CN101921819A - Expression and purification system using recombinant protein as label - Google Patents
Expression and purification system using recombinant protein as label Download PDFInfo
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- CN101921819A CN101921819A CN2009100529993A CN200910052999A CN101921819A CN 101921819 A CN101921819 A CN 101921819A CN 2009100529993 A CN2009100529993 A CN 2009100529993A CN 200910052999 A CN200910052999 A CN 200910052999A CN 101921819 A CN101921819 A CN 101921819A
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Abstract
The invention belongs to the technical field of biology and relates to a protein expression and purification system, in particular to an expression and purification system using a recombinant protein as a label. The protein expression and purification system is characterized in that the N end of a target protein is connected with a novel fluorescent protein sGFP (Green Fluorescent Protein) label, and the C end of the target protein is connected with histidine His peptide and is added with a polynucleotidase cutting site, thereby the protein is fused and expressed in full length; and meanwhile, a protease cutting site can be added between the target protein and the label as required. The protein expression efficiency of the system can be improved so that the yield of the target protein is improved, and the full-length protein is green in visible light so that the protein expression effect and a protein purification process can be monitored; and people can monitor the expression efficiency, the breaking degree and the purification process of the protein through naked eyes.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of protein expression and purification system, especially a kind of is the expression and purification system of label with the recombinant protein, and this expression and purification system can improve the target protein productive rate, implement monitoring protein expression effect, purge process.
Background technology
The production of known protein matter, separation and purifying have consequence in life science and polypeptide drug production.Searching makes the visual controlled good method that proteinic output improves, makes that production process and purge process become, and is one of research emphasis of this area researchist.Widespread use at present be that the used label of sophisticated prokaryotic expression system has maltose binding protein label (MBP), Thiadiazolidine isomerase (GST) label, Histidine His label etc., these labels are improving proteic solubleness, are helping to have shown certain effect aspect the protein folding.This main working method of expression and purification system comprises: the fusion fragment of design recombinant protein, structure has the segmental carrier of purpose, changes escherichia coli expression over to, roughing out, chromatography purification and the checking of race protein electrophoresis.
But practice shows that still there are the following problems for present technology: to albumen whether express, whether how, whether expression amount forms inclusion body precipitation, exist situation such as disconnected expression can not have one to get information about; When protein purification, the trend of target protein is known nothing simultaneously, can only have been judged whether the protein stream mistake from peak value; Whole process is finished in camera bellows, needs a large amount of financial resources of cost and material resources to go check or the like.Therefore the label that uses in the protein purification system of the prior art situation of indicating target albumen in expression, purifying in real time still, protein purification is long experimental period, a little less than the controllability.
Summary of the invention
The objective of the invention is the problem that runs in the present protein expression purifying for solving, providing a kind of is the expression and purification system of label with the recombinant protein.This expression and purification system can improve protein expression efficient, real-time follow-up protein expression purifying situation.
Particularly, the invention provides a kind of two labels " sandwich assay " protein expression and purification system based on fluorescin.
It is that label has made up two label " sandwich assay " protein expression system that the present invention adopts novel fluorescence albumen and His small peptide, it is characterized in that the two ends of target protein are connected label, connect a novel protein sGFP label at the N end, connect a Histidine His small peptide at the C end, and be added with the polynucleotide restriction enzyme site, albumen total length amalgamation and expression at fluorescin label two ends and target protein fragment two ends.The present invention can add protease cutting site simultaneously as required between target protein and label, the His label can shorten or prolong according to the purifying needs.
Among the present invention, target protein connected carry out amalgamation and expression behind the different labels and express, carry out purifying with affinity chromatography, wherein: fluorescin (superfold Green Fluorescence Protein, sGFP) be label one, Histidine His joint is a label two, is built into " sandwich assay " protein expression and purification system.
Among the present invention, fluorescin sGFP on target protein N termination, the small peptide of His6 and a terminator codon on target protein C termination, recombinant protein carries out the total length amalgamation and expression, and with Ni post affinitive layer purification.
