CN113278048A - Purification method of membrane protein - Google Patents
Purification method of membrane protein Download PDFInfo
- Publication number
- CN113278048A CN113278048A CN202110580012.6A CN202110580012A CN113278048A CN 113278048 A CN113278048 A CN 113278048A CN 202110580012 A CN202110580012 A CN 202110580012A CN 113278048 A CN113278048 A CN 113278048A
- Authority
- CN
- China
- Prior art keywords
- glycerol
- membrane protein
- buffer
- dodecyl
- maltoside
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108010052285 Membrane Proteins Proteins 0.000 title claims abstract description 72
- 102000018697 Membrane Proteins Human genes 0.000 title claims abstract description 63
- 238000000746 purification Methods 0.000 title claims abstract description 37
- 238000000034 method Methods 0.000 title claims abstract description 30
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims abstract description 105
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000006228 supernatant Substances 0.000 claims abstract description 47
- 239000003599 detergent Substances 0.000 claims abstract description 38
- NLEBIOOXCVAHBD-QKMCSOCLSA-N dodecyl beta-D-maltoside Chemical compound O[C@@H]1[C@@H](O)[C@H](OCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 NLEBIOOXCVAHBD-QKMCSOCLSA-N 0.000 claims abstract description 36
- 238000000464 low-speed centrifugation Methods 0.000 claims abstract description 20
- 238000001742 protein purification Methods 0.000 claims abstract description 17
- 239000002244 precipitate Substances 0.000 claims abstract description 16
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 13
- 239000003480 eluent Substances 0.000 claims abstract description 7
- 229910017053 inorganic salt Inorganic materials 0.000 claims abstract description 6
- 238000004090 dissolution Methods 0.000 claims abstract description 5
- 230000001939 inductive effect Effects 0.000 claims abstract description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 44
- 239000007983 Tris buffer Substances 0.000 claims description 28
- 210000004027 cell Anatomy 0.000 claims description 28
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 28
- 239000011780 sodium chloride Substances 0.000 claims description 22
- 239000012535 impurity Substances 0.000 claims description 19
- 239000000872 buffer Substances 0.000 claims description 13
- 239000012149 elution buffer Substances 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 12
- WLNARFZDISHUGS-MIXBDBMTSA-N cholesteryl hemisuccinate Chemical compound C1C=C2C[C@@H](OC(=O)CCC(O)=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 WLNARFZDISHUGS-MIXBDBMTSA-N 0.000 claims description 11
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 claims description 5
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 5
- 238000000108 ultra-filtration Methods 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- -1 n-dodecyl choline phosphate Chemical compound 0.000 claims description 4
- 102100026445 A-kinase anchor protein 17A Human genes 0.000 claims description 3
- 102000013128 Endothelin B Receptor Human genes 0.000 claims description 3
- 108010090557 Endothelin B Receptor Proteins 0.000 claims description 3
- 101000718019 Homo sapiens A-kinase anchor protein 17A Proteins 0.000 claims description 3
- 101710117021 Tyrosine-protein phosphatase YopH Proteins 0.000 claims description 3
- 241000607479 Yersinia pestis Species 0.000 claims description 3
- 229950004354 phosphorylcholine Drugs 0.000 claims description 3
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 claims description 2
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 38
- 108090000623 proteins and genes Proteins 0.000 abstract description 38
- 238000000703 high-speed centrifugation Methods 0.000 abstract description 17
- 238000000605 extraction Methods 0.000 abstract description 8
- 239000007853 buffer solution Substances 0.000 abstract description 3
- 238000004925 denaturation Methods 0.000 abstract description 2
- 230000036425 denaturation Effects 0.000 abstract description 2
- 238000001514 detection method Methods 0.000 description 23
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 17
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 17
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 17
- 239000008188 pellet Substances 0.000 description 16
- 241000588724 Escherichia coli Species 0.000 description 9
- 230000000052 comparative effect Effects 0.000 description 7
- 210000004962 mammalian cell Anatomy 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000005063 solubilization Methods 0.000 description 3
- 230000007928 solubilization Effects 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 101150015399 yopE gene Proteins 0.000 description 2
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 1
