, 4:1mL/g, 5:1mL/g, 6:1mL/g, 7:1mL/g, 8:1mL/g, 9:1mL/g or 10:1mL/g, preferably 10mL/g。
Preferably, the low temperature is 4-6 DEG C.
Preferably, the super-pressure is 70-90MPa, preferably 80MPa.
Preferably, the condition of the centrifugation is 30000-35000 × g, and 4 DEG C, 25-30min takes supernatant, preferably 35000 × g, 4 DEG C, 30min.
Preferably, the purifying is the following steps are included: successively pass through the affine layer of heparin for the crude product that thallus obtains is cracked Analysis, cation-exchange chromatography and anion-exchange chromatography purifying, obtain restriction enzyme MnlI.
Preferably, the cation exchange buffer includes the KH of final concentration of 5mmol/L~15mmol/L2PO4、 EDTA, 0.5mmol/L of 0.05mmol/L~0.15mmol/L~1.5mmol/L DTT, 5% glycerol and 40mmol/L~ The Nacl of 80mmol/L.
Preferably, the anion exchange buffer include final concentration of 5mmol/L~15mmol/L Tris-Hcl, EDTA, 0.5mmol/L of 0.05mmol/L~0.15mmol/L~1.5mmol/L DTT, 5% glycerol and 40mmol/L~ The Nacl of 80mmol/L.
It preferably, further include the Enzyme activity assay of restriction enzyme MnlI, specifically includes the following steps: to after purified After the restriction enzyme MnlI arrived carries out gradient dilution, in 1 × FlashOneTMUnder Buffer buffer condition, 20 μ l reactants In system, with substrate λ DNA in 37 DEG C of incubation 15min, then with the digestion situation of 1% agarose gel electrophoresis detection substrate.
The expression and purification operating process of restriction enzyme MnlI is shown in that Fig. 1, S110 are restriction enzyme in the present invention The expression screening of MnlI, specifically includes: correct recombinant expression plasmid pBAD-R.MnlI will be sequenced and convert to the methyl screened Change competent cell [pACYC184-M.MnlI, ER2566], is coated on the plate containing ampicillin and chloramphenicol antibiotics 37 DEG C of culture 12-16h are screened, the recombinant strains of restriction enzyme MnlI are obtained.Bacterium is recombinantly expressed to the MnlI of acquisition Strain carries out monoclonal screening, selects monoclonal bacterial strain into individual LB culture medium, 37 DEG C, 200rpm is cultivated to OD6000.8.With Arabinose induction, addition inducer arabinose to final concentration 0.2%, and 16h is cultivated with 200rpm in 25 DEG C of shaking tables.It collects The bacterium solution that 1.5ml has been induced into 2mlEP pipe, abandon supernatant and take precipitating by 15000 × g centrifugation, and 200 μ lB-PER are added, and (match is silent to fly Generation that), 15min is cracked, centrifugation takes supernatant, which is the crude enzyme liquid of MnlI.Thick enzyme activity inspection is carried out to the crude enzyme liquid of extraction It surveys: in 1XFlashOneTMUnder Buffer buffer condition, in 20 μ l reaction systems, with substrate λ DNA in 37 DEG C of incubation 15min, so Afterwards with the digestion situation of 1% agarose gel electrophoresis detection substrate；The enzyme activity of restriction enzyme MnlII is defined as: at 37 DEG C, In 20 μ l reaction systems, 1 μ l can digest completely 1 μ g λ DNA in 15min.
S120 is to carry out a large amount of Fiber differentiations to the restriction enzyme MnlI recombinant bacterial strain that screening obtains, and collect induction Thallus after culture, specifically includes the following steps: recombination engineering is inoculated into and contains by the ratio of 1:10000~15000 in proportion In the solid medium tablets for having ampicillin and chlorine enzyme penicillin resistance, 12h-16h is cultivated under conditions of 35 DEG C~38 DEG C. Resistance is selected according to the resistance locus contained in recombination engineering.In the present embodiment containing resistant culture medium be containing There is the LB culture medium of ampicillin and chlorine enzyme penicillin resistance, by resistance screening culture to determine the recombined engineering turned out The correctness of bacterium.Then the recombination engineering after recovery is added in LB culture solution according to the ratio of 1ml1 single colonie, In 2h-6h is cultivated under conditions of 35 DEG C~38 DEG C, obtains OD600In 0.7~0.9 activating solution.After recovery and amplification, obtain The growth vigor for activating recombination engineering in bacterium solution is preferable.It is 0.1%~0.3% that volume ratio is added into obtained activating solution Arabinose is cultivated under the conditions of 25 DEG C~30 DEG C, lower than the temperature of amplification cultivation recombination engineering.It is found in research process, The temperature of Fiber differentiation is lower, the expression of appropriate restriction restriction endonuclease MnlI, improves expression efficiency.Specifically, the above recovery is expanded It is 150rpm~250rpm in revolving speed during increasing and Fiber differentiation.Bacterium solution after Fiber differentiation is collected into receipts bacterium bottle Trim two-by-two, 4 DEG C, 5000 × g, 5min centrifugation are separated by solid-liquid separation, and are collected solids, are obtained thallus.
