CN103614310A - Recombinant pichia yeast for expressing UBC13 protein and construction method thereof - Google Patents
Recombinant pichia yeast for expressing UBC13 protein and construction method thereof Download PDFInfo
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- CN103614310A CN103614310A CN201310593987.8A CN201310593987A CN103614310A CN 103614310 A CN103614310 A CN 103614310A CN 201310593987 A CN201310593987 A CN 201310593987A CN 103614310 A CN103614310 A CN 103614310A
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Abstract
The invention discloses a recombinant pichia yeast for expressing a UBC13 protein and a construction method thereof. The recombinant pichia yeast is a recombinant bacteria for expressing the UBC13 protein and is obtained by introducing a synthetic UBC13 protein gene into a pichia yeast, wherein the nucleotide sequence of the UBC13 protein gene is shown as SEQ ID NO.1. The invention also discloses the construction method of the recombinant pichia yeast. The recombinant pichia yeast can be used for producing the UBC13 protein.
Description
Technical field
The invention belongs to gene engineering technology field, relate to a kind of recombinant yeast pichia pastoris that can express UBC13 albumen, and the construction process of this recombinant yeast pichia pastoris.
Background technology
UBC13 albumen is ubiquitin ligase E2N (Ubiquitin-conjugating enzyme E2N, UBE2N), and it is extensively present in human body various kinds of cell, as skin cells, lymphocyte etc., and brings into play critical function in these cells.The main biological function of UBC13 albumen comprises following aspect: 1, the heterodimer UBE2V1-UBE2N of UBC13 and UBE2V2-UBE2N can catalyze and synthesize the poly ubiquitin chain of non-normal Methionin-63, and such poly ubiquitination can not make proteasome degrade proteins; 2, UBC13 regulates the transcriptional activity of goal gene, and plays regulatory role in cell cycle and atomization; 3, UBC13 plays regulatory role in faultless DNA reparation approach, to the cells survival after DNA damage, plays a part certain; 4, UBC13 plays an important role to the lymphocytic activity of regulatory T, thereby affects Immunization Activities; Etc..
Pichia yeast expression system has that expression amount is high, can modify after protein expression, easily large-scale production, become to produce the features such as cost is lower, foreign protein genes genetic stability, and existing multiple foreign protein genes is expressed in pichia spp.Pichia spp gene expression system, except the feature that has general yeast and have, also has following advantage: 1, have the strict alcohol oxidase AOX1 gene promoter of Regulation Mechanism; 2, expression plasmid can be at genomic specific site the form stable bond with single copy or multiple copied; 3, yeast strain can carry out high density fermentation, and foreign aid to express output high.
There is not yet at present the report that utilizes Pichia anomala expression UBC13 albumen both at home and abroad.
Summary of the invention
The pichia spp and the construction process thereof that the object of this invention is to provide a kind of UBC13 of expression albumen.
The recombinant yeast pichia pastoris of expression UBC13 albumen provided by the present invention, is the pichia spp being transformed by the recombinant expression vector that carries UBC13 albumen, wherein, includes the nucleotide sequence of the UBC13 albumen shown in SEQ ID NO.1 in described recombinant expression vector.
Wherein, the gene source of described UBC13 albumen is in people.
Pichia yeast expression system has very high biological safety, obtains the extensive approval that comprises U.S. FDA.It can translate post-treatment to foreign protein, makes it conformation and activity closer to native protein, is a kind of eukaryotic expression system of widespread use.Pichia spp of the present invention is specially pichia spp X33.
The carrier that recombinant expression vector of the present invention is used is pwPICZalpha plasmid vector.
Another object of the present invention is to provide a kind of method that builds described recombinant yeast pichia pastoris, comprises the steps:
1) synthetic UBC13 protein gene;
2) in the multiple clone site of plasmid vector, insert UBC13 protein gene, build recombinant expression vector;
3) described recombinant expression vector is imported in pichia spp, obtain the recombinant yeast pichia pastoris of expressing UBC13 albumen.