Among the present invention, between target protein and sGFP, between target protein and His small peptide, has the polynucleotide sequence restriction enzyme site.
Among the present invention, between target protein and sGFP, between target protein and His small peptide, has insertable protease cutting site.
Among the present invention, His small peptide joint has different amino acid numbers.
The fusion rotein N end of vector expression of the present invention is a novel fluorescence albumen sGFP, this fluorescin self property is stable, the solubleness height, and can help the fusion rotein of its C end to carry out correct folding and expression, improve the solubility of target protein, be suitable as the albumen fusion tag.Simultaneously, sGFP excites green glow under visible light, therefore can only monitor proteic expression efficiency, breaking degree and purifying flow process by naked eyes to the whole process visual control when protein expression and purifying.
Among the present invention, provide to have this pair label " sandwich assay " recombinant protein fragment carrier pT7His, wherein, the open open reading-frame (ORF) orf that has host to discern on the recombinant protein fragment carrier pT7His, the AMP resistance marker screens, restriction endonuclease sites BamHI, NdeI, XhoI, EcoI, SamI, HindIII, and one section His small peptide gene.
Among the present invention, adopt the restriction enzyme zymotechnic, it can cut the site at specific nucleotide sequence place, helps to add or remove needed fragment.Designed the polynucleotide restriction enzyme site in the segmental both sides of fluorescin, can help replacement fluorescin.The present invention joins the left end of described carrier with the dna sequence dna of sGFP fluorescin, makes it can preferentially express the fluorescin with luminescent properties, makes whole expression and purification system reach visual effect in the very first time.
The present invention has designed a plurality of Nucleotide restriction enzyme sites in purpose fragment both sides, can insert the proteic fragment of various objectives as required, and is convenient to can carry out large-scale batch operation to the segmental replacement of target protein in commercial production.
Among the present invention, described His label has been placed on the end of recombinant protein full length fragment, and promptly the C end makes and have only when this recombinant protein is total length expressed to have the His label; Those disconnected expressed proteins then do not have the His label, then can obtain separating by nickel post affinity chromatography, and in effluent liquid, judge the disconnected expression of recombinant protein according to color.
Among the present invention, can shorten or prolong the amino acid number of Hi s small peptide as required, thus the effect of optimized Separation purifying.
Among the present invention, when being assembled into carrier, target protein designed protease cutting site at its C end, behind expression and purification with fluorescin label excision, the final protein product that obtains no macromolecule label, can satisfy the commercial production purpose complete, without any the protein product of joint.
The present invention is novel fluorescence albumen label sGFP on the gene fragment N of target protein termination, adds protease cutting site between fluorescin label and target protein.SGFP brightness height, physico-chemical property is stable, make the operator to judge whether target protein expresses by color, its expression degree how, in purge process, show the trend of combining target protein in real time, collect the elutriant that target protein is arranged according to color, need not can carry out subsequent experimental, reduce experimental implementation difficulty and experiment fees through electrophoresis step.SGFP also can help the folding of its C end fusion rotein simultaneously, can improve its folding efficiency and solvability for low expression, easy sedimentary albumen.Protease cutting site can be removed macromole fluorescin label, obtains not have the complete target protein of label, improves business-like relevance factor.His label on target protein fragment C termination is used for the affinity chromatography system of nickel post, is convenient to the purifying of recombinant protein.When containing the sample upper prop of target protein, disconnected expressed proteins since do not have the His label make that segment table reaches albumen can not upper prop, in effluent liquid, can indicate the fracture expression of indicator protein because of there being the fluorescin label to die.The present invention has designed a plurality of Nucleotide restriction enzyme sites in this carrier system, convenient different user selects for use and is familiar with or the processing of recombinating of the enzyme of preference, and therefore proteic replacement makes things convenient for to a large amount of various objectives, is applicable to broad scale research and production.
