CN108178790B - Method for separating cell membrane to screen transmembrane protein by using DNA coding compound library - Google Patents
Method for separating cell membrane to screen transmembrane protein by using DNA coding compound library Download PDFInfo
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- CN108178790B CN108178790B CN201810050638.4A CN201810050638A CN108178790B CN 108178790 B CN108178790 B CN 108178790B CN 201810050638 A CN201810050638 A CN 201810050638A CN 108178790 B CN108178790 B CN 108178790B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/726—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Abstract
The invention discloses a method for separating cell membranes so as to screen a transmembrane protein by a DNA coding compound library, which comprises the following steps: 1) physically disrupting the cells; 2) performing a first centrifugation cycle to separate soluble material and cell membranes and precipitate insoluble cellular structures; 3) carrying out high-speed centrifugation of a second round; 4) verifying the state of the separated cell membrane under a microscope; 5) and (3) screening a DNA coding compound library for transmembrane proteins by using cell membranes as carriers. The invention avoids the problem that the transmembrane protein loses the original structure in the purification process and simultaneously avoids the problem that the DNA coding compound library screened by living cells can not enter cells. Compared with the traditional high-throughput screening, the method improves the throughput of transmembrane protein drug screening, and simultaneously can improve the screening efficiency and reduce the screening cost.
Description
Technical Field
The invention belongs to the field of drug screening, and particularly relates to a method for screening a DNA coding compound library, in particular to a method for separating cell membranes so as to screen a transmembrane protein through the DNA coding compound library.
Background
As a novel small molecule drug screening method, the DNA coding compound library can greatly improve the flux and speed of drug screening, reduce the cost of manpower and material resources and improve the screening efficiency. Transmembrane proteins are known to be difficult to purify and maintain the original structure, so that screening methods for transmembrane proteins using compounds encoded by DNA are very few, and the prior art has no method for comprehensively screening a library of compounds encoded by DNA while maintaining the structure of transmembrane proteins. At present, only one affinity screening method using living cells as carriers exists. The limitation of this approach is that DNA-encoded compounds, due to molecular size and charge, are directed only to the cell surface and do not efficiently cross the cell membrane into the cell interior, thus failing to screen for the intracellular region of the transmembrane protein. In addition, this method requires a high expression level of the protein, and consequently, the signal-to-noise ratio (signal-to-interference ratio) is low, and it is difficult to obtain effective data. Alternatively, transmembrane proteins are purified and screened, which results in proteins that have difficulty maintaining their structure and biological activity and low success rates for obtaining effective compounds.
Chinese patent application CN1646917A discloses a method for identifying compounds that interact with transmembrane proteins by separating cell membrane components to determine the level of transmembrane proteins containing nuclear localization sequences in the cell membrane and confirming the interaction of candidate compounds with transmembrane proteins by changes in the level of transmembrane proteins. The method is used as a verification mode for identifying whether the screened compound has an effect or not by aiming at the traditional high screening form, and cannot improve the screening flux and reduce the screening cost.
Therefore, there is a need to develop a method for screening a library of transmembrane proteins for DNA-encoded compounds that overcomes the above-mentioned deficiencies.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for separating cell membranes so as to screen a DNA coding compound library for transmembrane proteins, and in order to solve the problems in the prior art, the method utilizes a physical method to break cells and separate out cell membrane parts so as to keep the original structure of the proteins as much as possible, and is used for screening the DNA coding compound library so as to effectively screen drugs aiming at extracellular and intracellular regions of the transmembrane proteins.
In order to solve the technical problems, the invention adopts the technical scheme that:
a method of separating cell membranes for screening a library of transmembrane proteins for DNA-encoded compounds, comprising the steps of:
(1) physically disrupting the cells;
(2) performing a first centrifugation cycle to separate soluble material and cell membranes and precipitate insoluble cellular structures;
(3) performing a second round of high speed centrifugation to precipitate cell membranes from soluble cellular material;
(4) verifying the state of the separated cell membrane under a microscope;
(5) and (3) screening a DNA coding compound library for transmembrane proteins by using cell membranes as carriers.