Wherein, described pichia spp is pichia spp X33, and described recombinant expression vector is used pwPICZalpha plasmid vector.The pichia spp called after pichia spp X33 of the expression UBC13 albumen that the present invention builds pwPICZalpha UBC13.
Recombinant yeast pichia pastoris of the present invention can be used to produce UBC13 albumen.
The present invention first in pichia yeast expression system secreting, expressing people UBC13 albumen, it is extremely low that secreting, expressing is cut in substratum foreign protein content without smudge cells, purification step is simple, suitability for industrialized is produced.Pichia spp is applicable to plant-scale high density fermentation, and substratum is cheap, and simple to operate, product has good biological safety, is applicable to the fields such as daily necessities, food and medicine.
Accompanying drawing explanation
Fig. 1 is that Xho I and BcoR I enzyme are cut the recombinant plasmid containing UBC13 protein gene of identifying synthetic.
Fig. 2 is that Xho I and BcoR I enzyme are cut the pwPICZalpha recombinant expression plasmid of identifying containing UBC13 protein gene.
Fig. 3 is the SDS-PAGE electrophoresis detection result after medium supernatant and supernatant liquor the first step purifying after expression of recombinant yeast (crossing nickel post).
Fig. 4 is the SDS-PAGE electrophoresis detection result after supernatant liquor second step purifying (crossing anion-exchange column).
Fig. 5 is the SDS-PAGE electrophoresis detection result before concentrated rear preservation of UBC13 albumen.
Specific implementation method
Embodiment 1: the structure that carries the recombinant expression vector of UBC13 protein gene.
1, the Preference to codon according to pichia spp, manually designs UBC13 protein gene, and adds restriction enzyme site Xho I at carbon teminal, and nitrogen end adds restriction enzyme site EcoR I, gives associated companies synthetic.The UBC13 protein gene sequence of artificial design is as shown in SEQ ID NO.1.
2, the intestinal bacteria that contain synthetic goal gene plasmid are being cultivated containing in the LB liquid nutrient medium of 0.1% ammonia benzyl.
With the Plasmid Mini Kit I test kit (D6943-01*) of OMEGA company, extract the plasmid containing goal gene of synthetic, extract plasmid and carry out double digestion evaluation by Xho I and EcoR I, enzyme is cut to product agarose gel electrophoresis and detect.Electrophoresis detection result as shown in Figure 1, detects synthetic UBC13 protein gene at the 500bp place of DNA molecular amount standard, and the object band that enzyme is cut product is 506bp, meets the base number of artificial design.
With the Gel Extraction Kit test kit (D2500-01) of OMEGA company, reclaim above-mentioned double digestion product.Reclaim product and be connected under the effect of T4 DNA ligase with the pwPICZalpha carrier being undertaken by same enzyme after enzyme is cut, 4 ℃ of condition of contacts, connect 12h, build recombinant expression vector pwPICZalpha-UBC13.
Recombinant expression vector pwPICZalpha-UBC13 is transformed in competent escherichia coli cell by heat shock method, under 37 ℃, 100-120rpm condition, cultivates 1h.The intestinal bacteria that import recombinant expression vector are screened under 37 ℃ of conditions with the LB flat board that contains 0.1%Zeocin.Select 6 positive bacterium colonies to be inoculated into respectively in the LB liquid nutrient medium that 2ml contains 0.1%Zeocin, under 37 ℃, 180rpm condition, cultivate 12-24h.Then use the Plasmid Mini Kit I test kit (D6943-01*) of OMEGA company to extract recombinant plasmid, and carry out double digestion evaluation by Xho I and EcoR I.Enzyme is cut product and is carried out agarose gel electrophoresis detection, and electrophoresis detection result as shown in Figure 2, detects the UBC13 protein gene of restructuring at the 500bp place of DNA molecular amount standard, and the object band that enzyme is cut product is 506bp, meets the base number of artificial design.And then obtain the positive intestinal bacteria contain recombinant expression vector pwPICZalpha-UBC13.
