CN101538579B - Method for constructing and producing restriction endonuclease Ecop15I - Google Patents
Method for constructing and producing restriction endonuclease Ecop15I Download PDFInfo
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Abstract
The invention provides a method for constructing and producing recombination III-type restriction endonuclease Ecop15I. The III-type restriction endonuclease Ecop15I plays an important role in serial analysis of gene expression, and the prior method is difficult to realize large-scale expression and purification. By utilizing a gene recombination method, two subunit fragments of the Ecop15I fused with a purification tag are artificially synthesized and are cloned into two independent expression units of a prokaryotic co-expression vector pACYCDuet-1 respectively; under IPTG induction, the expression units with independent T7 promoters, ribosome binding sites, start codon and stop codon simultaneously and independently express two subunits of enzyme in Escherichia coli, and are folded into a composite structure with biological activity; purification is performed through Ni affinity chromatography and DEAE ion-exchange chromatography; and the recombination-type Ecop15I restriction endonuclease with high yield and good activity and stability is finally obtained. Therefore, the method provides an effective way for mass production.
Description
Technical field
The present invention relates to structure, the expression and purification of the restricted Ecop15I restriction endonuclease of recombination III-type co-expression plasmid, belong to bioengineering field.By method provided by the present invention, obtained that purity is higher, vigor recombinant type Ecop15I restriction enzyme preferably, for scale operation provides effective approach.Simultaneously, described Ecop15I restriction endonuclease can be used as the instrument of expressed sequence analysis and genes involved identification, has good using value.
Background technology
" restriction enzyme " is commonly considered as identifying the specific sequence of 4 to 8 Nucleotide on double-stranded DNA, and the general name that can cut the restriction endonuclease of this sequence.At present, reported that more than 2900 kind restriction one (R/M) systems of modifying are differentiated, cut the factors such as position, recognition sequence, cofactor according to its subunit composition, catalyst mechanism, enzyme, restriction enzyme can be divided into three major types.I type restriction enzyme is that a class has restricted and multimeric protein complex body that methylate active concurrently, can be away from any cutting DNA chain in its recognition site place.II type restriction enzyme is a kind of restriction enzyme that is independent of corresponding methylase, it is among recognition site or close on locating point really and cut specifically the DNA chain, produce definite sequence end, restricted fragment, II type restriction enzyme major part all has strict substrate specificity, identifies the palindromic sequence of 4～6bp.III type restriction enzyme is the enzyme that has restriction concurrently, modifies two kinds of functions, mainly identifies inverted repeats, cuts by halves the DNA chain outside recognition site.
Restriction enzyme Ecop15I is the plasmid-encoded III type restriction enzyme of intestinal bacteria P15, is to be folded to form the oligomeric prozyme of 407KDa size by 2 Mod subunits and 2 Res subunits.The Mod subunit realizes identification and modifies dual-use function by restriction and the Competition of modifying, and its identification is unmethylated, reverse, sequence non-palindrome (5 '-CAGCAG-3 ').Ecop15I is in 25～26nt position, recognition site downstream cochain (Topstrand) and lower chain (Bottom strand) 27 or 28 positions, and under the effect of cofactor Mg2+ and ATP, enzyme is cut DNA sequence dna.Especially to DNA have a pair of unmethylated, reverse and that there is the certain space distance, the opposition position recognition site, enzyme is cut the most effective.Its main application is at gene expression analysis (Serial Analysis of Gene Expression, SAGE
TM) in there is vital role, traditional SAGE only can 14 base sequences of label, and at improved " longSAGE
TM" in scheme also
The label that only has 21 base sequences, and Ecop15I can surpass 25 base sequences by label, has improved efficiency and the accuracy of identified gene.Ecop15I has brought into play very large effect to the structure of gene and the modification of expression, thereby provides huge potentiality for various medical science, medicine and agricultural application.