- GUQQBLRVXOUDTN-XOHPMCGNSA-N 3-[dimethyl-[3-[[(4r)-4-[(3r,5s,7r,8r,9s,10s,12s,13r,14s,17r)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]pentanoyl]amino]propyl]azaniumyl]-2-hydroxypropane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CC(O)CS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 GUQQBLRVXOUDTN-XOHPMCGNSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 239000004135 Bone phosphate Substances 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- BACYUWVYYTXETD-UHFFFAOYSA-N N-Lauroylsarcosine Chemical compound CCCCCCCCCCCC(=O)N(C)CC(O)=O BACYUWVYYTXETD-UHFFFAOYSA-N 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 210000003000 inclusion body Anatomy 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a purification method of membrane protein, which comprises the following steps: inducing expression of the membrane protein of interest in the host cell; breaking host cells, centrifuging 490-1000 g at low speed, taking supernatant, centrifuging the supernatant at 30000g at high speed, and taking precipitate; resuspending the precipitate with buffer solution, adding detergent, shaking for dissolution, centrifuging, and collecting supernatant; purifying by affinity chromatography, eluting with eluent containing buffer solution, inorganic salt, dodecyl-beta-D-maltoside, imidazole and glycerol, and ultrafiltering and centrifuging the eluent to obtain the target membrane protein. Aiming at the technical problems of membrane protein denaturation caused by detergent, low extraction rate of target membrane protein, complex operation, high cost and the like existing in the conventional membrane protein purification method, the invention combines low-speed centrifugation and high-speed centrifugation, selects the detergent and effectively improves the content of the target membrane protein in the total protein, namely the invention provides the membrane protein purification method which is simple in operation, low in cost, high in extraction rate and wide in applicability.
Description
Technical Field
The invention belongs to the technical field of protein purification, and particularly relates to a membrane protein purification method.
Background
Membrane proteins are the major contributors to biofilm function, are expressed in relatively low amounts, and are usually purified as protein-lipid-detergent complexes. The solubility of this complex in an aqueous environment allows the use of essentially the same separation techniques as water-soluble proteins, with the difference that: where membrane protein purification is performed by adding detergents to the solution, the protein-detergent complex is dynamic and the detergent molecules will dissociate immediately in the absence of free detergent molecules. The 6 histidine-tagged water-soluble protein can be purified directly by reference to standard procedures, but the 6 histidine-tagged membrane protein binds weakly to immobilized metal ion affinity chromatography (IMAC), making purification less effective than water-soluble proteins, probably because detergent binding to the protein limits contact between the histidine tag and the IMAC ligand.
Detergents can be classified into ionic, nonionic and amphoteric detergents. Ionic detergents, including SDS, N-lauryl sarcosine, CTAB and sodium cholate, are effective in extracting proteins from membranes, and these detergents are harsh and tend to denature proteins because they disrupt intermolecular and intramolecular protein interactions. Nonionic detergents include maltosides, glucosides and polyoxyethylene glycols, which are used for purification and structural studies of membrane proteins. Amphoteric detergents include Zwittergents, Fos-Cholines, CHAPS/CHAPSO, and amine oxides, which are electrically neutral like nonionic detergents but generally disrupt protein-protein interactions like ionic detergents and are thus milder in nature.
Disclosure of Invention
Aiming at the technical problems of low extraction rate of target membrane protein, complex operation, high cost and the like of the existing membrane protein purification method, the invention provides the membrane protein purification method, which effectively improves the content of the target membrane protein in total protein and improves the operability and high cost of the membrane protein purification process by combining low-speed centrifugation and high-speed centrifugation and screening detergents.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a method for purifying a membrane protein, which comprises the following steps:
and 4, purifying the supernatant obtained in the step 3 by adopting affinity chromatography, eluting by using an elution buffer solution containing dodecyl-beta-D-maltoside, imidazole and glycerol, collecting eluent, and performing ultrafiltration and centrifugation to obtain the target membrane protein.
Further, in step 1, the target membrane protein is selected from the group consisting of: any one of endothelin receptor B protein, Yersinia pestis virulence protein, and B lymphocyte antigen.
Further, in step 1, the host cell is a prokaryotic cell and/or a eukaryotic cell.
Further, in step 2, when the host cell is a prokaryotic cell, the prokaryotic cell is centrifuged at 490g at a low speed for 15min at 4 ℃ and then the supernatant is taken; when the host cell is eukaryotic, centrifuging at 1000g for 15min at 4 deg.C, and collecting supernatant.