S130 is cracking thallus, obtains lysate, specifically includes the following steps: after inducing expression, is expressed in obtained thallus A large amount of restriction enzyme MnlI (albumen of purpose), by the way that cellular lysate, destination protein is released.With cracking The thallus is resuspended in buffer, obtains re-suspension liquid, wherein in the lysis buffer containing final concentration of 5mmol/L~ EDTA, 0.5mmol/L~1.5mmol/L of the Tris-Hcl of 15mmol/L, final concentration of 0.05mmol/L~0.15mmol/L DTT and final concentration of 150mmol/L~250mmol/L Nacl, 5% glycerol, the lysis buffer and thallus ratio are 10:1.The pH value of lysis buffer is about 7.5, and lysate is added into thallus, and the 5~15min that is vortexed is so that thallus is resuspended.Low temperature High pressure is crushed thallus, and cracking pressure is 60~90MPa；Degree of crushing be make bacterium solution is no longer sticky, subject to good fluidity.Broken bacterium Mixed liquor afterwards is centrifuged 20min~40min in 30000 × g~35000 × g, collects supernatant, obtains lysate.
S140 is that cracking passes through heparin affinity chromatography column purification, and it is affine that the destination protein in lysate is incorporated in Heparin Above chromatographic column, specifically includes the following steps: lysate is integrated on the heparin affinity chromatography column balanced, first with 3~5 times Bed volume combination buffer is rinsed.Linear ladder is carried out to Heparin affinity column with the Nacl buffer of concentration gradient again Degree elution.Occur collecting eluent when protein peak, SDS-PAGE electrophoresis is carried out to the eluent of collection, by SDS-PAGE electricity The position at the analytical judgment destination protein peak of swimming glue figure, collects the eluent 1 containing destination protein.Wherein, balance Heparin parent With the buffer components of chromatographic column are as follows: the Tris-Hcl of 5mmol/L~15mmol/L, final concentration of 0.05mmol/L~ The Nacl of EDTA, 0.5mmol/L of 0.15mmol/L~1.5mmol/L DTT and final concentration of 30mmol/L~60mmol/L, 5% glycerol, the pH value of equilibration buffer are 7.5；Nacl buffer components are the Tris-Hcl of 5mmol/L~15mmol/L, end Concentration is EDTA, 0.5mmol/L~1.5mmol/L DTT of 0.05mmol/L~0.15mmol/L and final concentration of The Nacl of 900mmol/L~1000mmol/L, 5%Glycerol, pH value are about 7.5.
S150 is that eluent 1 is successively carried out cation-exchange chromatography and anion-exchange chromatography, finely pure to eluent Change, obtain high-purity destination protein MnlI, specifically includes the following steps: the eluent 1 that S140 is collected carries out cation after diluting Displacement chromatography.Eluent 1 after dilution is integrated in the cation-exchange chromatography post balanced, first with 3~5 times of bed volumes The unbonded foreign protein of combination buffer rinsing, then line is carried out to cation-exchange chromatography post with the Nacl buffer of concentration gradient Property gradient elution.Occur collecting eluent when protein peak, SDS-PAGE electrophoresis is carried out to the eluent of collection, by SDS- The position at the analytical judgment destination protein peak of PAGE running gel figure, collects the eluent 2 containing destination protein.Wherein, balance sun The buffer components of ion exchange column are the KH of 5mmol/L~15mmol/L2PO4, final concentration of 0.05mmol/L~ The Nacl of EDTA, 0.5mmol/L of 0.15mmol/L~1.5mmol/L DTT and final concentration of 30mmol/L~60mmol/L, 5%Glycerol, pH value 6.0.High concentration Nacl buffer components are the KH of 5mmol/L~15mmol/L2PO4, it is final concentration of EDTA, 0.5mmol/L of 0.05mmol/L~0.15mmol/L~1.5mmol/L DTT and final concentration of 900mmol/L~ The Nacl of 1000mmol/L, 5%Glycerol.PH value is about 6.0.