III type restriction enzyme Ecop15I has vital role at serial analysis of gene expression, but lacks at present the method that realizes large-scale expression and purifying.Mode and the general restriction enzyme of producing at present this kind of enzyme there is no difference, as its step 1 are:
1,, by recombinant DNA technology, obtain goal gene and cloned from microorganism.
The normal clone who uses phage-infect to be used for identification and selectional restriction enzyme, but this method success ratio is lower.This method is that to comprise transfer limitations-modification system be initial feature, by plasmid, is loaded on the escherichia coli cloning carrier.This method is by the ever-increasing restriction modification system of clone, comprise by selection have methylase gene active clone, but this selective system does not always obtain restriction system completely, but only produces the methylase gene.After building the plasmid storehouse, then it is transformed the applicable sink Your Majesty, preparation, containing the crude extract of restriction endonuclease, filters out required clone by activation analysis.
2, adopt some Technologies to carry out expression to the clone who contains restriction endonuclease gene.
These technology can be by separately or be combined with to obtain the expression of Restriction Enzyme maximum, and mainly contain: (1), promotor can well be identified by intestinal bacteria, directly are inserted into the upstream start-up portion; (2), insert a ribosome bind site that is positioned at upstream region of gene; (3) thereby, change the DNA sequence dna of gene by directed mutagenesis or self synthetic gene of son that accesses to your password; (4), use the hybridized primer of polymerase chain reaction (PCR) and synthetic gene 5 ' end and 3 ' end and amplify expression.
3, in suitable medium, carry this restriction endonuclease modified and restrictive clone with the fermentation container increment, to produce this restriction endonuclease, then through centrifugal cell harvesting and through sonic oscillation, destroy it, make the cell crude extract containing the restriction enzyme enzymic activity;
The cell crude extract of 4, purifying containing the restriction enzyme enzymic activity as affinity chromatography or ion exchange chromatography with the purified technology of protein of standard.
Adopt above-mentioned traditional method, for scale operation III type restriction enzyme, there is following problem in EcoP15I:
1.Ecop15I restriction enzyme is that wild-type e. coli P15 is plasmid-encoded, in the situation that do not have EcoP15I gene clone source or source more difficult, need synthetic to obtain goal gene.
2.Ecop15I be the oligomeric prozyme of a 407KDa size, there is Mod and Res2 fragment gene.In E.coli p15 plasmid, there are two open reading frame of Mod and Res (ORF), the activated restriction enzyme of tool is in fact Mod
2Res
2Such tetramer.And adopt the enzyme that above-mentioned technology can't a kind of like this structure of effective expression III type restriction enzyme Ecop15I, therefore need novel method to improve expression system.
3. adopt traditional method to carry out purifying to EcoP15I, product is generally DNA methyltransferase (Mod
2) and restriction endonuclease (Mod
2Res
2) mixture.Purifying 1mg enzyme, generally need 7-8 to rise inoculum, and whole purge process needs 10-14 days, and the output obtained thus is the 0.1mg/g cell.Another problem is that on limited storage stability, especially technique, freezing EcoP15I is non-activity after melting.
Than above-mentioned traditional method, the present invention has following characteristic:
At first the present invention utilizes the method for overlapping PCR, and the synthetic Long fragment gene obtains goal gene.The method can be used for some difficult genes of originating, or the synthetic of the gene of source dangerous (such as virogene).The Ecop15I restriction enzyme is that intestinal bacteria P15 is plasmid-encoded, in the situation that do not have gene clone source or source more difficult, adopted the method for synthetic, obtain goal gene.And analyzed the codon frequency of Ecop15I genes encoding, compared with e. coli bl21 (DE3) codon frequency, the frequency ratio that rare codon occurs is lower.