Further, in step 3, the detergent is selected from: the volume ratio is 1: 0.1 dodecyl- β -D-maltoside and cholesterol hemisuccinate; or the volume ratio of 1: 0.2 of 1-octadecanoyl-sn-glycerol-3-phosphate- (1' rac glycerol) and cholesterol hemisuccinate; or the volume ratio of 1: 0.2 of n-dodecyl choline phosphate and cholesterol hemisuccinate.
Preferably, the detergent is a mixture of 1: 0.1 dodecyl-beta-D-maltoside and cholesterol hemisuccinate tribasic
Further, in step 3, the resuspension Buffer is Buffer a, which comprises: tris buffer, inorganic salts, glycerol and protease inhibitors.
Further, the buffer includes: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 1-10% volume fraction glycerol and protease inhibitor.
Further, the affinity chromatography purification steps are specifically as follows: and (3) using a Buffer B equilibrium chromatographic column containing Tris Buffer solution, inorganic salt, dodecyl-beta-D-maltoside and glycerol, then adding the supernatant obtained in the step (3) into the chromatographic column, and sequentially removing impurities and eluting with an elution Buffer solution containing Tris Buffer solution, inorganic salt, dodecyl-beta-D-maltoside, glycerol and 20-500 mM imidazole.
Further, the Buffer B includes: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside and 1-10% glycerol.
The invention also provides a membrane protein purification kit, which comprises:
resuspending Buffer a: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 1-10% volume fraction glycerol and protease inhibitor;
and (3) detergent: the volume ratio is 1: 0.1 dodecyl- β -D-maltoside and cholesterol hemisuccinate;
balance Buffer B: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside and 1-10% glycerol;
washing impurity Buffer C: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 20mM imidazole;
elution Buffer D: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 60mM imidazole;
elution Buffer E: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 100mM imidazole;
elution Buffer F: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 500mM imidazole.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention firstly uses low-speed centrifugation to precipitate impurities (including dead cells, cell fragments, inclusion bodies or incorrectly folded target protein) in broken cells in the membrane protein extraction process, wherein the incorrectly folded target protein cannot be dissolved by mild detergent in the subsequent purification step. The supernatant is centrifuged at high speed to obtain a precipitate, which includes endoplasmic reticulum membrane, plasma membrane, etc., wherein the target membrane protein is attached to the cell membrane fragments. By combining low-speed centrifugation and high-speed centrifugation, impurities are effectively removed and target membrane protein is enriched, so that the extraction content of the target membrane protein is obviously improved, and the method does not depend on expensive ultrahigh-speed centrifugation equipment.
(2) According to the invention, through optimizing the detergent, the membrane protein is fully dissolved by the mild detergent, and meanwhile, the glycerol capable of improving the solubility of the membrane protein is added into the buffer solution, so that the concentration of inorganic salt is reduced, the reduction of the solubility of the membrane protein is avoided, and the content of the membrane protein finally obtained by purification is obviously improved, namely, the purification effect of the membrane protein is obviously improved.
(3) The membrane protein purification method provided by the invention has wide applicability, and can be directly applied to a prokaryotic escherichia coli expression system, a eukaryotic mammalian expression system or other recombinant protein expression systems; the method is simple and quick, does not depend on expensive equipment, has low cost and high success rate, improves the operability of membrane protein purification while improving the yield of the target membrane protein, and has wide application prospect.
Drawings
FIG. 1 is a graph showing the results of SDS-PAGE detection of the EDNRB protein in example 1 of the present invention after 490g low-speed centrifugation and 30,000g high-speed centrifugation;
FIG. 2 is a graph showing the results of SDS-PAGE detection of the Yope protein after 490g low-speed centrifugation and 30,000g high-speed centrifugation in example 1 of the present invention;
FIG. 3 is a graph showing SDS-PAGE detection results after dissolution of EDNRB protein using three different detergents in example 1 of the present invention;
FIG. 4 is a graph showing the results of SDS-PAGE detection of the Yope protein solubilized with three different detergents in example 1 of the present invention;
FIG. 5 is a SDS-PAGE detection result of EDNRB protein in example 1 of the present invention after affinity chromatography purification;
FIG. 6 is a SDS-PAGE result of the Yope protein purified by affinity chromatography in example 1 of the present invention;
FIG. 7 is a graph showing SDS-PAGE detection results of the CD20 protein after 1000g low-speed centrifugation and 30,000g high-speed centrifugation in example 2 of the present invention;
FIG. 8 is a SDS-PAGE result of CD20 protein purified by affinity chromatography in example 2;
FIG. 9 is a diagram showing the result of SDS-PAGE detection of the purified Yope protein in comparative example 1 of the present invention;
FIG. 10 is a diagram showing the result of SDS-PAGE detection of the purified CD20 protein in comparative example 2 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 Membrane protein purification of E.coli expression System
The membrane proteins expressed in the E.coli expression system of this example: the optimization of the purification steps for the endothelin receptor B protein (EDNRB) and yersinia pestis virulence protein (YopE) was as follows:
1. using methods routine in the art, expression vectors were constructed according to the following table:
2. culturing the expression host cell E.coli BL21(DE3) in LB culture medium at 37 deg.C for 4h, inducing the expression of target membrane protein with 0.5mM IPTG, cooling to 18 deg.C, culturing overnight, and collecting bacteria.