It will be added in the cation-exchange chromatography post balanced after the dilution of obtained eluent 2, the purpose in eluent 2 Then protein binding is first not associated with 3~5 times of bed volume combination buffer rinsings miscellaneous on cation-exchange chromatography post Albumen.Linear gradient elution is carried out to cation-exchange chromatography post with the Nacl buffer of concentration gradient again.When there is protein peak Eluent is collected, SDS-PAGE electrophoresis is carried out to the eluent of collection, passes through the analytical judgment mesh to SDS-PAGE running gel figure Protein peak position, collect the eluent 3 containing destination protein.Wherein, the buffer of balance anion displacement chromatography column at Be divided into, the Tris-Hcl of 5mmol/L~15mmol/L, final concentration of 0.05mmol/L~0.15mmol/L EDTA, The Nacl of the DTT of 0.5mmol/L~1.5mmol/L and final concentration of 30mmol/L~60mmol/L, 5%Glycerol, pH value 7.5.Tris-Hcl, the final concentration of 0.05mmol/L that high concentration Nacl buffer components are 5mmol/L~15mmol/L~ EDTA, 0.5mmol/L of 0.15mmol/L~1.5mmol/L DTT's and final concentration of 900mmol/L~1000mmol/L Nacl, 5%Glycerol.PH value is about 7.5.
Obtained eluent 3 carries out buffer exchange, and the protein liquid after displacement is stored under the conditions of -20 DEG C.Specifically, Displaced buffer components include Tris-Hcl, the final concentration of 0.05mmol/L~0.15mmol/ of 5mmol/L~15mmol/L The Nacl of EDTA, 0.5mmol/L of L~1.5mmol/L DTT and final concentration of 250mmol/L~350mmol/L, 50% Glycerol.PH value is about 7.5.
Specifically, eluent 3 is added in bag filter, is placed in displacement buffer, it is slowly stirred 12 at 4 DEG C~ 16h.Taking-up is stored under the conditions of -20 DEG C.
It should be noted that in practical application, when expression and purification restriction enzyme MnlI, it is not limited to S110-S150 Sequence.Those skilled in the art, which can according to need, to be adjusted.
Second aspect, the present invention provide a kind of restriction enzyme that expression and purification method is prepared as described in relation to the first aspect Enzyme MnlI.
Compared with prior art, the invention has the following beneficial effects:
(1) expression of restriction enzyme MnlI of the invention is easy to operate, quickly, at low cost, high income, expression It is high to obtain destination protein expression quantity, it is soluble good, it is convenient for later-period purification.
(2) purification process of restriction enzyme MnlI of the invention is purified only with 3 steps, easy to operate, quickly, cost Low, high income, the destination protein activity height obtained after purification can reach 80U/ μ l, and it is pure that purity of protein height can reach 95% or more Degree.
(3) the purification process first step of restriction enzyme MnlI of the invention is captured using Heparin, is utilized The affine property of Heparin is combined a large amount of capture destination proteins and separates foreign protein, is conducive to subsequent purification, which can answer Purifying for most no label restriction enzyme.
(4) restriction enzyme MnlI of the invention is carried out pure in such a way that cation exchange and anion exchange are used in conjunction Change, using different medium property protein isolates, can achieve better separating effect, which can be applied in most The purifying of property albumen.
(5) the expression and purification mode of restriction enzyme MnlI of the invention is easy to operate, quickly, at low cost, and yield is high, It can be applied to the mass production of restriction enzyme MnlI.
Detailed description of the invention
Fig. 1 is the flow chart of the expression and purification method of restriction enzyme MnlI；
Fig. 2 is 1 restriction enzyme MnlI of embodiment expression bacterial strain screening figure, wherein the 1st swimming lane is albumen Marker, the 2 swimming lanes are lysate supernatant, and the 3rd swimming lane is cracking liquid precipitate, and the 4th swimming lane is Heparin affinity chromatography destination protein eluent, 5th swimming lane is cationic switching purpose protein eluate, and the 6th swimming lane is anion exchange destination protein eluent；
Fig. 3 is the SDS-PAGE electrophoresis result comparison of the sample in each stage during restriction enzyme MnlI is purified Figure, wherein the 1st swimming lane is λ DNA, the 2nd swimming lane is to mark product (NEB) cleavage map, 3-20 swimming lane MnlI expression screening monoclonal 1-18 cleavage map, the 21st swimming lane are Marker；