Secondly, the topmost characteristic of the present invention is the mode of having selected the intestinal bacteria coexpressions, has like this protein complexes of 2 and 2 above monomers to express the Ecop15I enzyme.III type Restriction Enzyme Ecop15I is the prozyme of a 407KDa size, has 2 Mod subunits and 2 Res subunits.If merge merely 2 fragment genes, express the fusion rotein of higher molecular weight, expression effect is bad, and may cause the change of pleated sheet structure due to the fusion of 2 sections protein monomers, has affected the biologic activity of protein complexes.And the present invention utilizes the method for coexpression to address this problem, and easy to operation.
Purification efficiency is higher, and purifying 1mg enzyme only needs the 36ml inoculum, and the whole purifying time needs 5-7 days, and EcoP15I output is about the 5.6-6.6mg/g cell, and output is higher.In addition, the EcoP15I that the present invention produces, be positioned over 4 ℃ and remain unchanged with-20 ℃ of long-time vigor, and stability is higher.
Summary of the invention
The invention provides a kind of effective means and expression system, can be used for scale operation III type restriction enzyme Ecop15I.The present invention utilizes bionic method, according to the Ecop15I gene order in Genebank, designs tens ofly to primer, utilizes the method (Overlapping PCR) of overlapping extension PCR, and synthetic has obtained target sequence (seeing accompanying drawing 1).Two synthetic fragment genes are subcloned into respectively in the co-expression carrier pACYCDuet-1 with two independent T7 promotors, ribosome bind site again, at Res subunit C end and Mod subunit N end, have respectively merged respectively 6 * His Tag.By nickel affinity chromatography, ion exchange layer analysis method, prepared purity up to more than 95%, the vigor recombinant type restriction enzyme similar to commercialization Ecop15I.
The bright main characteristics of this law builds III type restriction endonuclease Ecop15I co-expression plasmid exactly.The approach that realizes of coexpression mainly contains two/multichip carrier cotransformation method at present, two/polycistron method and two/multiple promoter support methods etc.For two/multichip carrier cotransformation method, find and to possess with different antibiotic markers, the compatible good expression vector of plasmid comparatively difficultly, and be difficult to effective means and maintain the copy number balance between plasmid, the expression amount of restive different subunits is how many.Selection utilizes polycistron simple substance grain to express, although mRNA is equivalent when genetic transcription, translation process is but relatively to oppose; And in the bicistronic mRNA carrier, there are some researches show, translation skill can not balance, be placed in IRES (internal ribosome entry site, internal ribosome entry site) expression amount of the foreign gene in downstream is 20%～50% of its upstream cap startup translation gene, and the translation efficiency that the translation efficiency started with IRES does not have cap to rely on is high.
Such III type restriction enzyme for Ecop15I, select the multiple promoter co-expression carrier can express the characteristics of 2 or a plurality of protein monomers simultaneously, two monomers of restriction enzyme are cloned into respectively to two and independently express in unit, express 2 subunits of desirable proteins simultaneously.In the process of expressing, 2 subunits interact, and are folded into and have bioactive quaternary structure, and this coexpression mode has unrivaled advantage.Because Ecop15I is different from II type restriction enzyme, independent Res subunit is not have enzyme to cut activity, and need and Mod form the protein complexes of quaternary structure could exercise its biological function.This just requires to express and two subunits of this albumen of purifying simultaneously, and controls and keep the relative equilibrium of the expression amount of 2 subunits.Do not have the Preference of translation for these 2 subunits of Ecop15I coexpression due to intestinal bacteria itself, thereby no matter its gene order how, all can balance expression amount between the two.
Such phraseology, in other II type restriction enzyme cloning and expression, also has lot of advantages: in II type restriction enzyme cloning and expression before, need screen in advance and have anti-this restriction enzyme, the Host Strains that contains the homology methylase.If utilize multilist to reach carrier, express 2 enzymes in the R-M system simultaneously, the methylase given expression to has just been modified the DNA of Host Strains and plasmid, prevents the restriction enzyme that the gives expression to toxic action to Host Strains.Wherein restriction enzyme is merged purification tag, and just separation and purification easily goes out required enzyme.
