CN115074336B - Protein with phospholipidosis enzyme activity and purification method thereof - Google Patents
Protein with phospholipidosis enzyme activity and purification method thereof Download PDFInfo
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- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 99
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000000746 purification Methods 0.000 title claims abstract description 33
- 230000000694 effects Effects 0.000 title claims abstract description 29
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 9
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 9
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 23
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 15
- 238000001742 protein purification Methods 0.000 claims abstract description 15
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 12
- 239000013604 expression vector Substances 0.000 claims abstract description 12
- 238000000926 separation method Methods 0.000 claims abstract description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 36
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 102000010175 Opsin Human genes 0.000 claims description 22
- 108050001704 Opsin Proteins 0.000 claims description 22
- 210000004027 cell Anatomy 0.000 claims description 22
- HATKUFQZJPLPGN-UHFFFAOYSA-N 2-phosphanylethane-1,1,1-tricarboxylic acid Chemical compound OC(=O)C(CP)(C(O)=O)C(O)=O HATKUFQZJPLPGN-UHFFFAOYSA-N 0.000 claims description 18
- 108010039627 Aprotinin Proteins 0.000 claims description 18
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 claims description 18
- 229960004405 aprotinin Drugs 0.000 claims description 18
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 claims description 18
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 claims description 18
- 108010052968 leupeptin Proteins 0.000 claims description 18
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 18
- 238000005406 washing Methods 0.000 claims description 16
- WRDABNWSWOHGMS-UHFFFAOYSA-N AEBSF hydrochloride Chemical compound Cl.NCCC1=CC=C(S(F)(=O)=O)C=C1 WRDABNWSWOHGMS-UHFFFAOYSA-N 0.000 claims description 14
- -1 hydroxyethyl piperazine ethylsulfanilic acid Chemical compound 0.000 claims description 14
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000000872 buffer Substances 0.000 claims description 12
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](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 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 12
- 238000011534 incubation Methods 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 11
- 108010052285 Membrane Proteins Proteins 0.000 claims description 10
- 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 description 10
- 239000000243 solution Substances 0.000 claims description 10
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 9
- 241000238631 Hexapoda Species 0.000 claims description 9
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 8
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- 102000018697 Membrane Proteins Human genes 0.000 claims description 6
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- 235000012000 cholesterol Nutrition 0.000 claims description 6
- 238000001976 enzyme digestion Methods 0.000 claims description 6
- OVIISSOUXFJLSM-KPNWGBFJSA-N butanedioic acid;(3s,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-ol Chemical compound OC(=O)CCC(O)=O.C1C=C2C[C@@H](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 OVIISSOUXFJLSM-KPNWGBFJSA-N 0.000 claims description 5
- 229940089468 hydroxyethylpiperazine ethane sulfonic acid Drugs 0.000 claims description 5
- 229920002472 Starch Polymers 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 4
- 230000005484 gravity Effects 0.000 claims description 4
- 239000001103 potassium chloride Substances 0.000 claims description 4
- 235000011164 potassium chloride Nutrition 0.000 claims description 4
- 235000019698 starch Nutrition 0.000 claims description 4
- 239000008107 starch Substances 0.000 claims description 4
- 238000002270 exclusion chromatography Methods 0.000 claims description 3
- 108091005706 peripheral membrane proteins Proteins 0.000 claims description 3
- 108010076818 TEV protease Proteins 0.000 claims description 2
- 239000012535 impurity Substances 0.000 claims description 2
- 239000000463 material Substances 0.000 claims description 2
- 210000001322 periplasm Anatomy 0.000 claims description 2
- 238000000605 extraction Methods 0.000 claims 1
- 239000001573 invertase Substances 0.000 abstract description 7
- 108010051210 beta-Fructofuranosidase Proteins 0.000 abstract description 6
- 235000011073 invertase Nutrition 0.000 abstract description 6
- 150000003904 phospholipids Chemical class 0.000 abstract description 6
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- 101000831825 Homo sapiens Transmembrane protein 41B Proteins 0.000 description 3
- 102000015439 Phospholipases Human genes 0.000 description 3
- 108010064785 Phospholipases Proteins 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
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- 238000007877 drug screening Methods 0.000 description 2
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- 150000002632 lipids Chemical class 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 102100036546 Salivary acidic proline-rich phosphoprotein 1/2 Human genes 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 230000008436 biogenesis Effects 0.000 description 1
