CN110982839A - Method for biotin labeling protein at cellular level - Google Patents
Method for biotin labeling protein at cellular level Download PDFInfo
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- CN110982839A CN110982839A CN201911352980.0A CN201911352980A CN110982839A CN 110982839 A CN110982839 A CN 110982839A CN 201911352980 A CN201911352980 A CN 201911352980A CN 110982839 A CN110982839 A CN 110982839A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
Abstract
The invention discloses a method for biotin labeling protein at a cellular level, which comprises the following steps: 1) constructing biotin ligase plasmids and target protein plasmids with AVI-tag; 2) the cell density is 1.2X106cells with cell/mL above and the survival rate more than or equal to 95 percent are centrifuged, and the density is adjusted to 1.2 multiplied by 10 by using a fresh culture medium6cells/mL; adding biotin to the mixture to a final concentration of 50 μ M; 3) co-transfecting the cells obtained in the step 2) with the two plasmids constructed in the step 1); 4) and collecting cells or proteins after 2-5 days, and detecting the labeling condition of the cells. By the method, the marking efficiency of the cells reaches over 90 percent; the biotin labeling is carried out while the protein is expressed, so that the in vitro labeling link is saved, and the time of about 1 week is shortened in the process.
Description
Technical Field
The invention relates to the technical field of protein expression and purification, in particular to a method for biotin labeling a protein at a cellular level.
Background
Biotin, a small molecule naturally occurring in a living body, has a strong binding with high affinity and a multi-stage amplification effect with a Biotin-Avidin System (BAS) formed from Avidin, and is increasingly used in the fields of biomedicine and the like. After being activated, biotin can mark biomacromolecules such as proteins (antigens, antibodies) and the like, so that the bioactivity of the macromolecules is maintained, and the macromolecules have the advantages of high affinity, high sensitivity, strong specificity, good stability and the like of BAS. The method plays an irreplaceable role in the research of the location and detection of cells and antigens, antibody affinity screening and the like, and the production of high-quality biotin labeled protein is a great challenge at present.
Biotin Ligase (Biotin Ligase) is encoded by the birA gene and can activate Biotin to form biotinyl-5' -adenylate, and specifically transfer Biotin to a Biotin receptor tag protein (such as AviTag fusion protein) to biotinylate the tag protein, wherein Avi-tag is a short peptide tag consisting of 15 amino acid residues, and Biotin is linked to a lysine residue by Biotin Ligase in vivo or in vitro, thereby achieving biotinylation of the protein.
Currently, the method for labeling biotin on the market is generally to perform random labeling or site-directed labeling on in vitro purified proteins by using a commercial kit. However, such labeling methods tend to suffer from poor reproducibility, inefficient labeling, harsh reaction conditions, high cost, and the possibility of unpredictable effects on protein properties. Therefore, the development of a method for site-directed biotin labeling of biological macromolecules at the cellular level is very important for biomedical development.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for carrying out biotin labeling on protein at a cell level, so that the protein can be efficiently subjected to site-specific biotin labeling at the cell level.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for biotin labeling a protein at the cellular level comprising the steps of:
1) constructing biotin ligase plasmids and target protein plasmids with AVI-tag;
2) the cell density is 1.2X106cells with cell/mL above and the survival rate more than or equal to 95 percent are centrifuged, and the density is adjusted to 1.2 multiplied by 10 by using a fresh culture medium6cells/mL; adding biotin to the mixture to a final concentration of 50 μ M;
3) co-transfecting the cells obtained in the step 2) with the two plasmids constructed in the step 1);
4) and collecting cells or proteins after 2-5 days, and detecting the labeling condition of the cells. The labeling efficiency of the cells by this method reaches over 90%.
Specifically, in the step 2), the cells comprise 293F cells or CHO cells.
Specifically, in the step 2), the cell density is adjusted to 0.6-0.8 multiplied by 10 the day before transfection6cells/mL; on the day of transfection, F cell density reached 1.2X106About cells/mL, the activity rate is more than or equal to 95 percent.
Specifically, in the step 2), the centrifugation mode is centrifugation at 800rpm for 5 min.
Specifically, in the step 3), the mass ratio of the target protein plasmid to the biotin ligase plasmid is (8-10): 1.
Preferably, in the step 3), the mass ratio of the target protein plasmid to the biotin ligase plasmid is 9: 1.
Specifically, the protein includes cell surface membrane proteins and secreted proteins.
In one embodiment, the protein is a cell surface membrane protein. This cell surface membrane protein in said step 4), the cells or proteins were collected after 48 hours, and the labeling of the cells was examined by FACS.
In a specific embodiment, the protein is a secreted protein. The secreted protein was purified by collecting cell supernatants after 5 days of transient expression in said step 4), and biotin-labeling was performed by SDS-PAGE.
The invention provides a method for labeling cell surface membrane protein and secretory protein with biotin, which realizes the efficient site-specific biotin labeling of the cell surface membrane protein and secretory protein at the cell level.
Compared with the prior art, the invention has the following advantages:
1. the method of the invention leads the cell to carry out biotin labeling while expressing the protein, saves the in vitro labeling link and shortens about 1 week in the process.