The invention provides the structure of III type restriction enzyme Ecop15I co-expression plasmid, the method for expression and purification, its basic step is (seeing accompanying drawing 2):
1. gene synthesizes and the clone
(1) design amalgamation and expression sequence: obtain Ecop15I gene order (Genbank AJ634453) from the NCBI website, consider and facilitate downstream zymoprotein purifying requirement, Res subunit C end and Mod subunit N end merge respectively separately with 6 * His Tag, and add special restriction enzyme site at every section sequence two ends;
(2) design primer: tens of to primer according to the principle design of Overlapping Oligonucleotides,
(3) synthetic full-length gene clone: the method synthetic full-length gene of Overlapping PCR, flush end is cloned in the pUC57 plasmid respectively, the right-on full-length gene clone of Selective sequence after order-checking;
(4) subclone of gene: Res and Mod subunit are taken up in order of priority and are installed in two expression regulation districts in pACYCDuet-1, obtained the pACYC_P15 co-expression plasmid.
2.Ecop15I conversion and expression
(1) the pACYC_P15 co-expression plasmid is converted in intestinal bacteria competence BL21 (DE3), coats in the LB plate that contains chlorampenicol resistant, choose bacterium and cultivate, induce rear centrifugal receipts bacterium;
(2) whole bacterial protein electrophoretic analysis, by the comparative electrophoresis band, select correction Ecop15I enzyme band and expression amount mono-clonal relatively preferably, as the engineering bacteria of enlarged culturing;
(3) optimization expression, by bacteria concentration (OD600) gradient, IPTG induced concentration gradient, induction time gradients etc., as optimizing factors, are carried out orthogonal experiment;
(4), by the SDS-PAGE electrophoresis, compare the expression amount of soluble proteins under different parameters, to determine the reference conditions of purifying in enormous quantities.
3.Ecop15I enlarged culturing and purifying
(1) connect the Ecop15I engineering bacteria, be incubated in the LB liquid tube with chlorampenicol resistant, shaking culture is spent the night;
(2) the bacterium liquid that will spend the night is forwarded in the 2L triangular flask and is cultured to about OD=0.6, adds IPTG to induce, last centrifugal collection thalline;
(3) the resuspended thalline of Ni post binding buffer liquid, the ultrasonic disruption thalline; Centrifuging and taking supernatant crude protein; Supernatant crude protein solution is crossed to the nickel affinity chromatography purification column, and substep is collected.Getting the collection sample that purity and concentration are higher mixes, dialyses;
(4) protein solution of having dialysed is crossed to negatively charged ion DEAE ion exchange column, the stepped start-stop system wash-out, substep is collected.Getting the collection sample that purity and concentration are higher mixes, dialyses.And use the Bradford method, survey the Ecop15I total concn.
The accompanying drawing explanation
Fig. 1. with the synthetic Ecop15I target sequence of overlapping PCR method.
Fig. 2. produce III type restriction enzyme Ecop15I process flow sheet.
Synthetic and the clone of Fig. 3 .Ecop15I Mod and Res subunit.
The subclone of Fig. 4 .Ecop15I obtains co-expression carrier pACYC_P15 plasmid.
Fig. 5 .Ecop15I nickel affinity column purifying rear electrophoresis figure.
Fig. 6 .Ecop15I DEAE ion-exchange purification rear electrophoresis figure.
Fig. 7 .Ecop15I enzyme is cut pUC19 plasmid electrophorogram.
Embodiment
1. material:
Plasmid and cell strain pUC19, pUC57 are hundred biotechnology difficult to understand (Nantong) company limited (Nantong hundred Austria) products, co-expression carrier pACYCDuet-1 is purchased from Novagen company, clone strain Top10 and expression strain BL21 (DE3) all are purchased from Beijing Tian Gen Bioisystech Co., Ltd, according to molecular cloning CaCl
2Method self-control competence, guarantee to tire more than 106 (CFU/ug pUC19).