Fig. 4 is restriction enzyme MnlI viability examination electrophoretogram, and wherein swimming lane 1 is λ DNA, and swimming lane 2 is to mark product (NEB The MnlI of company's production), swimming lane 3-10 is to be respectively by MnlI stoste dilution 10 times, 20 times, 40 times, 80 times, 160 times, 320 Times, 640 times, 1280 times of specific cleavage map, final vigor is set to 80U/ μ l.
Specific embodiment
To make above-mentioned purpose of the invention, feature and advantage can be more obvious and easy to understand, combined with specific embodiments below and Specific embodiments of the present invention will be described in detail for attached drawing.Be explained in the following description many details in order to Fully understand the present invention.But the present invention can be implemented in many other ways than those described herein, art technology Personnel can do similar improvement without violating the connotation of the present invention, therefore the present invention is not by following public specific implementation Limitation.
Experimental material
1) bacterial strain and plasmid
B-PER is purchased from Thermo Fisher Scientific Inc. (ThermoFisherScientific)；
PBAD, pACYC184, pUC19 are purchased from the biological plasmid platform of vast spirit；
Enzyme activity assay substrate λ DNA is purchased from Thermo Fisher Scientific Inc. (ThermoFisherScientific)；
2) instrument and reagent
Low-temperature ultrahigh-pressure continuous flow cell is crushed instrument: being purchased from the biotech inc Yong Lian；
Protein Marker: PAGE-MASTERProteinStandardPlus is purchased from the limited public affairs of Jin Sirui biotechnology Department；
Restriction enzyme MnlI is purchased from NewEnglandBiolabs (NEB) to mark product；
10×FlashOneTMBuffer is purchased from Mo Na Biotechnology Co., Ltd.
Embodiment 1
(1) recombination engineering containing MnlI segment is screened
Correct recombinant expression plasmid pBAD-R.MnlI will be sequenced to convert to the methylation competent cell screened [pACYC184-M.MnlI, ER2566] is coated on screening and culturing on the plate containing ampicillin and chloramphenicol antibiotics, obtains Obtain the recombinant strains of restriction enzyme MnlI；The bacterial strain is induced after expanding culture with arabinose, is obtained restricted The recombinant protein of restriction endonuclease MnlI, thick enzyme extract, and carry out digestion verification, and using λ DNA as substrate, NEB company is produced restricted interior Enzyme cutting MnlI is control, carries out digestion experiment respectively to positive colony, as a result sees Fig. 2, and screening obtains the optimal monoclonal of enzyme activity Bacterial strain, and save.
In Fig. 2, the 1st swimming lane is λ DNA；2nd swimming lane is to mark product (NEB) cleavage map；3-20 swimming lane MnlI expression screening Monoclonal 1-18 cleavage map；21st swimming lane is Mark.As shown in Figure 2, the digestion that selected monoclonal bacterial strain all has MnlI is lived Power finally selects No. 16 best monoclonal bacterial strains of digestion vigor and carries out conservation by contrasting with control group.
(2) Expression of Activated of strain
The 1 above-mentioned recombination engineering of μ l is taken to carry out 4 on solid medium of the 15ml containing ampicillin and chlorine enzyme penicillin Ride.12~16h is cultivated in 37 DEG C of constant incubators.By cultured recombinant bacterial strain according to the ratio of 1ml1 single colonie It is transferred in fresh LB liquid medium.37 DEG C, 200rpm shake culture 5h obtains OD600For 0.5 activating solution；It will activation Good strain is transferred to fresh LB Liquid Culture in the ratio of 1:50 and concentrates.37 DEG C, 200rpm shakes 6h, and final concentration is added 0.2% arabinose carries out Fiber differentiation 16h, thalline were collected by centrifugation then at 30 DEG C under the conditions of 200rpm.
(3) restriction enzyme MnlI is purified
The thallus frozen is placed in thaw at RT, with 150ml lysis buffer (10mmol/LTris-Hcl, pH7.5, 0.1mmol/L EDTA, 1mmol/L DTT, 50mmol Nacl) thallus is resuspended, obtain re-suspension liquid.It is broken by cryogenic high pressure (80MPa, 2.5min) cracks thallus.Then it is centrifuged 30min under the conditions of 35000 × g, collects supernatant, obtains 150ml cracking Liquid.
A) heparin affinity chromatography