3. Taking the Escherichia coli cells, carrying out ultrasonic cell disruption, carrying out low-speed centrifugation at 490g for 15 minutes at 4 ℃, separating a supernatant and a precipitate, respectively carrying out SDS-PAGE detection, then taking 30,000g of the supernatant, carrying out high-speed centrifugation for 1 hour, separating the supernatant and the precipitate, and respectively carrying out SDS-PAGE detection. The detection results of EDNRB are shown in FIG. 1, and the detection results of Yope are shown in FIG. 2.
According to fig. 1 and fig. 2, the Marker is, from top to bottom: 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4kDa, lane 1 is BL21(DE3) control containing no membrane protein of interest, lane 2 is the result of direct detection after disruption of E.coli cells, lanes 3 and 4 are the supernatant and pellet of 490g of disrupted cells after low speed centrifugation, respectively, and lanes 5 and 6 are the supernatant and pellet of 30,000g of supernatant after high speed centrifugation, respectively. The arrow points to the membrane protein of interest EDNRB or YopE.
The results in FIG. 1 show that EDNRB can be expressed in E.coli with a protein size of 50.3kDa, and that EDNRB can be detected in both the supernatant (lane 3) and the pellet (lane 4) after 490g low speed centrifugation for 15min, but the pellet may contain impurities such as incorrectly folded protein, and the pellet is discarded. After taking the supernatant and continuing the high speed centrifugation at 30,000g for 1h, EDNRB was mainly concentrated in the pellet (lane 6) and almost not in the supernatant (lane 5). I.e., after low and high speed centrifugation in sequence, the EDNRB content in total protein was significantly increased compared to the initial lane 2.
Similarly, Yope can be expressed in E.coli with a protein size of 26.9kDa and can be detected in both the supernatant (lane 3) and the pellet (lane 4) after 490g low speed centrifugation for 15min, but the pellet may contain impurities such as incorrectly folded protein and is discarded. After taking the supernatant and continuing the 30,000g high speed centrifugation for 1h, Yope was mainly enriched in the pellet (lane 6) and almost none in the supernatant (lane 5). I.e., after low and high speed centrifugation in sequence, the content of Yope in total protein was significantly increased compared to the initial lane 2.
4. Solubilization and purification of Membrane proteins
Resuspending Buffer a: 25mM Tris, pH7.5, 150mM NaCl, 5% glycerol, cOmplete EDTA-free protease-inhibitor cocktail tables (Roche);
balance Buffer B: 25mM Tris, pH7.5, 150mM NaCl, 0.05% dodecyl- β -D-maltoside (DDM), 5% glycerol;
washing impurity Buffer C: 25mM Tris, pH7.5, 150mM NaCl, 0.05% DDM, 20mM imidazole, 5% glycerol;
elution Buffer D: 25mM Tris, pH7.5, 150mM NaCl, 0.05% DDM, 60mM imidazole, 5% glycerol;
elution Buffer E: 25mM Tris, pH7.5, 150mM NaCl, 0.05% DDM, 100mM imidazole, 5% glycerol;
elution Buffer F: 25mM Tris, pH7.5, 150mM NaCl, 0.05% DDM, 500mM imidazole, 5% glycerol.