As a preferred embodiment of the present invention, in step (1), the physical methods include homogenerizer (homogenizer, high speed stirrer), bead beater, and sonication (sonication).
As a preferable technical scheme of the invention, the following steps are added before the step (1): seeding the cells on a culture plate to grow a uniform monolayer of cells; cells were scraped into buffer with a spatula.
As a preferred embodiment of the present invention, the buffer solution comprises 20mM HEPES (hydroxyethylpiperazine ethanesulfonic acid), 250mM Sucrose, 10mM KCl, 2mM MgCl21mM EDTA (ethylenediaminetetraacetic acid), 1mM EGTA (ethylene glycol bis (2-aminoethylether) tetraacetic acid), 1mM DTT (dimercaptothreitol) and protease inhibitor cocktail (protease inhibitor cocktail) were added prior to use, pH 7.4.
As a preferred technical scheme of the invention, in the step (2), the first round of centrifugation is performed at the centrifugal force of 10,000g for 10 minutes at the temperature of 4 ℃.
In a preferred embodiment of the present invention, in step (3), the second round of high speed centrifugation is performed at a centrifugal force of 100,000g for 1 hour at a temperature of 4 ℃.
As a preferred technical scheme of the present invention, in the step (5), the screening process specifically comprises in sequence: cleaning, heating and high-speed centrifuging; in the screening process, high-speed centrifugation is used to separate soluble and cell membrane components so as to separate compounds which are not combined with protein; denaturing the protein by heating, allowing the bound compound to fall off the protein, into solution, and into the next round of screening; in total, one to three rounds of selection were performed until the total amount of extracted DNA-encoding compound was at 1X107To 1X109Within the range.
As a preferable technical scheme of the invention, in the step (5), the high-speed centrifugation is carried out, wherein the speed is 100,000g of centrifugal force, the time is 45 minutes, and the temperature is 4 ℃; the heating temperature is 80 ℃, and the heating time is 10 minutes.
As a preferable technical solution of the present invention, in the step (5), the cleaning specifically includes: adding the DNA coding compound library into a screening solution containing cell membranes, reacting, centrifuging, and washing with the screening solution; the screening medium consisted of 50mM Tris-HCl (Tris HCl), 150mM NaCl, 0.1% v/v Tween20 (Tween 20), 250mM Sucrose (Sucrose), 0.5mM EDTA, 1mM DTT (dimercaptothreitol), 1mg/mL fish sperm DNA (protamine DNA), 1mg/mL BSA (bovine serum albumin), pH 7.5.
As a preferable technical scheme of the invention, the following steps are added after the step (5):
(6) after a total of 1-3 rounds of screening of step (5), performing second-generation sequencing on the results to decode the structure of the compound;
(7) functional tests are carried out to verify the function of the compound.
Compared with the prior art, the invention has the beneficial effects that: the invention provides an effective screening method aiming at transmembrane proteins, which avoids the problem that the transmembrane proteins lose the original structure in the purification process and simultaneously avoids the problem that the DNA coding compound library screened by living cells cannot enter cells. The method can effectively apply the screening of the DNA coding compound library to the transmembrane protein target, the separated cell membrane is used for preparing the target protein before screening, the cell membrane is used as a carrier for fixing the target protein in the method to play a role, compared with the traditional high-throughput screening, the method improves the flux of screening transmembrane protein drugs, increases the flux from 1000 ten thousand compounds to more than 100 hundred million compounds, and can cover larger structural space. Meanwhile, the screening efficiency can be improved and reduced to 3-6 months from 9-18 months. The screening cost, including the cost of manpower and material, is reduced.
Drawings
The invention is further illustrated with reference to the following figures and examples.
FIG. 1 is a schematic flow diagram of the process of the present invention.
FIG. 2 is a graph showing the results of qPCR in example 1 of the present invention.
FIG. 3 is a graph showing the results of data analysis of the sequencing results in example 2 of the present invention.
FIG. 4 is a graphical representation of the IC50 results for Compound A of example 2 of the present invention.
Detailed Description
The present invention will now be described in further detail with reference to the accompanying drawings. These drawings are simplified schematic views illustrating only the basic structure of the present invention in a schematic manner, and thus show only the constitution related to the present invention.