Lysate is added on the Heparin affinity column balanced with lysis buffer, with 3 times of bed volumes Lysis buffer washes unbonded foreign protein.Then linear gradient elution is carried out with the Nacl buffer solution of concentration gradient to obtain To the eluent containing destination protein, collection sample Nacl concentration is 200~300mmol/L.It is flat with lysis buffer before loading Weighing apparatus chromatographic column reaches baseline, conductance to UV280, and pH stablizes.When upper Heparin affinity column, loading flow velocity is 1cm/min, Elution flow rate is 2cm/min, and applied sample amount is every ml matrix loading mycoprotein 50mg, and temperature maintains 4 DEG C during upper prop.This Step purifying purpose is to capture destination protein.
B) cation-exchange chromatography
The Heparin eluent of collection carries out moderate with cation exchange and purifies.First cleaned with the NaOH of 3 times of bed volumes Cation-exchange chromatography post, system and pipeline are cleaned with NaOH to ensure that albumen contact site is sterile.It is handed over before loading with cation Change low salt buffer (10mmol/L KH2PO4, pH7.5,0.1mmol/L EDTA, 1mmol/L DTT, 50mmol Nacl) and to layer Analysis column is balanced 3 times of bed volumes, until UV280 reaches baseline, conductance, pH stablizes.1cm/min loading, with 3 after loading The cation exchange low salt buffer of times bed volume washes unbonded foreign protein.Then it is buffered with the Nacl of concentration gradient Solution 2cm/min carries out linear gradient elution and obtains the eluent containing destination protein, collect sample Nacl concentration be 100~ 200mmol/L.Collection vessel and period sample apparatus are both needed to by sterilization treatment.Temperature maintains 4 DEG C during upper prop.The step Purifying obtains higher degree destination protein.
C) anion-exchange chromatography
The cation-exchange eluate of collection is subjected to polishing purification with small particle anion exchange chromatography.First with 3 times The NaOH of bed volume cleans anion exchange chromatography, system and pipeline, cleaned with NaOH with ensure albumen contact site without Bacterium.Anion exchange low salt buffer (10mmol/LTris-Hcl, pH7.5,0.1mmol/L EDTA, 1mmol/L are used before loading DTT, 50mmol Nacl) 3 times of bed volumes are balanced to chromatographic column, until UV280 reaches baseline, conductance, pH stabilization is It can.1cm/min loading washes unbonded foreign protein with the cation exchange low salt buffer of 3 times of bed volumes after loading. Then linear gradient elution is carried out with the Nacl buffer solution 2cm/min of concentration gradient obtain the eluent containing destination protein, Collection sample Nacl concentration is 100~200mmol/L.Collection vessel and period sample apparatus are both needed to by sterilization treatment.Upper prop Temperature maintains 4 DEG C in the process.The purity of protein that the step purifies is high, dialysis to Storage buffer (10mmol/L Tris-Hcl pH7.4,1mmol/L DTT, 0.1mmol/L EDTA, 300mmol/L Nacl) it is stored under the conditions of -20 DEG C.
The SDS-PAGE analysis of the sample in each stage during purifying restriction enzyme MnlI
By to lysate supernatant, precipitating, heparin affinity chromatography destination protein eluent, cation-exchange chromatography purpose egg The SDS-PAGE of white eluent and anion-exchange chromatography destination protein eluent analysis, each stage sample of control purification process As a result quality is shown in Fig. 3, wherein the 1st swimming lane: albumen Marker；2nd swimming lane: lysate supernatant；3rd swimming lane: cracking liquid precipitate； 4th swimming lane: Heparin affinity chromatography destination protein eluent；5th swimming lane: cationic switching purpose protein eluate；6th swimming Road: anion exchange destination protein eluent, the molecular weight of albumen Marker be followed successively by from top to bottom 120KD, 80KD, 60KD, 50KD, 40KD, 30KD, 20KD and 10KD.
From figure 3, it can be seen that lysate is largely enriched with destination protein by Heparin affinity chromatography；In cation exchange Degree purifying, largely removes foreign protein；Anion exchange polishing purification improves purity of protein；High-purity destination protein is finally obtained, The 6th swimming lane in Fig. 3 is analyzed by Gel-Pro analyzer4 strip analysis software, gained purity of protein is greater than 95%.
Restriction enzyme MnlI enzyme activity determination after purification
