CN102010875A - Method for preparing biotinylated protein - Google Patents
Method for preparing biotinylated protein Download PDFInfo
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- CN102010875A CN102010875A CN200910192139XA CN200910192139A CN102010875A CN 102010875 A CN102010875 A CN 102010875A CN 200910192139X A CN200910192139X A CN 200910192139XA CN 200910192139 A CN200910192139 A CN 200910192139A CN 102010875 A CN102010875 A CN 102010875A
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Abstract
The invention relates to a method for preparing a biotinylated protein. The method comprises the following steps of: (a) structuring a protein expression vector having a multifunctional united label, wherein the united label at least comprises a label convenient to purify a recombination protein, a label comprising a specific protease cleavage site and a biotinylated label; (b) cloning a coding sequence of the target protein to the protein expression vector described in the step (a) and carrying out fusion expression on the united label with the interest protein to obtain the recombination protein which carries out biotinylation on the biotinylated label in the fusion protein internally or externally; and (c) adopting specific protease suitable for the united label to cut off the label convenient to purify the recombination protein in the fusion protein and cutting off the specific protease cleavage site in the united label so as to obtain the high-purity biotinylated protein. The invention has the advantages of integrated process, simple process, high biotinylated efficiency, no impact on the protein structure and activity by biotinylation, and the like.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to the preparation technology of biotinylation recombinant protein.
Background technology
Vitamin H can very closely combine with avidin (Avidin), Streptavidin (Streptavidin) specifically, each avidin or Streptavidin can be in conjunction with the vitamin Hs of 4 molecules, and avidity is extremely strong, avidin constant (K) is 1015L/mol, than the avidity between antigen and antibody (K=10.5～11L/mol) high at least 100 times, the two binding specificity height and good stability.Utilize this kind biotin-avidin system to have the affinity interaction of high specificity, can be with behind the vitamin H on the protein labeling (being biotinylated protein), can be by the specific combination of avidin/Streptavidin, thus realize that identification detects, application such as biomacromolecule catches, protein-interacting detection.Biotinylated protein has been widely used in the experimental study field of immunology, Celluar and Molecular Biology, for example in high throughput protein interacts screening, the AlphaScreen technology (www.perkinelmer.com) of finding broad application, as donor, the acceptor microballon then utilizes the albumin A (proteinA) of bead surface to fix another albumen to the Streptavidin that utilizes bead surface exactly biotinylated proteopexy.Under the irradiation of laser (wavelength is 680nm), photosensitizers on the donor microballon is more active free oxygen with the oxygen conversion in the surrounding environment, free oxygen diffuses to the acceptor microballon, with the chemoluminescence agent reaction on it, activated the fluorophor on the acceptor microballon, (transformation period of free oxygen is 4 μ Sec, and the diffusion length in solution is about 200nm to make it to send the fluorescence that wavelength is 520～620nm.If there is not interaction in biomolecules, free oxygen can't be diffused into the acceptor microballon, does not then have fluorescent signal and produces).
The method that prepare at present biotinylated protein matter has: (1) obtains biotinylated protein by chemical-biological element labelling method after with the target protein purifying, usually the carboxyl that utilizes vitamin H is the chemically modified derivative that can be made into various active groups in addition, carries out chemical reaction with target protein matter then.This kind method is because reaction conditions is more violent, and in the chemical process biotinylation process, some are the easy inactivation of albumen of sensitivity relatively; Simultaneously, because the activatory vitamin H does not have the ability of identification specified protein, thereby chemical process biotinylation right and wrong are special.(2) by biotinylation label and target protein amalgamation and expression, in vivo or external biotin label is carried out biotinylation, thus the biotinylation of realization recombinant protein.As form by intestinal bacteria vitamin H ligase enzyme (Biotin Ligase) activation vitamin H biotin acyl-5 '-adenylic acid (AMP), vitamin H is transferred to specifically on the Methionin of vitamin H acceptor label protein-AviTag, make Biotinization; Intestinal bacteria vitamin H acyl carrier protein (biotin carboxylcarrier protein; BCCP) be a subunit of intestinal bacteria CoA carboxylase; can under the catalysis of vitamin H synthase, on its C end Methionin, connect a vitamin H, make Biotinization.This species specific pointed decoration, little to proteic activity influence, product is single-minded, but this kind method must be carried out amalgamation and expression to target protein and biotin label (as AviTag etc.), carry out purifying then and obtain highly purified recombinant protein, but vitamin H acceptor label protein-AviTag can not be applicable to protein purification, and can't improve Recombinant Protein Expression efficient and solubility, often cause with the AviTag fusion rotein can not soluble-expression or be difficult to purifying.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of biotinylated protein matter, this method is easy and simple to handle, biotinylation efficient height, protein structure and activity are not influenced by biotinylation.