Example 1: screening for positive compounds at the ETa receptor to confirm feasibility of the method
Firstly, background: ETa (receptor) belongs to GPCR, and is a protein with multiple transmembrane domains, which is difficult to purify while maintaining biological activity and structure
Secondly, the implementation method comprises the following steps:
as shown in fig. 1, the method specifically includes the following steps:
1. seeding cells expressing ETa receptor in T75 flasks to grow a uniform monolayer of cells;
2. cells were scraped into 3 ml of buffer with a spatula;
3. the buffer component was 20mM HEPES (hydroxyethylpiperazine ethanesulfonic acid), 250mM Sucrose, 10mM KCl, 2mM MgCl21mM EDTA (ethylene diamine tetraacetic acid), 1mM EGTA (ethylene glycol bis (2-aminoethylether) tetraacetic acid), 1mM DTT (dimercaptothreitol) and protease inhibitor cocktail (protease inhibitor cocktail) added prior to use, pH 7.4;
4. the cells were broken by back-and-forth aspiration 10 times with 5mL 27Gauge diameter needle tubing (Gauge is the unit of needle diameter);
5. the disrupted cells were placed on ice for 20 minutes;
6. a first centrifugation round was performed to separate out soluble material and cell membranes, precipitating insoluble cellular structures: centrifuging at 4 deg.C for 10 min with a centrifugal force of 10,000g to remove the pellet (containing nuclei, mitochondria) and retain the supernatant (containing cytoplasm and cell membrane);
7. transferring the supernatant to a new tube, and performing a second round of high speed centrifugation with a high speed centrifuge for 1 hr (centrifugal force of 100,000g, 4 deg.C), wherein the cell membrane is heavy and can be precipitated from soluble cell material;
8. the supernatant was removed and the pellet was washed through a 25 gauge diameter needle using 2 ml of buffer;
9. verifying the state of the separated cell membrane under a microscope; then centrifuging for 45 minutes again (100,000 g, 4 ℃), the cell membrane precipitated during the washing process is broken up, and the cell membrane needs to be centrifuged again to precipitate again;
10. removing the supernatant, and adding 3 ml of screening solution;
11. the screening medium consisted of 50mM Tris-HCl (Tris-HCl), 150mM NaCl, 0.1% v/v Tween20 (Tween 20), 250mM Sucrose (Sucrose), 0.5mM EDTA, 1mM DTT (dimercaptothreitol), 1mg/mL fish sperm DNA (protamine DNA;), 1mg/mL BSA (bovine serum albumin), pH 7.5;
12. adding about 1nmol of a DNA-encoding compound library (containing 50,000 compound structures) and 2pmol of a positive compound BQ-123 (enrichment degree of 100: 1) ligated with a DNA sequence to a cell membrane-containing screening solution;
13. reacting on a shaker at 4 ℃ overnight;
14. centrifugation for 45 minutes (100000 g of centrifugal force, 4 ℃);
15. removing the supernatant, washing the precipitate with 5ml of screening solution;
16. three washes in total, 10 minutes each, during which 45 minutes (100, 000g, 4 ℃) are centrifuged;
17. the remaining cell membranes were heated with 3 ml of the selection solution at 80 ℃ for 10 minutes;
18. centrifugation for 45 minutes (100000 g, 4 ℃);
19. the supernatant is reserved;
20. after each round of screening, approximately 20 μ L of supernatant was retained for qPCR to determine the enrichment of positive compound BQ-123;
21. one to three rounds of screening were performed until the enrichment degree of the positive compound BQ-123 reached 2000:1 or more;
22. the enrichment degree of the positive compound BQ-123 was determined by qPCR for the products after each round of screening. The qPCR results are schematically shown in figure 2. FIG. 2 shows the degree of enrichment of BQ-123 relative to compounds in the pool of other DNA-encoding compounds after two rounds of screening, 100:1 before screening, one round of screening increasing to 1000: 1, after two screening rounds, the ratio is increased to 2500:1, and the standard of more than 2000:1 is achieved, so that the screening is considered to be successful so far.