The enzyme activity of restriction enzyme MnlI is defined as: at 37 DEG C, in 20 μ l reaction systems, 1 μ l can be in 15min 1 μ g λ DNA of digestion completely.After the present invention carries out gradient dilution to the restriction enzyme MnlI obtained after purified, 1 × FlashOneTMUnder Buffer (Mo Na Biotechnology Co., Ltd) buffer condition, in 20 μ l reaction systems, with substrate λ DNA 37 DEG C incubate 15min, then with the digestion situation (Fig. 4) of 1% agarose gel electrophoresis detection substrate.Swimming lane 1 is λ DNA in Fig. 4； Swimming lane 2 is to mark product (MnlI of NEB company production)；Swimming lane 3-10 be that MnlI stoste is subjected to different gradient dilutions respectively Specific cleavage map, final vigor is set to 80U/ μ l, specifically, 3: stoste dilutes 10 times；4: stoste dilutes 20 times；5: stoste is dilute Release 40 times；6: stoste dilutes 80 times；7: stoste dilutes 160 times；8: stoste dilutes 320 times；9: stoste dilutes 640 times；10: stoste 1280 times of dilution.
As shown in Figure 4, the restriction enzyme MnlI for purifying acquisition has good digestion vigor, and Rate activity is about 66666.6U/mg induction bacterium solution.
In conclusion by carrying out Fiber differentiation to the recombination engineering for expressing segment containing restriction enzyme MnlI, And collect the thallus after Fiber differentiation.Then cellular lysate obtains the cracking containing destination protein (restriction enzyme MnlI) Liquid.Preliminary purification is carried out to the lysate using Heparin affinity column.Obtain the elution containing destination protein (MnlI) Liquid, foreign protein largely reduces in eluent, and volume is less, reduces the loading time of subsequent purification.Then cation is successively carried out Exchange and anion-exchange chromatography carry out polishing purification to the eluent containing destination protein, finally obtain the restricted of high-purity Restriction endonuclease MnlI.This expression and purification mode is easy to operate, quickly, at low cost, high income, and expression and purification obtains restricted interior Enzyme cutting purity is high, meets in the market to the purity requirement of restriction enzyme.
The Applicant declares that the present invention is explained by the above embodiments method detailed of the invention, but the present invention not office Be limited to above-mentioned method detailed, that is, do not mean that the invention must rely on the above detailed methods to implement.Technical field Technical staff it will be clearly understood that any improvement in the present invention, equivalence replacement and auxiliary element to each raw material of product of the present invention Addition, selection of concrete mode etc., all of which fall within the scope of protection and disclosure of the present invention.
SEQUENCE LISTING
<110>Mo Na (Wuhan) Biotechnology Co., Ltd
<120>a kind of restriction enzyme MnlI and its expression and purification method
<130> 20190816
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 386
<212> PRT
<213>artificial synthesized
<400> 1
Met Asp Phe Asn Asn Phe Leu Asn Lys Ala Thr Leu Asn Ala Leu Leu
1 5 10 15
Val Ala Phe Ala Asn Ala Lys Pro Ile Thr Lys Pro Phe Ile Gly Phe
20 25 30
Val Ile Glu Ser Ser Tyr Lys Asn Ser Ala Tyr Ser Pro Ile Glu Leu
35 40 45
Thr Asp Asp Ile Tyr Gln Gln Asn Asn Gln Leu Leu Ser Gln Phe Thr
50 55 60
Asn Leu Asp Phe Gln Leu Asn Gly Lys Lys His Gln Phe Phe Ile Gln
65 70 75 80
Ser Asp Leu Ser Cys Asp Asp Phe Phe Ala Leu Val Tyr Lys Asn Ile
85 90 95
Leu Ile Lys Gly Ser Thr Ile Asp Asn Asp Glu Phe Ser Lys Leu Ile
100 105 110
Leu Leu Ser Phe Phe Ala Leu Arg Gly Ser Pro Asp Phe Lys Leu Asn
115 120 125
Phe Tyr Ser Leu Asp Leu Pro Arg Gln Ile Val Ser Lys Asn Tyr Leu
130 135 140
Asp Asn Leu Phe Lys Leu Ser Thr Asn Val Ser Asp Leu Arg Gln Leu
145 150 155 160
Asn Leu Asn Phe Arg Glu Leu Gln Glu Gln Phe Ile Thr Gly Glu Asn
165 170 175
Glu Arg Asn Thr Gln Phe Arg Ile Asn Leu Arg Tyr Phe Tyr Asp Asn
180 185 190
Phe Ser Asp Asp Leu Ala Lys Ile Asn Leu Phe Lys Ser Asn Ile Leu
195 200 205
Lys His Asn Ala Asn Leu Ile Lys Thr Lys Asn Ile Ala Gln Glu Ser
210 215 220
Arg Thr Phe Ile Glu Arg Leu Asn Phe Tyr Arg Asp Asn Val Leu Asn
225 230 235 240
Gln Thr Lys Thr Gln His Glu Ile Glu Gln Leu Arg Lys Asn Leu Gly
245 250 255
Phe Ile Tyr Asp Asp Thr Ile Asp Glu Lys Thr Thr Arg Asn Gln Gly
260 265 270
Ile Val Gln Tyr Val Arg Ala Tyr Phe Pro Asp Glu Cys Ala Cys Cys
275 280 285
Lys Asn Gln Tyr Asn Ile Lys Asp Arg Ser Phe Thr Tyr Arg His Ser
290 295 300
Asp Arg Tyr Tyr Leu Glu Val His His Val Ile Ser Phe Ala Ser Asp
305 310 315 320
Arg Thr Leu Asp Gln Ile Asp Asn Leu Val Lys Val Cys Pro Thr Cys
325 330 335
His Arg Ala Leu Ser Lys Asn Arg Ala Asp Glu Gln Tyr Gln Lys Glu
340 345 350
Leu Ile Ser Glu Ile Leu Ile Asn Ala Pro Lys Ala Lys Glu Phe Cys
355 360 365
Leu Asn Phe Thr Asp Glu Asn Asp Cys Ile Gln Phe Ile Tyr Asp Arg
370 375 380
Leu Arg
385