The preparation method of a kind of biotinylated protein matter of the present invention may further comprise the steps:
(a) make up a kind of protein expression vector with multi-functional associating label, described associating label comprises at least: be convenient to the label of recombinant protein purification, contain the label of differential protein restriction enzyme site, and the biotinylation label;
(b) encoding sequence of target protein is cloned in the described protein expression vector of step (a), described associating label and target protein carry out amalgamation and expression, and acquisition can be in vivo or external biotinylation label in the fusion rotein carried out biotinylated recombinant protein; And
(c) specific protease that adopts and unite label to adapt excises the label of being convenient to recombinant protein purification in the fusion rotein, and cuts off the differential protein restriction enzyme site in the associating label, obtains biotinylated protein matter.
According to the preparation method's of biotinylated protein matter of the present invention further feature, the described label of being convenient to recombinant protein purification comprises at least: six Histidines (6His) label, Thiadiazolidine isomerase (GST) label, maltose binding protein (MBP) label, Trx (Trx) label.
Further feature according to the preparation method of biotinylated protein matter of the present invention, the described label that contains the differential protein restriction enzyme site comprises at least: and little ubiquitin relevant modifications thing (Small Ubiqitin-related Modifer, SUMO), marmor erodens proteolytic enzyme (Tobacco etch virus Protease) recognition sequence, enteropeptidase (Enterokinase) recognition sequence, zymoplasm (Thrombin) recognition sequence, Xa factor (Factor Xa) recognition sequence.
Further feature according to the preparation method of biotinylated protein matter of the present invention; described biotinylation label comprises at least: AviTag (GLNDIFGAQKIEWHE) and derivative thereof, intestinal bacteria vitamin H acyl carrier protein (biotincarboxyl carrier protein; BCCP) and derivative, yeast acceptor peptide (yeast acceptor peptide, yAP) (TTNWVAQAFKMTFDP) and derivative thereof.
The present invention also provides a kind of protein expression vector, this carrier contains and has multi-functional associating label, and regulatable promotor, replication initiation, resistance screening identification element, described associating label comprises at least: the label of being convenient to recombinant protein purification, the label that contains the differential protein restriction enzyme site, and biotinylation label.
The present invention also provides a kind of test kit, comprising: foregoing protein expression vector, specific proteolytic enzyme, the biotinylation label can be carried out biotinylated cell or enzyme.
According to the further feature of test kit of the present invention, described specific proteolytic enzyme comprises at least: little ubiquitin relevant modifications thing proteolytic enzyme, marmor erodens proteolytic enzyme, enteropeptidase, zymoplasm, Xa factor.
According to the further feature of test kit of the present invention, describedly the biotinylation label can be carried out biotinylated cell and comprise at least: express the plain ligase enzyme of bacterium living beings cell, express the plain synthetic enzyme of bacterium living beings cell, express the cell of the plain ligase enzyme of yeast bio.
According to the further feature of test kit of the present invention, describedly the biotinylation label is carried out biotinylated enzyme comprise at least: the plain ligase enzyme of bacterium living beings, the plain synthetic enzyme of bacterium living beings, yeast bio ligase enzyme.