2. reagent:
(1) toolenzyme: Taq archaeal dna polymerase, Pfu archaeal dna polymerase (Nantong hundred Austria); Restricted enzyme, (New England Biolabs) such as SmaI, BamHI; T4DNA ligase enzyme (New EnglandBiolabs);
(2) biochemical reagents: Yeast, Typtone, Tris alkali, glycine, SDS, agarose etc. (work is given birth in Shanghai);
(3) DNA purification kit: the coventional type plasmid reclaims test kit, and sepharose DNA reclaims test kit (Nantong hundred Austria);
(4) chromatographic stuffing: the affine filler of Ni sepharose; DEAE sepharose anion exchange filler; CM sepharose anion exchange filler (Beijing Webster is rich intelligent).
1. the synthetic of gene
Obtain Ecop15I gene order (Genbank AJ634453) from the NCBI website, design amalgamation and expression sequence.Consider and facilitate downstream zymoprotein purifying requirement, merge respectively separately with 6 * His Tag at the Res of Ecop15I subunit C end and Mod subunit N end.
Tens of to primer according to the principle design of overlapping oligonucleotides, the Res of two ends primer and Mod fragment two ends have end to end been added respectively the restriction enzyme site of KpnI, XhoI and BamHI, SalI, and add a G base after the BamHI restriction enzyme site, to guarantee enzyme sequence reading frame correct in translation process.To nearly 3kb Res gene fragment, the unique BglII restriction enzyme site by centre, be divided into 2 sections by the Res fragment, then, after cutting with KpnI and BglII and enzyme, connect complete Res gene fragment with the T4 ligase enzyme.After two fragment genes synthesize respectively, flush end is cloned in carrier pUC57,42 ℃ of heat shock methods transform intestinal bacteria TOP10 competence, carry out blue hickie screening, then examine PCR method by bacterium, tentatively obtain positive colony, further utilize alkaline lysis extraction agent box extracting positive colony plasmid to be checked order, for point mutation and the base deletion of gene, carry out the PCR reparation, to obtain the right-on plasmid of sequencing result.
2. the subclone of gene
The restriction enzyme site that utilization designs, the method for employing double digestion, successively install to Res and Mod subunit respectively in two expression regulation districts in pACYCDuet-1, obtained the pACYC-P15 co-expression plasmid.
3.Ecop15I expression and purification
(1) Ecop15I transforms and optimization expression
The pACYC_P15 co-expression plasmid is converted in intestinal bacteria competence BL21 (DE3) by 42 ℃ of heat shock methods.Coat in the LB plate that contains chlorampenicol resistant, be inverted overnight incubation for 37 ℃.In the LB test tube that after 6 hours, 8 single bacterium colonies of picking contain chlorampenicol resistant in 4ml respectively, transfer unconverted BL21 (DE3) in the LB without chlorampenicol resistant, 37 ℃, 220rpm shaking culture simultaneously.After 6 hours, after bacterium grows to finite concentration, inhale 0.8ml bacterium liquid, mix with 0.2ml sterile glycerol (80%) and preserve bacterial classification.The bacterium liquid final concentration that remains about 3ml is that 1mM IPTG induces after 3 hours and (stays a pipe not induce in contrast simultaneously).2 contrast and induce simultaneously centrifugal receipts bacterium of bacterium, with the resuspended centrifuge washing of 20mM Tris-Cl (pH8.0) of 1/10 volume of original bacteria liquid amount, precipitate, and repeat once.Get suspension 20ul and add 5ul SDS-PAGE sample-loading buffer, boil 10 minutes rear electrophoresis.Comparative electrophoresis band of expression, and then definite correction Ecop15I enzyme and expression amount mono-clonal relatively preferably, as the engineering bacteria of enlarged culturing.And then again by bacteria concentration (OD600) gradient, IPTG induced concentration gradient, induction time gradients etc., as optimizing factors, are carried out orthogonal experiment.By the SDS-PAGE electrophoresis, compare the expression amount of soluble proteins under different parameters, to determine the condition of purifying in enormous quantities.