Containing the LB liquid nutrient medium of 0.1% ammonia benzyl, by following material, formed: 1.0g 100ml peptone, 0.5g 100ml yeast extract powder, 0.5g 100ml NaCl, 0.1g 100ml ampicillin.
Containing the LB flat board of 0.1% Zeocin, by following material, formed: 1.0g 100ml peptone, 0.5g 100ml yeast extract powder, 0.5g 100ml NaCl, 1.5g 100ml agar, 0.1ml 100ml Zeocin.
Containing the LB liquid nutrient medium of 0.1% Zeocin, by following material, formed: 1.0g 100ml peptone, 0.5g 100ml yeast extract powder, 0.5g 100ml NaCl, 0.1ml 100ml Zeocin.
Embodiment 2: the structure of expressing the recombinant yeast pichia pastoris of UBC13 albumen.
One, the preparation of competence pichia spp X33.
1, get 1.0ml pichia spp X33 Jun liquid and be added in 50ml YPD liquid nutrient medium, under 30 ℃, 250rpm condition, cultivate 12-24h.
2, by above-mentioned pichia spp Jun liquid centrifugal 5min under 4 ℃, 1500g condition.
3, discard substratum, add 0 ℃ of frozen water of 50ml resuspended, then centrifugal 5min under 4 ℃, 1500g condition.
4, discard above-mentioned frozen water, add 25ml frozen water resuspended, then centrifugal 5min under 4 ℃, 1500g condition.
5, discard above-mentioned frozen water, add 2ml ice sorbyl alcohol resuspended, then centrifugal 5min under 4 ℃, 1500g condition.
6, discard above-mentioned sorbyl alcohol, add appropriate (0.5ml left and right) ice sorbyl alcohol resuspended, prepared competence pichia spp X33.
Two, the linearizing of recombinant expression vector and the foundation of recombinant yeast pichia pastoris.
The intestinal bacteria Jun liquid that 1ml is contained to recombinant expression vector pwPICZalpha-UBC13 is added in 50ml LB substratum, under 37 ℃, 160-180rpm condition, cultivates 12-24h.With the Endotoxin-Free Plasmid Isolation Mini Kit II test kit of OMEGA company, extract recombinant expression vector pwPICZalpha-UBC13.
By Sac I, recombinant expression vector pwPICZalpha-UBC13 is carried out to linearizing, reaction conditions: 37 ℃, enzyme is cut 3h.
Linearizing product carries out agarose gel electrophoresis, with Gel Extraction Kit test kit (D2500-01), reclaims electrophoresis product.
Get 80 μ l competence pichia spp X33, reclaim product with the linearizing of 20-50 μ l recombinant expression plasmid and mix, put into immediately electric revolving cup (RIO-RAD) the ice bath 5min of ice bath.With gene importing equipment (the biological company limited of the new sesame in Ningbo, SCIENTZ-2C) at U=1500V, C=25 μ F, carries out pulse under R=400 Ω condition; After pulse, add immediately 1ml ice sorbyl alcohol also this solution to be moved on in sterile tube in electric revolving cup, under 30 ℃ of conditions, hatch 1h.The pichia spp Sorbitol Solution USP that imports recombinant expression vector is screened under 30 ℃ of conditions with the YPD flat board that contains 0.1% Zeocin.
Select 6 positive bacterium colonies to be inoculated into respectively in the YPD liquid nutrient medium that 5ml contains 0.1% Zeocin, under 30 ℃, 250rpm condition, cultivate 24h; The centrifugal 5min of 4000rmp, abandons YPD substratum, adds 5ml YPG substratum, under 30 ℃, 250rpm condition, cultivates 24h; The centrifugal 5min of 4000rmp, abandons YPG substratum, adds 2ml BMMYC substratum, under 27 ℃, 225rpm condition, cultivates 48h, during every 12h, add 0.5-1.0% methyl alcohol; The centrifugal 5min of 4000rmp, collects supernatant.Carry out Westen Blot checking, prove and in supernatant, contain target protein, and then proof recombinant pichia yeast strain successfully constructs.