The invention has the advantages that: (1) utilizes biotin label, as AviTag, can be in vivo or external by the vitamin H ligase enzyme the efficient and biotinylated characteristic of high specific, carry out amalgamation and expression with target protein, the biotinylated protein that is obtained is to carry out biotinylation at the biotinylation label, rather than on target protein, carry out biotinylation, therefore very little to the activity and the structure influence of target protein; (2) utilize other label that the purification process of convenient and efficient is provided, and then utilize proteolytic cleavage to remove other label, only keep biotinylation label and target protein, thereby obtain biotinylated protein.The biotinylated protein mass-energy that obtains by preparation method of the present invention enough at utmost guarantees biotinylated homogeneity and proteic activity, and can directed be fixed on the magnetic bead surfaces of avidin bag quilt, during can be widely used in that immunodetection, mixture are caught, AlphaScreen etc. used.
Description of drawings
Fig. 1 is the schematic diagram that utilizes " associating label " preparation biotinylated protein;
Fig. 2 utilizes the proteic schema of " TrxHisSUMOAvi " biotinylated Avi-AML1 of preparation;
Fig. 3 is a pReceiver-B62 carrier collection of illustrative plates;
Fig. 4 shows that TrxHisSUMOAvi-AML1 albumen carries out the result that protein immunoblotting (Western Bolt) detects with anti-6 Histidine monoclonal antibodies (Anti-His) and Streptavidin-horseradish peroxidase (Strepavtidin-HRP) respectively;
Fig. 5 demonstration utilizes the proteic SDS-PAGE detected result of Ni-NTA affinitive layer purification TrxHisSUMOAvi-AML1;
Fig. 6 shows that TrxHisSUMOAvi-AML1 albumen behind the SUMO proteolytic cleavage, utilizes the Ni-NTA affinity chromatography, the proteic SDS-PAGE detected result of purifying Avi-AML1, and albumen shown in the arrow is to have excised the Avi-AML1 albumen behind the TrxHisSUMO among the figure;
Fig. 7 shows the proteic SDS-PAGE detected result of Avi-AML1 behind MonoQ ion exchange chromatography purifying.
Embodiment
The preparation method of a kind of biotinylated protein matter of the present invention as shown in Figure 1, may further comprise the steps:
(a) make up a kind of protein expression vector with multi-functional associating label, described associating label comprises at least: be convenient to the label of recombinant protein purification, contain the label of differential protein restriction enzyme site, and the biotinylation label;
(b) encoding sequence of target protein is cloned in the described protein expression vector of step (a), described associating label and target protein carry out amalgamation and expression, and acquisition can be in vivo or external biotinylation label in the fusion rotein carried out biotinylated recombinant protein; And
(c) specific protease that adopts and unite label to adapt excises the label of being convenient to recombinant protein purification in the fusion rotein, and cuts off the differential protein restriction enzyme site in the associating label, obtains biotinylated protein matter.
The present invention will be further described below in conjunction with specific embodiment, and purpose is content of the present invention and the protection domain of unrestricted invention.The test method of unreceipted actual conditions among the following embodiment, usually according to normal condition, as people such as Sambrook work, molecular cloning: test guide (New York, Cold Spring Habour Laboratory Press) condition described in, or carry out according to manufacturer's suggestion condition.Test required bacterial classification, plasmid, reagent etc. and be commercial prod, can be purchased by biological factory and buy acquisition.
Embodiment: the preparation of human AML1 biotinylated protein
The preparation method: the encoding sequence that will unite label TrxHisSumoAvi and human AML1 gene is built into the amalgamation and expression clone, and in expression in escherichia coli, the Avi label is by vitamin H ligase enzyme biotinylation in the bacterium.Recombinant protein removes the TrxHisSUMO label through behind the chromatography purification with the SUMO proteolytic cleavage, obtains biotinylated Avi-AML1 albumen (as shown in Figure 2).