(2) enlarged culturing of Ecop15I and purifying
Inhale 40ul (1% connects the bacterium amount) and be kept at the engineering bacteria in glycerine, be incubated in the LB liquid tube of 4ml with chlorampenicol resistant.37 ℃, 220rpm shaken overnight.
Approximately after 4 hours, will spend the night bacterium by 1% the bacterium amount that connects, be forwarded in the triangular flask of the LB liquid that contains the paraxin resistance, 37 ℃, 220rpm shaking culture, when growing to the OD600=0.6 left and right, adding IPTG to make its final concentration is 0.5mM, induce 4 hours, bacterium liquid is placed in to mixture of ice and water cooling 30 minutes; Centrifugal 15 minutes of 4 ℃, 6000rpm, the precipitation thalline of collection, with the resuspended washing thalline of 20mM Tris-Cl (pH8.0) of 4 ℃ of precoolings of 1/10 volume, repeat once afterwards thalline to be stored in-20 degree.
With 80ml Ni post binding buffer liquid (50mM Tris-HCl, 500mM NaCl, the 10mM 2 mercapto ethanol, 0.1%TritonX-100, the 20mM imidazoles, pH8.0) resuspended thalline, ultrasonic disruption under 4 ℃ of conditions, work and within 2 seconds, suspend 6 seconds lasting 30min.4 ℃, 13000rpm, after centrifugal 20 minutes, are left and taken supernatant.Supernatant liquor is crossed to the good Ni post of Ni binding buffer liquid balance; Ni lavation buffer solution (containing the 50mM imidazoles) washing with 10 times of column volumes; Ni elution buffer (containing the 150mM imidazoles) wash-out, be in charge of the collection rear electrophoresis with the 1.5ml centrifuge tube.Choosing 4 pipes that concentration is higher mixes.By DEAE binding buffer liquid (50mMTris-HCl, 10mM 2 mercapto ethanol, pH8.0) dialysed overnight.
By the good DEAE anion-exchange column of 8.7ml albumen sample overbalance of having dialysed, with the DEAE lavation buffer solution washing of 10 times of column volumes.The stepped start-stop system wash-out, be in charge of the collection rear electrophoresis.Choose the high and purity of concentration preferably 3 pipes mix.With preserve damping fluid (10mM Tris-HCl, 100mM NaCl, 1mM DTT, 0.1mM EDTA, 50% glycerine, pH7.4) dialysis, obtain 3.7ml albumen and collect liquid, is stored in-20 ℃.And use the Bradford method, and 30 times of diluted samples, BSA, as standard substance, surveys the Ecop15I total concn.
(1) the pACYC_P15 co-expression plasmid builds result
The Res subunit of Ecop15I and Mod subunit be synthetic 3 fragment genes of artificial PCR altogether, are respectively Eco_Mod, Eco_ResI and Eco_ResII (seeing shown in accompanying drawing 3).Three fragment genes are cloned by flush end respectively, are loaded in pUC57, obtain pMod, tri-plasmids of pRes_I and pRes_II; By after pRes_I and pRes_II use BglII and NotI double digestion, T4 ligase enzyme enzyme connects, and obtains the plasmid of complete Res fragment.
Enzyme after pMod plasmid and pACYCDuet-1 plasmid BamHI and SalI double digestion is connected, the pACYC_Mod plasmid that acquisition comprises the Mod subunit, again with pRes_All plasmid KpnI and XhoI double digestion after enzyme connect, obtain and comprise two subunit pACYC_P15 plasmids of complete Ecop15I (seeing shown in accompanying drawing 4).