2. The biotinylation is realized in cells through the reaction of enzyme and a substrate, the reaction condition is mild, the protein conformation can be maintained, and the labeling efficiency and the labeling specificity are greatly improved.
3. The method of the invention saves the cost of using the labeling kit.
Drawings
FIG. 1 is a flow chart of biotin labeling of the cell surface membrane protein of the present invention.
FIG. 2 is a flow chart of biotin labeling of the secreted protein of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely below, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Example one
1. Material preparation
1.1 Biotin configuration
Biotin powder was dissolved in DMSO at a concentration of 100mM and stored at-20 ℃.
1.2 plasmid preparation
Constructing a plasmid for expressing BirA and a plasmid for expressing a target protein with AVI-tag.
1.3 cell preparation
1) Rotating shaftThe day before staining, 293F cell density was adjusted to 0.6-0.8x106cells/mL。
2) On the day of transfection, 293F cells reached a density of 1.2X106cells/mL, with a viability of 95% or more, at which time the cells were centrifuged at 800rpm for 5min and the density was adjusted to 1.2X10 with fresh medium6cells/mL, volume 90% of the final volume. A Biotin stock was added thereto to give a final concentration of 50M.
2. Transfection (taking 20ml transfection system as an example)
2.1 labelling of cell surface Membrane proteins
1) 20mg of the plasmid (objective gene plasmid: 18mg, BirA plasmid: 2mg, mass ratio 9: 1).
2) Mu.l lipofectamine 2000 was diluted with 950. mu.l Opti-MEM.
3) Standing for 5min, mixing the two solutions, and standing for 20min to obtain liposome-DNA complex.
4) The liposome-DNA complex was added dropwise to the cells of adjusted density, and the cells were cultured on a shaker.
5) FACS detection of Biotin labelling efficiency after 48 h: detecting the expression level of the target protein by using a specific primary antibody, and selecting PE by using a secondary antibody; the level of Biotinylation was measured using Alexa647-SA at a dilution ratio of 1: 1000; finally, the rate of double positive is used as the marking efficiency of Biotin.
6) As a result: the labeling efficiency of the cells by this method reaches over 90%.
2.2 secreted protein markers
1) 20mg of the plasmid was diluted with 1ml of Opti-MEM (target gene: 18mg, BirA: 2mg in a mass ratio of 9: 1);
2) 50l lipofectamine 2000 was diluted with 950l Opti-MEM;
3) standing for 5min, mixing the two solutions, and standing for 20min to form liposome-DNA complex;
4) adding the liposome-DNA complex dropwise into the cells with adjusted density, and culturing the cells in a shaking table
5) After 5 days, the supernatant was collected and purified, and the labeling efficiency was checked by SDS-PAGE.
6) As a result: the labeling efficiency of the cells by this method reaches over 90%.
In summary, the above embodiments are merely preferred embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (11)
1. A method for biotin labeling a protein at the cellular level, comprising the steps of:
1) constructing biotin ligase plasmids and target protein plasmids with AVI-tag;
2) the cell density is 1.2X106cells with cell/mL above and the survival rate more than or equal to 95 percent are centrifuged, and the density is adjusted to 1.2 multiplied by 10 by using a fresh culture medium6cells/mL; adding biotin to the mixture to a final concentration of 50 μ M;
3) co-transfecting the cells obtained in the step 2) with the two plasmids constructed in the step 1);
4) and collecting cells or proteins after 2-5 days, and detecting the labeling condition of the cells.
2. The method of claim 1, wherein in step 2), the cells are 293F cells or CHO cells.
3. The method according to claim 1, wherein in the step 2), the cell density is adjusted to 0.6 to 0.8X10 the day before transfection6cells/mL; on the day of transfection, F cell density reached 1.2X106About cells/mL, the activity rate is more than or equal to 95 percent.
4. The method of claim 1, wherein the centrifugation is at 800rpm for 5 min.
5. The method according to claim 1, wherein the mass ratio of the target protein plasmid to the biotin ligase plasmid is (8-10): 1.
6. The method of claim 5, wherein the mass ratio of the target protein plasmid to the biotin ligase plasmid is 9: 1.
7. The method of claim 1, wherein the proteins comprise cell surface membrane proteins and secreted proteins.
8. The method of claim 7, wherein the protein is a cell surface membrane protein.
9. The method according to claim 8, wherein the cell surface membrane proteins are collected after 48 hours in step 4), and the labeling of the cells is detected by FACS.
10. The method of claim 7, wherein the protein is a secreted protein.
11. The method of claim 10, wherein the secreted protein is purified by collecting cell supernatants after 5 days of transient expression in step 4) and biotin-labeling is performed by SDS-PAGE.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103374068A (en) * | 2012-04-27 | 2013-10-30 | 中国科学院上海生命科学研究院 | Preparation method and application of novel monoclonal antibody |
| CN107207609A (en) * | 2014-11-20 | 2017-09-26 | 豪夫迈·罗氏有限公司 | Common light chain and application method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103374068A (en) * | 2012-04-27 | 2013-10-30 | 中国科学院上海生命科学研究院 | Preparation method and application of novel monoclonal antibody |
| CN107207609A (en) * | 2014-11-20 | 2017-09-26 | 豪夫迈·罗氏有限公司 | Common light chain and application method |
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