Idiographic flow is as follows:
1. (the GenBank accession number: encoding sequence L21756) is cloned into and is obtained the pTrxHisSumoAvi-AML1 cloning by expression in plasmid pRecevier-B62 (Fig. 3) carrier, carries out sequence verification with people AML1 gene;
2. pTrxHisSumoAvi-AML1 is transferred in BL21 (DE3) bacterial strain, picking list bacterium colony incubated overnight in the LB substratum was transferred in the fresh LB substratum by 1% in second day, and 37 ℃ when being cultured to OD600 and being 0.6-0.8, concentration is the IPTG of 0.4mM in the adding, continues to cultivate 4hrs; Get 1ml bacterium liquid subsequently, centrifugal collection thalline, behind the broken cell, centrifugally get cleer and peaceful precipitation respectively and carry out SDS-PAGE, carry out Western Blot detection with anti-6 Histidine monoclonal antibodies (anti-His) and Streptavidin-horseradish peroxidase (Strepavtidin-HRP) respectively after changeing film, result all positive (Fig. 4) illustrates that the recombinant protein of expressing contains His simultaneously and by biotinylation;
(annotate: fusion rotein is when expressing, and contained vitamin H ligase enzyme can effectively be linked to vitamin H on the AviTag in the bacterial body, thereby recombinant protein is biotinylated albumen)
3. will contain pTrxHisSumoAvi-AML1BL21 (DE3) bacterial strain and carry out abduction delivering, centrifugal collection thalline, lysate (the 50mM Tris that adds 4ml by every gram bacterium, 50mM NaCl, 2.5% Glycerol, pH 8.0) resuspended thalline, ultrasonication cell: amplitude: 21% working hour: 5.5sec intermittent time: 9.9sec, each ultrasonic time: 5min repeats 3 times, weaken bubble to liquid till;
4. it is centrifugal to get bacterial cell disruption liquid, 12,000rpm 30min, 4 ℃, get supernatant to beaker, on one side stir, slowly add pulverous ammonium sulfate on one side, to concentration be 50%, continue to stir 2 hours,
5. centrifugal, 12,000rpm 30min, 4 ℃, abandon supernatant, precipitation is used 50mM Tris, 50mM NaCl, 2.5%Glycerol, pH8.0 dissolving;
6. centrifugal, 12,000rpm 30min, 4 ℃, after getting supernatant, be 0.45 μ m membrane filtration, carry out the Ni-NTA affinity chromatography then with the aperture, adopt the continuous gradient type of elution, balance liquid: 50mM Tris (pH8.0), 50mM NaCl, 2.5%Glycerol, elutriant: 50mM Tris (pH8.0), 50mM NaCl, 2.5%Glycerol, 500mM Imidazol presses the peak and collects, each is collected sample and carries out the SDS-PAGE electrophoresis detection, the result as shown in Figure 5, swimming lane 1 is the supernatant of lysis; Swimming lane 2 is protein standard references, from top to bottom band be respectively 100,80,70,60,50,40,30,25,20,15,10kDa; Swimming lane 3 and swimming lane 4 are penetrating of Ni-NTA chromatography; Swimming lane 5 is wash-outs of Ni-NTA chromatography to swimming lane 15;
7. the collection sample that will contain target protein merges and dialyses then to 50mM Tris (pH8.0), and 50mM NaCl is in the 2.5%Glycerol solution;
8. target protein is carried out the SUMO enzyme and cut, the TrxHisSumo part in the excision recombinant protein, the endonuclease reaction condition is according to the Coolcut of Genecopoeoia company
TMThe suggestion of protease product description is carried out;
9. carry out the Ni-NTA affinity chromatography then, balance liquid: 50mM Tris (pH8.0), 50mM NaCl, 2.5%Glycerol, elutriant: 50mM Tris (pH8.0), 50mM NaCl, 2.5% Glycerol, 500mM Imidazol, collection penetrates liquid, carry out the SDS-PAGE electrophoresis detection subsequently, the result as shown in Figure 6, swimming lane 1 is not carry out the protein sample that enzyme is cut; Swimming lane 2 and swimming lane 3 are the samples after cutting through enzyme; Swimming lane 4 is protein standard references, from top to bottom band be respectively 100,80,70,60,50,40,30,25,20,15,10kDa; Swimming lane 5 to swimming lane 9 is penetrating of Ni-NTA chromatography; Swimming lane 10 is wash-outs of Ni-NTA chromatography to swimming lane 15.The result shows, has obtained biotinylated Avi-AML1 albumen (albumen shown in the arrow is to have excised the Avi-AML1 albumen behind the TrxHisSUMO among Fig. 6);
10. the liquid that penetrates with the Ni-NTA affinity chromatography carries out the MonoQ ion exchange chromatography, be further purified, adopt the continuous gradient type of elution, balance liquid: 50mM Tris (pH8.0), 50mM NaCl, 2.5%Glycerol, elutriant: 50mMTris (pH8.0), 300mM NaCl, 2.5%Glycerol presses the peak and collects.