Claims (10)

1. a kind of expression and purification method of restriction enzyme MnlI, which is characterized in that the described method comprises the following steps:
Restriction enzyme MnlI recombinant expression carrier is constructed, conversion methylation competent cell obtains restriction enzyme MnlI Recombinant bacterial strain, Fiber differentiation crack thallus, and purification and recovery product is to get restriction enzyme MnlI.
2. expression and purification method according to claim 1, which is characterized in that the restriction enzyme MnlI recombinant expression The construction method of carrier includes that restriction enzyme MnlI genetic fragment is connected pBAD plasmid, obtains restriction enzyme MnlI Recombinant expression carrier pBAD-R.MnlI；
Preferably, the amino acid sequence of the restriction enzyme MnlI genetic fragment is as shown in SEQ ID NO.1.
3. expression and purification method according to claim 1 or 2, which is characterized in that the system of the methylation competent cell Preparation Method connects expression vector pACYC184 the following steps are included: screening MnlI methyl transferase gene segment, obtains methyl and turns It moves enzyme recombinant expression carrier pACYC184-M.MnlI and converts Escherichia coli ER2566, obtain methylation competent cell [pACYC184-M.MnlI, ER2566].
4. expression and purification method according to claim 1-3, which is characterized in that the inducer of the Fiber differentiation Including arabinose；
Preferably, the final concentration of 0.1-0.3% of the inducer, preferably 0.2%；
Preferably, the condition of the culture is 25-30 DEG C, 180-220rpm, cultivates 15-20h.
5. expression and purification method according to claim 1-4, which is characterized in that the cracking thallus includes following Step: collecting thallus after Fiber differentiation, thallus is resuspended with lysis buffer, obtains re-suspension liquid, carries out low-temperature ultrahigh-pressure to re-suspension liquid It is broken, lysate is obtained, crude product is centrifuged to obtain；
Preferably, the lysis buffer include final concentration of 5mmol/L~15mmol/L Tris-Hcl, 0.05mmol/L~ EDTA, 0.5mmol/L of 0.15mmol/L~1.5mmol/L DTT, 5% glycerol and 150mmol/L~250mmol/L Nacl；
Preferably, the volume mass of the lysis buffer and thallus ratio (2-10): 1mL/g；
Preferably, the low temperature is 4-6 DEG C；
Preferably, the super-pressure is 70-90MPa, preferably 80MPa；
Preferably, the condition of the centrifugation is 30000-35000 × g, and 4 DEG C, 25-30min takes supernatant, preferably 35000 × g, 4 DEG C, 30min.
6. expression and purification method according to claim 1-5, which is characterized in that the purifying includes following step It is rapid: the crude product that thallus obtains will be cracked and successively pass through heparin affinity chromatography, cation-exchange chromatography and anion-exchange chromatography Purifying, obtains restriction enzyme MnlI.
7. expression and purification method according to claim 6, which is characterized in that the cation exchange buffer includes dense eventually Degree is the KH of 5mmol/L~15mmol/L2PO4, 0.05mmol/L~0.15mmol/L EDTA, 0.5mmol/L~1.5mmol/ The Nacl of the DTT of L, 5% glycerol and 40mmol/L~80mmol/L.
8. expression and purification method according to claim 6 or 7, which is characterized in that the anion exchange buffer includes Tris-Hcl, 0.05mmol/L of final concentration of 5mmol/L~15mmol/L~0.15mmol/L EDTA, 0.5mmol/L~ The Nacl of the DTT of 1.5mmol/L, 5% glycerol and 40mmol/L~80mmol/L.
9. expression and purification method according to claim 1-8, which is characterized in that further include restriction enzyme The Enzyme activity assay of MnlI, specifically includes the following steps: carrying out gradient dilution to the restriction enzyme MnlI obtained after purified Afterwards, in 1 × FlashOneTMUnder Buffer buffer condition, in 20 μ l reaction systems, with substrate λ DNA in 37 DEG C of incubation 15min, Then with the digestion situation of 1% agarose gel electrophoresis detection substrate.
10. a kind of restriction enzyme MnlI that the expression and purification method as described in claim any one of 1-9 is prepared.
CN201910804225.5A 2019-08-28 2019-08-28 A kind of restriction enzyme MnlI and its expression and purification method Pending CN110438104A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910804225.5A CN110438104A (en) 2019-08-28 2019-08-28 A kind of restriction enzyme MnlI and its expression and purification method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910804225.5A CN110438104A (en) 2019-08-28 2019-08-28 A kind of restriction enzyme MnlI and its expression and purification method