The invention has the beneficial effects as follows:
1) fluorescin brightness height, and stable, help visual.
2) fluorescin stable in properties helps foldingly, can improve the target protein lyotropy.
3) the fracture expression of two labels " sandwich assay " design indicator protein.
4) the His joint length can be adjusted arbitrarily according to purification condition.
5) target protein replacement, simple operation are convenient in the design of polynucleotide restriction enzyme site.
6) monitoring is in real time saved time and funds.
7) protease cutting site helps to commercially produce target protein.
8) expression and purification system maturation is easy to large-scale promotion.
9) but mass-producing, industrialization, batch operation brings considerable economic benefit.
Be that this further specifies this experiment is novel with experimental example in conjunction with the accompanying drawings below.
Description of drawings
Fig. 1 is a vector construction synoptic diagram of the present invention, the full length DNA segment contents that has shown recombinant protein, wherein, the grey vertical line is represented restricted Nucleotide restriction enzyme site, the dna fragmentation of white expression fluorescin label, the small peptide of left side grey direction indication protease cutting site, the dna fragmentation of the circular expression of Dark grey target protein, the right side grey is square to be the dna fragmentation of His label.
Fig. 2 is the standard synoptic diagram with this pair label " sandwich assay " recombinant protein fragment carrier pT7His,
Wherein, the open open reading-frame (ORF) orf that has host to discern on the recombinant protein fragment carrier pT7His, the AMP resistance marker screens, restriction endonuclease sites BamHI, NdeI, XhoI, EcoI, SamI, HindIII, and one section His small peptide gene.
Fig. 3 changes the carrier that structure is finished in the intestinal bacteria over to screen, and gets correctly to be cloned in and expresses example in the automatic induction culture medium.
Fig. 4 is that the albumen that has a fluorescence labels can full-rangely show comprehensive in receiving bacterium, roughing out process,
Wherein, be centrifugal receipts bacterium, resuspended thalline, centrifugation and centrifugal supernatant from left to right.
The synoptic diagram that the label target protein moved towards in real time when Fig. 5 and Fig. 6 were the chromatography column separation and purification,
Wherein, Fig. 5 Oxford gray is partly represented the target protein of hanging column, shows the link that target protein carries out on stream, disconnected expression of albumen and proteic actual expression amount; Before the first from left is last sample among Fig. 6; The second from left is the effluent liquid of hanging column not, and no light tone can judge that the disconnected expression of this albumen is lower; Right two effluent liquid for the NiC cleaning illustrate that two label recombinant protein hanging columns are effective; Right one is the sample behind the NiB wash-out.
Fig. 7 combines effect and wash-out result showing during with DEAE ion exchange column gradient elution.
Fig. 8 is to use the enzyme activity determination result of the TEV enzyme of this pair label " sandwich assay " system.
Embodiment
Add restriction enzyme site according to the target protein dna sequence dna application round pcr that will study or produce at target protein sequence two ends, and adding protease cutting site at the N end as required, be used to remove joint.
In conjunction with shown in Figure 2, the target protein fragment behind the PCR is inserted in two label " sandwich assay " carrier systems.
The carrier that structure is finished changes in the intestinal bacteria and screens, and gets correctly to be cloned in the automatic induction culture medium and expresses.Shown in Figure 3, used whether to express albumen after this pair label " sandwich assay " system and had indicative function, wherein shown the protein expression that has fluorescence labels, be light tone, from color tangible protein expression is arranged as can be seen, and do not have the expression system of fluorescence labels, then can not infer the protein expression what state.
In conjunction with shown in Figure 4, the present invention has observed the albumen that has fluorescence labels in the whole process of receiving bacterium, roughing out process, and the result shows that centrifuged deposit has only dark color, has not had light tone, illustrates brokenly that bacterium is complete.