Resuspend the protein pellet with Buffer a, add different detergents:
(1) 2% (w/v) DDM and 0.2% (w/v) CHS; (ii) a
(2) 1% (w/v) n-dodecyl choline phosphate (FOS12) and 0.2% (w/v) CHS;
(3) 1% (w/v) 1-octadecanoyl-sn-glycerol-3-phosphate- (1' rac glycerol) (LMPG) and 0.2% (w/v) Cholesterol Hemisuccinate (CHS)
The mixture was dissolved overnight at 4 ℃ with gentle shaking, centrifuged at 30,000g for 30min the next day, and the supernatant and the precipitate were subjected to SDS-PAGE separately. The detection results of EDNRB are shown in fig. 3, and the detection results of YopE are shown in fig. 4. Wherein the Marker is respectively from top to bottom: 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4kDa, 2% (w/v) DDM and 0.2% (w/v) CHS solubilized pellet and supernatant in lanes 1 and 4, respectively, 1% (w/v) FOS12 and 0.2% (w/v) CHS solubilized pellet and supernatant in lanes 2 and 5, respectively, 1% (w/v) LMPG and 0.2% (w/v) CHS solubilized pellet and supernatant in lanes 3 and 6, respectively, with the arrows pointing to the membrane protein of interest EDNRB or Yope.
The results in FIGS. 3 and 4 show that the membrane proteins EDNRB and Yope enriched by the above separation method are soluble in the three detergents, and that the DDM solubility for the yopE protein is slightly weaker (the main band in lane 4 is thinner than those in lanes 5 and 6 in FIG. 4), so that the denaturation of the target membrane protein by the detergent can be avoided. Therefore, 2% (w/v) DDM and 0.2% (w/v) CHS-solubilized membrane protein were taken for further purification.
5. Affinity chromatography purification
Taking Ni-NTA resin (Qiagen), balancing the column by using Buffer B, then taking the dissolved membrane protein for sampling, collecting the flow-through liquid, washing impurities by using Buffer C, eluting by using Buffer D, Buffer E and Buffer F in sequence, and respectively carrying out SDS-PAGE detection on the eluates, wherein the detection result of EDNRB is shown in figure 5, and the detection result of yopE is shown in figure 6. Wherein lane 1 is the stock solution without column, lane 2 is the flow-through, lane 3 is the Buffer D eluate, lane 4 is the Buffer E eluate, and lane 5 is the Buffer F eluate. The results in fig. 5 and 6 show that other impurities are effectively removed after impurity washing and multiple elution, and pure target membrane protein is obtained. Centrifuging the protein obtained from the last elution to a proper volume by using an ultrafiltration tube (MWCO 30kDa, Millipore) at 3500rpm and 4 ℃ to obtain target membrane proteins EDNRB and Yope. And (3) measuring the EDNRB and Yope of the target membrane protein obtained by purification, and calculating the extraction rate, wherein the content of the EDNRB protein is as follows: 7.6mg/L, the purification rate is: 81.5 percent; the content of Yope protein was: 21.0mg/L, the purification rate is 85.5%.
Example 2 Membrane protein purification of mammalian cell expression System
This example demonstrates, on the basis of the optimization procedure of example 1, the following results for the membrane proteins of mammalian cell expression systems: the purification effect of the B lymphocyte antigen (CD20) is as follows:
1. using methods routine in the art, expression vectors were constructed according to the following table:
2. one day before transfection, cells were divided into 1X106cells/mL, optimizing the parameters of transfected cells, the optimal transfection parameters are: the ratio of plasmid DNA to PEI (MW 25000, Polysciences) was 1: 3. The plasmid was every 1X106cells added 1. mu.g of plasmid DNA. On the day of transfection, the plasmid and PEI were diluted separately with medium, and PEI was then poured into the plasmid. After incubation at room temperature for 10min, the mixed plasmid PEI mixture was poured into the cells. After 2 days of expression, cells were harvested.
3. The above mammalian cells were subjected to ultrasonication and then to low-speed centrifugation, but it was found that the removal effect of impurities was not good after low-speed centrifugation at 490g for 15 minutes at 4 ℃ as shown in example 1. Therefore, the rotating speed is adjusted, specifically: centrifuging at 4 deg.C for 15min at 1000g low speed, separating supernatant and precipitate, respectively performing SDS-PAGE detection, centrifuging the supernatant at 30,000g high speed for 1h, separating supernatant and precipitate, and respectively performing SDS-PAGE detection, with the detection results shown in FIG. 7. Wherein the Marker is respectively from top to bottom: 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, 14.4kDa, as measured directly after disruption of mammalian cells in lane 1, supernatant and pellet from 1000g of disrupted cells after low speed centrifugation in lanes 2 and 3, and supernatant and pellet from 30,000g of supernatant after high speed centrifugation in lanes 4 and 5, respectively. The arrow points to the membrane protein of interest CD 20.