Publications (1)

Publication Number Publication Date
CN110438104A true CN110438104A (en) 2019-11-12

Family

ID=68438089

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910804225.5A Pending CN110438104A (en) 2019-08-28 2019-08-28 A kind of restriction enzyme MnlI and its expression and purification method

Country Status (1)

Country Link
CN (1) CN110438104A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004793A (en) * 1998-08-18 1999-12-21 New England Biolabs, Inc. Method for cloning and producing the AvaI restriction endonuclease in E. coli and purification of the recombinant AvaI restriction endonuclease
US20050233432A1 (en) * 2003-07-10 2005-10-20 Morgan Richard D Novel type II restriction endonuclease, BtgZI, obtainable from bacillus thermoglucosidasius 36A and a process for producing the same
CN106480076A (en) * 2016-11-25 2017-03-08 淮海工学院 A kind of method of protecting high efficiency recombinant expressed restriction enzyme of utilization methylase
CN106755002A (en) * 2016-11-25 2017-05-31 江苏愚公生命科技有限公司 Using the method for the high efficiency recombinant expressed restriction enzyme of protection of methylase
CN107557372A (en) * 2017-08-30 2018-01-09 江苏愚公生命科技有限公司 For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme SacI

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6004793A (en) * 1998-08-18 1999-12-21 New England Biolabs, Inc. Method for cloning and producing the AvaI restriction endonuclease in E. coli and purification of the recombinant AvaI restriction endonuclease
US20050233432A1 (en) * 2003-07-10 2005-10-20 Morgan Richard D Novel type II restriction endonuclease, BtgZI, obtainable from bacillus thermoglucosidasius 36A and a process for producing the same
CN106480076A (en) * 2016-11-25 2017-03-08 淮海工学院 A kind of method of protecting high efficiency recombinant expressed restriction enzyme of utilization methylase
CN106755002A (en) * 2016-11-25 2017-05-31 江苏愚公生命科技有限公司 Using the method for the high efficiency recombinant expressed restriction enzyme of protection of methylase
CN107557372A (en) * 2017-08-30 2018-01-09 江苏愚公生命科技有限公司 For the screening technique for the protection bacterial strain that methylates for expressing restriction enzyme SacI

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KRIUKIENE, E.等: "MnlI restriction endonuclease [Moraxella nonliquefaciens]，ACCESSION NO：AAU87367.1", 《GENBANK》 *

Similar Documents

Publication Publication Date Title
Moks et al. Large–Scale Affinity Purification of Human Insulin–Like Growth Factor I from Culture Medium of Escherichia Coli
CN112209995B (en) Preparation method of SARS-CoV-2 surface protein receptor binding region
EP0408178A1 (en) Immobilized protein G variants and the use thereof
CN111117977B (en) Recombinant polypeptide linked zymogen, preparation method, activation method and application thereof
CN104593313B (en) For preparing bacteriocin durancin GL recombinant bacterium, preparation method and application
CN107849088A (en) The purification process of antibody-like protein matter
US20230203109A1 (en) Fusion polypeptides for target peptide production
CN101760466B (en) The preparation of A gene of recombined protein and expression product thereof
CN110862978B (en) Preparation method of recombinant halophilic archaea protease
CN110713545B (en) Human-derived programmed cell death factor receptor protein-1 and production method and application thereof
CN110387363A (en) A kind of Restriction enzyme Sma I and its expression and purification method
EP0587541A1 (en) Process to purify the big-endothelin protein
CN110438104A (en) A kind of restriction enzyme MnlI and its expression and purification method
CN114507293B (en) Fusion protein for expressing linaclotide in gene recombination tandem and method for expressing linaclotide
CN109942700A (en) A kind of recombinant type buckwheat trypsase inhibitor mutant and trypsase affinitive material
CN111019927B (en) Recombinant plasmid for expressing TEV protein, recombinant engineering bacterium and method for preparing and purifying TEV protein
CN111575314A (en) Application of stable urokinase receptor mutant suPARcc in eukaryotic extracellular protein expression
CN112522234B (en) Preparation method of restriction endonuclease FseI
Tabita et al. Synthesis and assembly of a novel recombinant ribulose bisphosphate carboxylase/oxygenase
CN103214561B (en) Human hepatitis c virus core antigen and preparation method and application thereof
CN102703490B (en) Process for preparing recombinant streptavidin
CN113481230B (en) Method for realizing recombinant protein purification by using anti-freeze protein-intein as purification tag and application
CN116375806A (en) Preparation method of B/Austria/1359417/2021 (B/Victoria linear) HA antigen
CN107849095A (en) The antibody-binding protein that antibody binding capacity in acid pH range reduces
CN116410266A (en) Preparation method of B/Washington/02/2019 (B/Victoria linear) HA antigen

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
RJ01 Rejection of invention patent application after publication

Application publication date: 20191112

RJ01 Rejection of invention patent application after publication