11. the MonoQ ion exchange chromatography is collected sample, SDS-PAGE detects, result as shown in Figure 7, and swimming lane 1 is biotinylated Avi-AML1 albumen, swimming lane 2 is protein standard references, from top to bottom band be respectively 100,80,70,60,50,40,30,25,20,15,10kDa.Subsequently albumen is dialysed to Storage Buffer:20mM Tris 50%Glycerol PH8.0.
Interpretation of result shows:
1, adopt TrxHisSumoAvi such " associating label " can be effectively and target protein ANML1 carry out amalgamation and expression;
2, adopt Stepavtidin-HRP antibody to carry out Western Blot and detect demonstration, fusion rotein is when expressing, and contained vitamin H ligase enzyme can effectively be linked to vitamin H on the AviTag in the bacterial body;
3, present embodiment only needs through two-step purifying: at first carry out the Ni-NTA affinity chromatography, carry out ion exchange chromatography subsequently, can obtain purity at 95% above biotinylated protein, and easy and simple to handle, efficient is high.
Claims (9)
1. the preparation method of a biotinylated protein matter is characterized in that, may further comprise the steps:
(a) make up a kind of protein expression vector with multi-functional associating label, described associating label comprises at least: be convenient to the label of recombinant protein purification, contain the label of differential protein restriction enzyme site, and the biotinylation label;
(b) encoding sequence of target protein is cloned in the described protein expression vector of step (a), described associating label and target protein carry out amalgamation and expression, obtain in vivo or external biotinylation label in the fusion rotein are carried out biotinylated recombinant protein; And
(c) specific protease that adopts and unite label to adapt excises the label of being convenient to recombinant protein purification in the fusion rotein, and cuts off the differential protein restriction enzyme site in the associating label, obtains highly purified biotinylated protein matter.
2. the preparation method of biotinylated protein matter according to claim 1 is characterized in that: the described label of being convenient to recombinant protein purification comprises at least: hexahistidine tag, Thiadiazolidine isomerase label, maltose binding protein label, Trx label.
3. the preparation method of biotinylated protein matter according to claim 1 is characterized in that: the described label that contains the differential protein restriction enzyme site comprises at least: little ubiquitin relevant modifications thing, marmor erodens protease recognition sequence, enteropeptidase recognition sequence, zymoplasm recognition sequence, Xa factor recognition sequence.
4. the preparation method of biotinylated protein matter according to claim 1 is characterized in that: described biotinylation label comprises at least: AviTag and derivative thereof, intestinal bacteria vitamin H acyl carrier protein and derivative thereof, yeast acceptor peptide and derivative thereof.
5. protein expression vector, contain and have multi-functional associating label, and regulatable promotor, replication initiation, resistance screening identification element, described associating label comprises at least: the label of being convenient to recombinant protein purification, the label that contains the differential protein restriction enzyme site, and biotinylation label.