The result shows that the CD20 can be expressed in mammalian cells, the protein size is 34.4kDa, CD20 is mainly enriched in the supernatant after 1000g of low-speed centrifugation for 15min, namely, the impurities can be effectively removed and the target membrane protein CD20 is enriched in the supernatant through 1000g of low-speed centrifugation. After taking the supernatant and continuing the high speed centrifugation at 30,000g for 1h, CD20 was mainly enriched in the pellet, but almost none in the supernatant. That is, after low-speed centrifugation and high-speed centrifugation in sequence, the content of CD20 protein expressed by mammalian cells in total protein was significantly increased, impurities were significantly reduced, and the main band was more significant, compared to the initial lane 1.
4. Solubilization and purification of Membrane proteins
The CD20 protein precipitate was resuspended in Buffer A, CD20 was solubilized directly with detergent 2% (w/v) DDM and 0.2% (w/v) CHS, and after overnight solubilization with gentle shaking at 4 ℃ 30,000g was centrifuged for 30 minutes the next day, and the supernatant was collected for further purification.
5. Affinity chromatography purification
Ni-NTA resin (Qiagen) is taken, the column is balanced by Buffer B, then the dissolved membrane protein CD20 is taken for sample loading, the flow-through liquid is collected, then the impurities are washed by Buffer C, after the Buffer D, the Buffer E and the Buffer F are sequentially eluted, the SDS-PAGE detection is carried out on the eluent, and the detection result is shown in figure 8. Wherein lane 1 is the raw CD20 without passing through the column, lane 2 is the flow-through, and lane 3 is the Buffer F eluate.
The result shows that other impurities are effectively removed to obtain pure target membrane protein after impurity washing and multiple times of elution, and the Buffer F eluent is centrifuged to a proper volume at 3500rpm and 4 ℃ by using an ultrafiltration tube (MWCO 30kDa, Millipore), so that the target membrane protein CD20 is obtained. The content of the purified target membrane protein CD20 is determined as follows: 8.2mg/L, the purification rate is: 92.2 percent.
In summary, the invention provides a membrane protein purification method with simple operation, low cost, high extraction rate and wide applicability, which comprises the following steps:
and 4, purifying the supernatant obtained in the step 3 by adopting affinity chromatography, balancing a column by using a Buffer B, then taking the supernatant for sampling, washing impurities by using a Buffer C, sequentially eluting a Buffer D, a Buffer E and a Buffer F, taking an eluent of the Buffer F, and carrying out ultrafiltration and centrifugation to obtain the target membrane protein.
Comparative example 1
This comparative example differs from example 1 in that: EDNRB and YopE proteins were purified using membrane protein purification methods conventional in the art, comprising the steps of: cells were harvested after protein expression. After the cells are broken or lysed, the cells are centrifuged at high speed to obtain a precipitate containing the target membrane protein. Then screening different detergents for dissolving membrane proteins, and reducing the concentration of the detergents for affinity chromatography purification after the proteins are dissolved.
Among them, the EDNRB protein is weakly expressed in original E.coli (as shown in lane 2 of FIG. 1), and after direct high-speed centrifugation, the EDNRB protein of interest cannot be successfully dissolved by mild detergent, and the EDNRB protein is also tried to be dissolved by 8M urea, but finally the purification fails.
The purification results of the Yope protein are shown in FIG. 9, in which lane 1 is the raw solution without passing through the column, lane 2 is the flow-through solution, lane 3 is the Buffer D eluate, lane 4 is the Buffer E eluate, and lane 5 is the Buffer F eluate. The result shows that the content and the extraction rate of the YopE obtained by the conventional purification method are both low, the content of the YopE protein obtained by purification is only 0.2mg/L, and the extraction rate is only 52.4%.
Comparative example 2
This comparative example differs from example 3 in that: the CD20 protein was purified using membrane protein purification methods conventional in the art, as in comparative example 1.
The results are shown in FIG. 10, and show that only a small amount of CD20 can be obtained by mild detergent after direct high-speed centrifugation, and the target protein cannot be purified after multiple elutions, i.e., the purification fails.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (10)
1. A method for purifying a membrane protein, the method comprising:
step 1, inducing the expression of target membrane protein in host cells;
step 2, breaking host cells, centrifuging 490-1000 g at low speed, taking the supernatant, centrifuging 30000g at high speed, and taking the precipitate;
step 3, resuspending the precipitate obtained in the step 2 by using a resuspension buffer, then adding a detergent, shaking for dissolution, centrifuging, and taking a supernatant;
and 4, purifying the supernatant obtained in the step 3 by adopting affinity chromatography, eluting by using an elution buffer solution containing dodecyl-beta-D-maltoside, imidazole and glycerol, collecting eluent, and performing ultrafiltration and centrifugation to obtain the target membrane protein.