Example 2: screening of the V1a receptor for libraries of DNA-encoding compounds
Firstly, background: the V1a receptor belongs to GPCR, is a protein with multiple transmembrane domains, is difficult to purify and maintains biological activity and structure
Secondly, the implementation method comprises the following steps:
as shown in fig. 1, the method specifically includes the following steps:
1. growing a uniform monolayer of cells from cells expressing the V1a receptor on a 100mm plate;
2. scraping into 2 ml of buffer;
3. the buffer component was 20mM HEPES (hydroxyethylpiperazine ethanesulfonic acid), 250mM Sucrose, 10mM KCl, 2mM MgCl21mM EDTA (ethylene diamine tetraacetic acid), 1mM EGTA (ethylene glycol bis (2-aminoethylether) tetraacetic acid), 1mM DTT (dimercaptothreitol) and protease inhibitor cocktail (protease inhibitor cocktail) added prior to use, pH 7.4;
4. the cells were broken by back-and-forth aspiration 10 times with a 5mL 27gauge needle;
5. the disrupted cells were placed on ice for 20 minutes;
6. a first centrifugation round was performed to separate out soluble material and cell membranes, precipitating insoluble cellular structures: centrifuging at 4 deg.C for 10 min with a centrifugal force of 10000g to remove the precipitate (including nuclei, mitochondria) and retain the supernatant (including cytoplasm and cell membrane);
7. transferring the supernatant into a new tube, and performing a second round of high speed centrifugation with a high speed centrifuge for 1 hr (100000 g, 4 deg.C), wherein the cell membrane is heavy and can be precipitated from soluble cell material;
8. the supernatant was removed and the pellet was washed through a 25 gauge needle with 2 ml of buffer;
9. verifying the state of the separated cell membrane under a microscope; then centrifuging for 45 minutes again (100000 g, 4 ℃), wherein the cell membrane precipitated in the washing process is broken up and needs to be centrifuged again to precipitate again;
10. removing the supernatant, and adding 2 ml of screening solution;
11. the screening medium consisted of 50mM Tris-HCl (Tris-HCl), 150mM NaCl, 0.1% v/v Tween20 (Tween 20), 250mM Sucrose (Sucrose), 0.5mM EDTA, 1mM DTT (dimercaptothreitol), 1mg/mL fish sperm DNA (protamine DNA;), 1mg/mL BSA (bovine serum albumin), pH 7.5;
12. adding about 2.5nmol of a pool of DNA-encoding compounds to a cell membrane-containing selection solution;
13. reacting on a shaker at 4 ℃ overnight;
14. centrifugation for 45 minutes (100000 g, 4 ℃);
15. removing the supernatant, washing the precipitate with 5ml of screening solution;
16. three washes in total, 10 minutes each, during which 45 minutes of centrifugation (100000 g, 4 ℃) were performed;
17. the remaining cell membranes were heated with 2 ml of the selection solution at 80 ℃ for 10 minutes;
18. centrifugation for 45 minutes (100000 g, 4 ℃);
19. reserving the supernatant and carrying out the next round of screening;
20. one to three rounds of selection were performed until the total amount of extracted DNA encoding the compound was at 1X107To 1X109Within the range;
21. the final product is amplified by PCR and decoded by second-generation sequencing to obtain an effective compound structure A; the data analysis results of the sequencing results are schematically shown in FIG. 3. Each axis in FIG. 3 represents a DNA label, with three different DNA labels per compound. Each dot in the graph represents a compound screened out, and the size of the dot represents the degree of enrichment of the compound in the product. The most enriched compound structure (exemplified as compound a) was selected from figure 3 and synthesized separately.
22. And performing biological function verification on the compound A. Since V1a is a GPCR, FLIPR assay can be used to test the inhibitory effect of compounds on V1a activity. The IC50 results for compound a are shown schematically in figure 4. This figure shows that the inhibitory activity of the compound against the V1a receptor is 0.59 nmol.
The above is only a specific application example of the present invention, and the protection scope of the present invention is not limited in any way; other variations and modifications will be apparent to persons skilled in the art in light of the above description. All embodiments need not be described or illustrated herein. The technical solutions similar to the above embodiments formed by equivalent transformation or equivalent replacement fall within the scope of the claims of the present invention.