(2) Ecop15I expression and purification
Utilize the induction time gradient, IPTG induced concentration gradient, the bacteria concentration gradient, culture temperature etc. are as the optimization expression factor, orthogonal experiment, and having obtained cultivation and inducing temperature is 37 ℃, inducing bacteria concentration is logarithmic phase mid-term, while being the OD600=0.6 left and right, the IPTG final concentration of take is induced as 0.5mM, centrifugal receipts bacterium after 3 hours.
Through the Ni affinity chromatography, the protein liquid that single tube is collected carries out the purification effect preferably (seeing accompanying drawing 3) that the SDS-PAGE electrophoresis has obtained Ecop15I.In 74KDa and 110Kda left and right, and estimate that result is consistent from the molecular weight of 5,2 band of expression of accompanying drawing, prove that this band is the Ecop15I of purifying.Wherein swimming lane 1～4 protein content will be chosen this 4 pipe and mix dialysis generally higher than other swimming lanes, further carries out DEAE ion exchange chromatography chromatography purification (seeing accompanying drawing 4).Protein content and purity of protein from accompanying drawing 6,3～5 swimming lanes will, higher than other swimming lanes, be selected rear-20 ℃ of preservations of this 3 effective preservation damping fluid dialysis.Utilize Bandscan software to be analyzed the SDS-PAGE picture, the expression amount of Ecop15I (two subunit total amounts) accounts for 15% left and right of E.coli total protein.Through after Ni affinitive layer purification for the first time, just reached the purity more than 95%, then, after DEAE anion-exchange chromatography purifying, the purity of 2 subunits is all more than 97%.
Use the Bradford method, the concentration that after the Ni affinity chromatography, albumen is collected liquid is at 6.6mg/ml, the nearly 60mg of total protein concentration; After the DEAE ion exchange chromatography, albumen is collected the concentration of liquid at 5.6mg/ml.800ml bacterium liquid obtains the albumen of 22mg left and right altogether.
(3) activity of Ecop15I detects
The Ecop15I gradient enzyme of purifying is cut to the pUC19 plasmid, after 37 ℃ of enzymes are cut 30min, 65 ℃ of 20min deactivations.The Ecop15I of NEB is as positive control, and as shown in Figure 7, the Ecop15I enzyme of purifying and the enzyme of NEB, when 0.5ul, have same enzyme activity.
(4) Ecop15I stability analysis
By the Ecop15I enzyme be stored in store buffer liquid, be positioned over 4 ℃ and-20 ℃, after 4 weeks, through the protein SDS-PAGE electrophoresis, do not find degraded.Enzyme is cut pUC19 and is detected, and vigor remains unchanged substantially.
Aminoacid sequence table SEQUENCE LISTING
Claims (5)
1. a method that builds III type restriction enzyme EcoP15I co-expression plasmid, the Res subunit C end of described Ecop15I and Mod subunit N hold and merge respectively separately with 6 * His Tag chromatography label, and its construction step comprises:
(1) synthetic primer, add respectively BamHI, SaII restriction enzyme site by the two ends primer end to end in the Mod fragment, and add the G base after the BamHI restriction enzyme site, obtains the Eco_Mod gene; Adding respectively end to end KpnI, XhoI restriction enzyme site in the Res fragment, by the Bg1II restriction enzyme site in the middle of the Res gene fragment, the Res fragment is divided into to two sections, is Eco_ResI and Eco_ResII gene; Obtain comprising 3 fragment gene Eco_Mod, Eco_ResI and the Eco_ResII of Res subunit and Mod subunit, 3 fragment genes are cloned by flush end respectively, are loaded in pUC57, obtain pMod, pRes_I and tri-plasmids of pRes_II; By after pRes_I and pRes_II use Bg1II and NotI double digestion, T4 ligase enzyme enzyme connects, and obtains the pRes_All plasmid of complete Res fragment;
(2) enzyme after pMod plasmid and pACYCDuet-1 plasmid BamHI and SalI double digestion is connected, the pACYC_Mod plasmid that acquisition comprises the Mod subunit, again with pRes_All plasmid KpnI and XhoI double digestion after enzyme connect, obtain the Ecop15I co-expression plasmid that comprises two subunits of complete EcoP15I.