2. The purification method according to claim 1, wherein in step 1, the membrane protein of interest is selected from the group consisting of: any one of endothelin receptor B protein, Yersinia pestis virulence protein, and B lymphocyte antigen.
3. The purification method according to claim 1, wherein in step 1, the host cell is a prokaryotic cell and/or a eukaryotic cell.
4. The purification method according to claim 1, wherein in step 2, when the host cell is a prokaryotic cell, the supernatant is obtained by low-speed centrifugation at 490g for 15min at 4 ℃; when the host cell is eukaryotic, centrifuging at 1000g for 15min at 4 deg.C, and collecting supernatant.
5. The purification process according to claim 1, wherein in step 3, the detergent is selected from the group consisting of: the volume ratio is 1: 0.1 dodecyl- β -D-maltoside and cholesterol hemisuccinate; or the volume ratio of 1: 0.2 of 1-octadecanoyl-sn-glycerol-3-phosphate- (1' rac glycerol) and cholesterol hemisuccinate; or the volume ratio of 1: 0.2 of n-dodecyl choline phosphate and cholesterol hemisuccinate.
6. The purification method according to claim 1, wherein in step 3, the resuspension Buffer is Buffer A, comprising: tris buffer, inorganic salts, glycerol and protease inhibitors.
7. The purification method according to claim 6, wherein the BufferA comprises: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 1-10% volume fraction glycerol and protease inhibitor.
8. The purification method according to claim 1, wherein in step 4, the affinity chromatography purification step is specifically: and (3) using a Buffer B equilibrium chromatographic column containing Tris Buffer solution, inorganic salt, dodecyl-beta-D-maltoside and glycerol, then adding the supernatant obtained in the step (3) into the chromatographic column, and sequentially removing impurities and eluting with an elution Buffer solution containing Tris Buffer solution, inorganic salt, dodecyl-beta-D-maltoside, glycerol and 20-500 mM imidazole.
9. The purification method according to claim 8, wherein the Buffer B comprises: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside and 1-10% glycerol.
10. A membrane protein purification kit, comprising:
resuspending Buffer a: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 1-10% volume fraction glycerol and protease inhibitor;
and (3) detergent: the volume ratio is 1: 0.1 dodecyl- β -D-maltoside and cholesterol hemisuccinate;
balance Buffer B: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside and 1-10% glycerol;
washing impurity Buffer C: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 20mM imidazole;
elution Buffer D: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 60mM imidazole;
elution Buffer E: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 100mM imidazole;
elution Buffer F: 20-50 mM Tris, pH7.0-7.5, 50-200 mM NaCl, 0.01-0.1% dodecyl-beta-D-maltoside, 1-10% glycerol and 500mM imidazole.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110580012.6A CN113278048B (en) | 2021-05-26 | 2021-05-26 | Purification method of membrane protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110580012.6A CN113278048B (en) | 2021-05-26 | 2021-05-26 | Purification method of membrane protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113278048A true CN113278048A (en) | 2021-08-20 |
| CN113278048B CN113278048B (en) | 2023-03-10 |
Family
ID=77281895
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110580012.6A Active CN113278048B (en) | 2021-05-26 | 2021-05-26 | Purification method of membrane protein |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113278048B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112625111A (en) * | 2019-09-24 | 2021-04-09 | 四川大学华西医院 | Preparation method of synaptozectin SV2A |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102850447A (en) * | 2012-08-06 | 2013-01-02 | 天津耀宇生物技术有限公司 | Method for purifying natural membrane protein PRMC2 of bombyx |
| CN108623662A (en) * | 2017-03-23 | 2018-10-09 | 周界文 | Phytopathogen antigenic compound, antibody, detection kit and its application |
| CN109371051A (en) * | 2018-11-15 | 2019-02-22 | 华南农业大学 | A kind of extracting method and application suitable for obtaining a large amount of high-purity salmonella outer membrane proteins |