Claims (8)
1. A method of separating cell membranes for screening a library of transmembrane proteins for DNA encoding compounds, wherein the transmembrane protein is a GPCR receptor, comprising the steps of:
(1) physically disrupting the cells;
(2) performing a first centrifugation cycle to separate a supernatant containing soluble cellular material and precipitate insoluble cellular structures; the soluble cellular material comprises cell plasma and cell membrane; the insoluble cellular structure comprises a nucleus and mitochondria;
(3) performing a second round of high speed centrifugation to precipitate cell membranes from soluble cellular material;
(4) verifying the state of the separated cell membrane under a microscope;
(5) using cell membrane as carrier, screening transmembrane protein with DNA coding compound library, the screening process includes: adding the DNA coding compound library into a screening solution containing cell membranes, reacting, centrifuging at a high speed, removing supernatant, and washing the precipitate with the screening solution; heating the remaining cell membranes and the screening solution together; then high-speed centrifugation is carried out, supernatant is reserved, and the next round of screening is carried out; in the screening process, high-speed centrifugation is used for separating soluble components and cell membrane components so as to separate compounds which are not combined with the protein; denaturing the protein by heating, allowing the bound compound to fall off the protein, into solution, and into the next round of screening; a total of one to three rounds of selection were performed until the total amount of extracted DNA-encoding compounds was at 1X107To 1X109Within the range; in step (5), the composition of the screening solution was 50mM Tris-HCl, 150mM NaCl, 0.1% v/vTween20, 250mM sucrose, 0.5mM EDTA, 1mM DTT, 1mg/mL protamine DNA, 1mg/mL LBSA, pH 7.5.
2. The method of claim 1, wherein in step (1), the physical method comprises using a homogenizer, homogenizer or high speed stirrer, using a bead beater, or using sonication.
3. The method of claim 1, wherein the following steps are added before step (1): seeding the cells on a culture plate to grow a uniform monolayer of cells; cells were scraped into buffer with a spatula.
4. The method of claim 3, wherein the buffer composition is: 20mM HEPES, 250mM Sucrose, 10mM KCl, 2mM MgCl21mM EDTA, 1mM EGTA, 1mM DTT and protease inhibitor cocktail, pH 7.4, were added prior to use.
5. The method of claim 1, wherein in step (2), the first round of centrifugation is performed at a centrifugal force of 10,000g for 10 minutes at a temperature of 4 degrees celsius.
6. The method of claim 1, wherein in step (3), the second round of high speed centrifugation is performed at a centrifugal force of 100,000g for 1 hour at a temperature of 4 degrees celsius.
7. The method of claim 1, wherein in step (5), the high speed centrifugation is at a centrifugal force of 100,000g for 45 minutes at a temperature of 4 degrees Celsius; the heating temperature is 80 ℃, and the heating time is 10 minutes.
8. The method of claim 1, wherein the following steps are added after step (5):
(6) after a total of 1-3 rounds of screening of step (5), performing second-generation sequencing on the results to decode the structure of the compound;
(7) functional tests are carried out to verify the function of the compound.
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| CN112430598A (en) * | 2019-08-26 | 2021-03-02 | 成都先导药物开发股份有限公司 | Method for screening living cell membrane protein of DNA coding compound library |
| CN110548304A (en) * | 2019-09-07 | 2019-12-10 | 天津药明康德新药开发有限公司 | centrifugal concentrator for removing boiling point solvent |
| CN111621463A (en) * | 2020-06-02 | 2020-09-04 | 英文特生物技术(北京)有限公司 | Method for separating total membrane from cells by column method |
| CN112210019A (en) * | 2020-10-15 | 2021-01-12 | 广东省科学院生物工程研究所 | Purification method of transmembrane region of membrane protein containing single transmembrane region |
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| CN101531997A (en) * | 2008-03-13 | 2009-09-16 | 中国科学院上海生命科学研究院 | Method and model for high throughout screening of G protein-coupled receptor ligand |
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| CN1646917A (en) * | 2002-04-12 | 2005-07-27 | 布赖恩·F·奥当德 | Method of identifying transmembrane protein-interacting compounds |
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