Label of the present invention can show that when the chromatography column separation and purification target protein moves towards (Fig. 5 and Fig. 6) in real time, the operator is according to colour-change, can judge the target protein of institute's hanging column, understand the link that target protein carries out on stream, can effectively check simultaneously the albumen expression that breaks, can also be from the actual expression amount of the color guestimate target protein of the finished product.All right basis is the effluent liquid of the no light tone of hanging column not, and it is lower to judge the disconnected expression of albumen; According to the effluent liquid that NiC cleans, it is effective to judge two label recombinant protein hanging columns.
Showing during with DEAE ion exchange column gradient elution combines effect and the wash-out result shows, under the experiment condition that does not have the ultraviolet watch-dog, can according to the color in every pipe determine target protein be present in which pipe and concentration how, it is high more that color is deeply felt bright this tubulin concentration more, can correspondingly collect target protein wherein easily.The present invention measures the enzyme work of the TEV enzyme that uses this pair label " sandwich assay " system, compares with traditional MBP label, and experimental result shows that the present invention is to the not influence of living of proteolytic enzyme enzyme.
Claims (5)
1. one kind is the expression and purification system of label with the recombinant protein, it comprises, target protein connected carry out amalgamation and expression behind the different labels and express, carry out purifying with affinity chromatography, it is characterized in that: the two ends of described target protein are connected to label, its N end connects fluorescin (sGFP) label, and the C end connects Histidine His small peptide label, is built into " sandwich assay " protein expression and purification system.
2. expression and purification according to claim 1 system, it is characterized in that: described target protein C termination has small peptide and terminator codon of His6, and recombinant protein carries out the total length amalgamation and expression, and with Ni post affinitive layer purification.
3. expression and purification according to claim 1 system is characterized in that: between described target protein and fluorescin (sGFP), have the polynucleotide sequence restriction enzyme site between described target protein and His small peptide.
4. expression and purification according to claim 1 system is characterized in that: between described target protein and fluorescin (sGFP), have insertable protease cutting site between described target protein and His small peptide.
5. expression and purification according to claim 1 system, it is characterized in that: described His small peptide joint has the different aminoacids number.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312096A (en) * | 2017-07-17 | 2017-11-03 | 武汉博欧特生物科技有限公司 | Recombinant protein and its application for detecting the tri-methylated modification in histone site |
| CN109971776A (en) * | 2017-12-28 | 2019-07-05 | 中粮集团有限公司 | Method for purifying proteins based on light cutting motif |
| CN115074336A (en) * | 2022-06-29 | 2022-09-20 | 电子科技大学 | Protein with phospholipid flippase activity and purification method thereof |
| CN117659212A (en) * | 2023-12-11 | 2024-03-08 | 吉林省中科康的科技有限公司 | Fusion protein of epidermal cell growth factor and preparation method and application thereof |
-
2009
- 2009-06-12 CN CN2009100529993A patent/CN101921819A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107312096A (en) * | 2017-07-17 | 2017-11-03 | 武汉博欧特生物科技有限公司 | Recombinant protein and its application for detecting the tri-methylated modification in histone site |
| CN107312096B (en) * | 2017-07-17 | 2019-12-20 | 湖北大学 | Recombinant protein for detecting trimethylation modification of histone locus and application thereof |
| CN109971776A (en) * | 2017-12-28 | 2019-07-05 | 中粮集团有限公司 | Method for purifying proteins based on light cutting motif |
| CN109971776B (en) * | 2017-12-28 | 2021-04-09 | 中粮集团有限公司 | Protein purification method based on photocleavage motif |
| CN115074336A (en) * | 2022-06-29 | 2022-09-20 | 电子科技大学 | Protein with phospholipid flippase activity and purification method thereof |
| CN115074336B (en) * | 2022-06-29 | 2024-03-22 | 电子科技大学 | Protein with phospholipidosis enzyme activity and purification method thereof |
| CN117659212A (en) * | 2023-12-11 | 2024-03-08 | 吉林省中科康的科技有限公司 | Fusion protein of epidermal cell growth factor and preparation method and application thereof |
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Application publication date: 20101222 |