6. test kit comprises: protein expression vector as claimed in claim 5, specific proteolytic enzyme, the biotinylation label can be carried out biotinylated cell or enzyme.
7. test kit according to claim 6 is characterized in that: described specific proteolytic enzyme comprises at least: little ubiquitin relevant modifications thing proteolytic enzyme, marmor erodens proteolytic enzyme, enteropeptidase, zymoplasm, the xa factor.
8. test kit according to claim 6 is characterized in that: describedly the biotinylation label can be carried out biotinylated cell and comprise at least: express the plain ligase enzyme of bacterium living beings cell, express the plain synthetic enzyme of bacterium living beings cell, express the cell of the plain ligase enzyme of yeast bio.
9. test kit according to claim 6 is characterized in that: describedly the biotinylation label is carried out biotinylated enzyme comprise at least: the plain ligase enzyme of bacterium living beings, the plain synthetic enzyme of bacterium living beings, yeast bio ligase enzyme.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864938A (en) * | 2014-03-24 | 2014-06-18 | 北京博翱泰生物技术有限公司 | Target-specificity double-mutant fused protein and preparation process thereof |
| CN106520835A (en) * | 2016-10-31 | 2017-03-22 | 山西医科大学第医院 | Slow virus single-plasmid in-vivo biotinylation vector and preparation method |
| CN106978429A (en) * | 2017-03-16 | 2017-07-25 | 中国人民解放军第四军医大学 | A kind of bovine enterokinase light chain load magnetic bead and its preparation method and application |
| CN107916243A (en) * | 2016-10-10 | 2018-04-17 | 中国科学院微生物研究所 | For handling microbial cell and albumen, the corresponding processing method and kit of heavy metal pollution |
| CN113061589A (en) * | 2021-03-17 | 2021-07-02 | 领诺(上海)医药科技有限公司 | Preparation method and application of cell strain for stably expressing biotin ligase Bir A |
| CN114891086A (en) * | 2022-06-01 | 2022-08-12 | 恺佧生物科技(上海)有限公司 | Biotin-labeled GDF15 renaturation method |
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2009
- 2009-09-08 CN CN200910192139XA patent/CN102010875A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103864938A (en) * | 2014-03-24 | 2014-06-18 | 北京博翱泰生物技术有限公司 | Target-specificity double-mutant fused protein and preparation process thereof |
| WO2015143581A1 (en) * | 2014-03-24 | 2015-10-01 | 北京博翱泰生物技术有限公司 | Target-specific double-mutant fusion protein and preparation process therefor |
| CN107916243A (en) * | 2016-10-10 | 2018-04-17 | 中国科学院微生物研究所 | For handling microbial cell and albumen, the corresponding processing method and kit of heavy metal pollution |
| CN106520835A (en) * | 2016-10-31 | 2017-03-22 | 山西医科大学第医院 | Slow virus single-plasmid in-vivo biotinylation vector and preparation method |
| CN106978429A (en) * | 2017-03-16 | 2017-07-25 | 中国人民解放军第四军医大学 | A kind of bovine enterokinase light chain load magnetic bead and its preparation method and application |
| CN106978429B (en) * | 2017-03-16 | 2019-11-15 | 中国人民解放军第四军医大学 | A kind of bovine enterokinase light chain-loaded magnetic beads and its preparation method and application |
| CN113061589A (en) * | 2021-03-17 | 2021-07-02 | 领诺(上海)医药科技有限公司 | Preparation method and application of cell strain for stably expressing biotin ligase Bir A |
| CN114891086A (en) * | 2022-06-01 | 2022-08-12 | 恺佧生物科技(上海)有限公司 | Biotin-labeled GDF15 renaturation method |
| CN114891086B (en) * | 2022-06-01 | 2024-03-26 | 恺佧生物科技(上海)有限公司 | Renaturation method of biotin-marked GDF15 |
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Application publication date: 20110413 |