The recombinant pichia yeast strain successfully constructing is inoculated in 250ml YPD substratum, under 30 ℃, 250rpm condition, cultivates 24h; The centrifugal 5min of 4000rmp, abandons YPD substratum, adds 250ml YPG substratum, under 30 ℃, 250rpm condition, cultivates 24h; The centrifugal 5min of 4000rmp, abandons YPG substratum, adds 125ml BMMYC substratum, under 27 ℃, 225rpm condition, cultivates 48h, during every 12h, add 0.5% methyl alcohol; The centrifugal 5min of 4000rmp, collects supernatant liquor.
Containing the YPD flat board of 0.1% Zeocin, by following material, formed: 2.0g 100ml peptone, 1.0g 100ml yeast extract powder, 1.5g 100ml agar, 5ml 100ml 20% glucose, 0.1ml 100ml Zeocin.
The YPD liquid nutrient medium that contains 0.1% Zeocin is comprised of following material: 2.0g 100ml peptone, 1.0g 100ml yeast extract powder, 0.1ml 100ml Zeocin, 5ml 100ml 20% glucose.
YPD liquid nutrient medium is comprised of following material: 2.0g 100ml peptone, 1.0g 100ml yeast extract powder, 5ml 100ml 20% glucose.
YPG liquid nutrient medium is comprised of following material: 2.0g 100ml peptone, 1.0g 100ml yeast extract powder, 5ml 100ml 10% glycerine.
BMMYC liquid nutrient medium is comprised of following material: 2.0g 100ml peptone, 1.0g 100ml yeast extract powder, 10ml 100ml 10% TYR hydrolyzate, 10ml 100ml 5% methyl alcohol, 10ml 100ml phosphate buffered saline buffer, 10ml 100ml YNB, 200 μ l 100ml vitamin H.
The purifying of embodiment 3:UBC13 albumen.
Process supernatant liquor, in the supernatant liquor of processing, contain 0.3M NaCl, 0.05M NaH
2pO
4, 0.01M Imidazole, 0.01M Tris-HCl, volume is not enough uses ultrapure water polishing, then crosses 0.4 μ l filter.
Sample preparation liquid the first step purifying---cross Ni-NTA resin post (Quan Shijin, F0014,10ml).
1, cross at least 100ml balance liquid (0.3M NaCl, 0.05M NaH
2pO
4, 0.01M Imidazole, 0.01M Tris-HCl), balance Ni post.
2, cross sample preparation liquid, and collected the liquid (flowing through liquid) after Ni post.
3, cross 80ml washings (0.3M NaCl, 0.05M NaH
2pO
4, 0.01M Imidazole, 0.01M Tris-HCl), washing does not have the albumen of the upper Ni post of absorption, and minute 8 pipes are collected washings, every pipe 10ml.
4, cross 80ml elutriant (0.3M NaCl, 0.05M NaH
2pO
4, 0.5M Imidazole, 0.01M Tris-HCl), wash-out is adsorbed onto the target protein of Ni post, and minute 8 pipes are collected elutriant, every pipe 10ml.
5, cross 50ml 20% Alcohol Protection Ni post.
6, through polyacrylate hydrogel electrophoresis, verified Ni post result, and as shown in Figure 3, in expressing supernatant, flowing through liquid, elutriant the 2nd, 3 pipes, all detected expressing protein, wherein in elutriant the 2nd pipe, protein concentration is the highest.
Use 3.5kD dialysis tubing (spectrum, article No. 132725) through dialyzate (20mm Tris-HCl, 1mm EDTA, 5% glycerine) dialysis 8-12h in the albumen of the first step purifying.
The second step purifying of target protein---cross anion-exchange column.
1, cross at least 100ml balance liquid (20mm Tris-HCl, 1mm EDTA, 5% glycerine), balance anion exchange column.
2, cross the protein sample after dialysis, and collected the liquid after anion-exchange column, obtain albumen treatment solution.
3, cross 80ml washings (20mm Tris-HCl, 1mm EDTA, 5% glycerine), washing does not have the albumen of the upper anion-exchange column of absorption, and divides 2 pipes to collect, every pipe 40ml.