2. method according to claim 1, wherein said chromatography label 6 * His Tag is Histidine nickel (Ni
2+) the affinity chromatography label.
3. method according to claim 1, wherein said Ecop15I co-expression plasmid was expression plasmid.
4. a method of producing III type restriction enzyme EcoP15I, step comprises:
(1) with the synthetic EcoP15I gene of overlapping PCR method, wherein Res subunit C end and Mod subunit N end merge respectively separately with 6 * His Tag chromatography label;
(2) build the EcoP15I co-expression plasmid, comprising:
A. synthetic primer, add respectively BamHI, SaII restriction enzyme site end to end in the Mod of two ends primer fragment, and add the G base after the BamHI restriction enzyme site, obtains the Eco_Mod gene; Adding respectively end to end KpnI, XhoI restriction enzyme site in the Res fragment, by the Bg1II restriction enzyme site in the middle of the Res gene fragment, the Res fragment is divided into to two sections, is Eco_ResI and Eco_ResII gene; Obtain comprising 3 fragment gene Eco_Mod, Eco_ResI and the Eco_ResII of Res subunit and Mod subunit, 3 fragment genes are cloned by flush end respectively, are loaded in pUC57, obtain pMod, pRes_I and tri-plasmids of pRes_II; By after pRes_I and pRes_II use Bg1II and NotI double digestion, T4 ligase enzyme enzyme connects, and obtains the pRes_All plasmid of complete Res fragment;
B. enzyme after pMod plasmid and pACYCDuet-1 plasmid BamHI and SalI double digestion is connected, the pACYC_Mod plasmid that acquisition comprises the Mod subunit, again with pRes_All plasmid KpnI and XhoI double digestion after enzyme connect, obtain the Ecop15I co-expression plasmid that comprises two subunits of complete EcoP15I;
(3) conversion of EcoP15I and expression;
(4) EcoP15I large scale culturing and expression;
(5) EcoP15I purifying.
5. method according to claim 4, wherein the step of purifying EcoP15I-comprises:
(1) the resuspended centrifugal rear thalline of nickel post binding buffer liquid, ultrasonic disruption thalline, centrifuging and taking supernatant crude protein;
(2) cross the nickel affinity chromatography purification column, substep is collected;
(3) getting the collection sample that purity and concentration are higher mixes, dialyses;
(4) cross negatively charged ion DEAE ion exchange column, the stepped start-stop system wash-out, substep is collected;
(5) get the collection sample that purity and concentration are higher and mix, dialysis, survey the Ecop15I total concn.
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|---|---|---|---|---|
| EP1584677A1 (en) * | 2004-04-07 | 2005-10-12 | Charité - Universitätsmedizin Berlin | Ecop151 - Process conditions and an efficient method for its large-scale purification |
| CN100584957C (en) * | 2003-05-09 | 2010-01-27 | 岩手县 | Use of a type III restriction enzyme to isolate identification tags comprising more than 25 nucleotides |
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| CN100584957C (en) * | 2003-05-09 | 2010-01-27 | 岩手县 | Use of a type III restriction enzyme to isolate identification tags comprising more than 25 nucleotides |
| EP1584677A1 (en) * | 2004-04-07 | 2005-10-12 | Charité - Universitätsmedizin Berlin | Ecop151 - Process conditions and an efficient method for its large-scale purification |
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| Hümbelin M等.Type III DNA restriction and modification systems EcoP1 and EcoP15. Nucleotide sequence of the EcoP1 operon, the EcoP15 mod gene and some EcoP1 mod mutants.《journal of molecular biology》.1988,第200卷(第1期),23-29. * |
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