| CN112062820A (en) * | 2020-08-24 | 2020-12-11 | 黑龙江八一农垦大学 | Renaturation and purification method of colibacillus recombinant outer membrane protein A inclusion body protein |
| CN112625111A (en) * | 2019-09-24 | 2021-04-09 | 四川大学华西医院 | Preparation method of synaptozectin SV2A |
-
2021
- 2021-05-26 CN CN202110580012.6A patent/CN113278048B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102850447A (en) * | 2012-08-06 | 2013-01-02 | 天津耀宇生物技术有限公司 | Method for purifying natural membrane protein PRMC2 of bombyx |
| CN108623662A (en) * | 2017-03-23 | 2018-10-09 | 周界文 | Phytopathogen antigenic compound, antibody, detection kit and its application |
| CN109371051A (en) * | 2018-11-15 | 2019-02-22 | 华南农业大学 | A kind of extracting method and application suitable for obtaining a large amount of high-purity salmonella outer membrane proteins |
| CN112625111A (en) * | 2019-09-24 | 2021-04-09 | 四川大学华西医院 | Preparation method of synaptozectin SV2A |
| CN112062820A (en) * | 2020-08-24 | 2020-12-11 | 黑龙江八一农垦大学 | Renaturation and purification method of colibacillus recombinant outer membrane protein A inclusion body protein |
Non-Patent Citations (1)
| Title |
|---|
| 宫彩霞: "去污剂的性质及其在膜蛋白结构生物学中的应用", 《中国生物化学与分子生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112625111A (en) * | 2019-09-24 | 2021-04-09 | 四川大学华西医院 | Preparation method of synaptozectin SV2A |
| CN112625111B (en) * | 2019-09-24 | 2023-09-15 | 四川大学华西医院 | Preparation method of synaptic vesicle protein SV2A |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113278048B (en) | 2023-03-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hwang et al. | Targeted expression, purification, and cleavage of fusion proteins from inclusion bodies in Escherichia coli | |
| Jeon et al. | High-throughput purification and quality assurance of Arabidopsis thaliana proteins for eukaryotic structural genomics | |
| US8293876B2 (en) | Method of purification of hydrophobic proteins | |
| CN113286810A (en) | Protein purification method | |
| Li et al. | Characterization of metal affinity of green fluorescent protein and its purification through salt promoted, immobilized metal affinity chromatography | |
| CN113278048B (en) | Purification method of membrane protein | |
| EP3448876A1 (en) | Streptavidin muteins and methods of using them | |
| Zhang et al. | Fusion partners as a tool for the expression of difficult proteins in mammalian cells | |
| Holland et al. | ENTEROVIRAL RIBONUCLEIC ACID: II. Biological, Physical, and Chemical Studies | |
| Fodor et al. | Modular broad-host-range expression vectors for single-protein and protein complex purification | |
| CN100432230C (en) | Fusion expression method for metallothionein and use thereof | |
| CN113004375B (en) | Small molecular protein for efficiently mediating recombinant polypeptide to form inclusion body | |
| WO2013068602A2 (en) | Method for the production of polypeptides | |
| KR20140037726A (en) | Method for production, extraction and purification of soluble recombinant protein | |
| US20220025355A1 (en) | Method for screening protease variant and obtained protease variant | |
| US10927149B2 (en) | Industrially scalable process for recovering biologically active recombinant carrier proteins | |
| McCann et al. | Polyacrylic acid based plasma fractionation for the production of albumin and IgG: Compatibility with existing commercial downstream processes | |
| EP4143331A2 (en) | Fusion polypeptides for target peptide production | |
| Besir | A generic protocol for purifying disulfide-bonded domains and random protein fragments using fusion proteins with SUMO3 and cleavage by SenP2 protease | |
| Butler et al. | Check Chapter 20 updates | |
| CN108164593B (en) | Protein purification method based on calmodulin property | |
| US11919936B2 (en) | TEV protease variant, fusion protein thereof, preparation method therefor and use thereof | |
| EP3239164B1 (en) | Method for purifying target protein | |
| CN106755034B (en) | Expression, extraction and purification method of human ribosomal protein S5 | |
| CN117305268A (en) | Purification method of recombinant protein MMLV-RT |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A purification method for membrane proteins Granted publication date: 20230310 Pledgee: Guanggu Branch of Wuhan Rural Commercial Bank Co.,Ltd. Pledgor: CUSABIO BIOTECH Co.,Ltd. Registration number: Y2024980009781 |