4, cross 80ml elutriant I (20mm Tris-HCl, 1mm EDTA, 5% glycerine, 100mm borax), wash-out is adsorbed onto the target protein of anion column, and minute 8 pipes are collected elutriant, every pipe 10ml.
5, cross 80ml elutriant II (20mm Tris-HCl, 1mm EDTA, 5% glycerine, 200mm borax), wash-out is adsorbed onto the target protein of anion column, and minute 8 pipes are collected elutriant, every pipe 10ml.
6, cross 80ml elutriant III (20mm Tris-HCl, 1mm EDTA, 1M NaCl), wash-out residue is adsorbed on all albumen on anion-exchange column, and minute 2 pipe collections, every pipe 40ml.
7, cross 50ml 20% Alcohol Protection anion column.
8, through polyacrylate hydrogel electrophoresis, verified anion column result, as shown in Figure 4, in sample preparation liquid, washings the 1st pipe, elutriant I 2-4 pipe, elutriant II 2-4 pipe, all detect expressing protein, wherein in elutriant I the 3rd pipe, protein concentration is the highest.
Concentrated and the preservation of UBC13 target protein.
Use the super filter tube of solution 3.5kD at 4 ℃, lower than centrifugal concentrating under the condition of 5000g in the UBC13 albumen that contains after second step purifying.
Albumen after concentrated is contained to the PBS dialyzate dialysis 8-12h of 5% glycerine with dialysis tubing (spectrum, the article No. 132725) warp with 3.5kD.
Dialyzate after concentrated is crossed to 0.22 μ m filter and inject aseptic EP pipe, prolonged preservation under-80 ℃ of conditions.
Concentrated effect before polyacrylate hydrogel electrophoresis checking UBC13 albumen-80 ℃ preservation, expressing protein concentrated effect as shown in Figure 5, every electrophoresis hole applied sample amount is followed successively by 1,2,4,6,8,10,12,14,16,18,20 μ l UBC13 protein samples, SDS-PAGE electrophoresis detection result shows: along with the rising of applied sample amount, the protein content detecting is also increasing gradually.
Claims (7)
1. expressing a recombinant yeast pichia pastoris for UBC13 albumen, is the pichia spp being transformed by recombinant expression vector, includes the nucleotide sequence of the UBC13 albumen shown in SEQ ID NO.1 in described recombinant expression vector.
2. recombinant yeast pichia pastoris according to claim 1, is characterized in that: described pichia spp is pichia spp X33.
3. recombinant yeast pichia pastoris according to claim 1, is characterized in that: the carrier that described recombinant expression vector is used is pwPICZalpha plasmid vector.
4. a method that builds arbitrary described recombinant yeast pichia pastoris in claims 1 to 3, comprises the steps:
1) synthetic UBC13 protein gene;
2) in the multiple clone site of plasmid vector, insert UBC13 protein gene, build recombinant expression vector;
3) described recombinant expression vector is imported in pichia spp, obtain the recombinant yeast pichia pastoris of expressing UBC13 albumen.
5. method according to claim 4, is characterized in that: described recombinant expression vector is used pwPICZalpha plasmid vector.
6. method according to claim 4, is characterized in that: described pichia spp is pichia spp X33.
7. the application of arbitrary described recombinant yeast pichia pastoris in producing UBC13 albumen in claims 1 to 3.
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| CN112877330A (en) * | 2020-03-02 | 2021-06-01 | 山东农业大学 | Method for efficiently expressing target gene in protoplast cell |
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| CN1322821A (en) * | 2000-05-09 | 2001-11-21 | 上海博德基因开发有限公司 | New polypeptide human ubiquitin conjugated enzyme 13 and its encoding polynucleotides |
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| CN101864426A (en) * | 2010-05-27 | 2010-10-20 | 陈世敏 | Method for preparing ubiquitin conjugated enzyme UbcH10 by using eukaryotic expression system |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112877330A (en) * | 2020-03-02 | 2021-06-01 | 山东农业大学 | Method for efficiently expressing target gene in protoplast cell |
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