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- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
Classifications
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
-
- 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
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/14011—Baculoviridae
- C12N2710/14041—Use of virus, viral particle or viral elements as a vector
- C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore
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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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/103—Plasmid DNA for invertebrates
- C12N2800/105—Plasmid DNA for invertebrates for insects
Abstract
The invention belongs to the technical field of protein purification, and particularly discloses a protein purification method with phospholipideverase activity and a protein with phospholipideverase activity obtained based on the purification method. The method for purifying the protein with the phospholipidosis enzyme activity comprises the following steps: constructing a 2MBP tag protein expression vector of the target protein to form recombinant protein; carrying out protein purification after expressing recombinant proteins; and (3) directly removing the 2MBP label on the semi-finished product protein after the semi-finished product protein is obtained by utilizing affinity chromatography separation, so as to obtain the target protein. The purification method provided by the invention selects 2MBP label to add, further obtains a larger yield of protein with phospholipid invertase activity, and can provide conditions for in vitro functional activity of macromolecular machine recombination. The invention also has the characteristics of simple purification method and easy industrialization realization.
Description
[ field of technology ]
The invention belongs to the technical field of protein purification, and particularly relates to a protein purification method with phospholipidosis enzyme activity and a protein with phospholipidosis enzyme activity obtained based on the purification method.
[ background Art ]
The asymmetry of the phospholipid bilayer in the biological membrane is the basis of important physiological activities such as maintenance of homeostasis of cells, signal transduction and the like, and ensures highly ordered performance of vital activities. Numerous studies have shown that rational sorting and distribution of phospholipids is a necessary condition for biogenesis and viral infection. Phospholipases are molecular machines capable of transporting macromolecules such as lipids across membranes, and they participate in and play a vital role in a variety of physiological and pathological processes including viral infections, metabolic diseases and neurodegenerative diseases by transporting phospholipids from one side of the plasma membrane to the other.
Phosphoeverse proteins are typically derived from the purification of natural membrane proteins. However, the existing protein purification method has the defects of low content, low yield, complex living purification process and the like. Therefore, the establishment of a purification system with higher protein yield and protein activity can lay a good foundation for deeply exploring the physiological functions of the purification system and taking the purification system as a drug target to carry out small-molecule drug screening and treating related diseases, and provide a reliable tool for application research.
[ invention ]
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a protein with phospholipideverase activity and a purification method thereof, so as to solve the problems of low content, low yield and complex purification process of the existing phospholipideverase.
To achieve the above object, a first aspect of the present invention discloses a method for purifying a protein having phospholipidinvertase activity. The purification method comprises the following steps:
(1) Constructing a 2MBP tag protein expression vector of the target protein to form recombinant protein;
(2) Carrying out protein purification after expressing recombinant proteins;
(3) Directly after affinity chromatography, the target protein was obtained by digestion overnight on a column and removal of the 2MBP tag.
Wherein the target protein is a protein with phospholipid invertase activity.
According to some embodiments of the invention, the forming a recombinant protein comprises:
(1) Providing original protein, and connecting a TEV enzyme cutting site and 2MBP labels in a gene C section of the original protein to form a recombinant protein gene; the original protein includes TMEM41B protein.
(2) Transferring the recombinant protein gene to a pFastBacDual expression vector;
(3) The pFastBacDual expression vector was transferred to Sf9 insect cells.
According to some embodiments of the invention, the expressing recombinant protein comprises: culturing the Sf9 insect cell under conditions suitable for expression of the protein. Further, the temperature suitable for expression is 25-28 ℃, and the time suitable for expression is 48-52 hours.
According to some embodiments of the invention, the performing protein purification comprises: at least one incubation of the expressed recombinant protein is performed, centrifugation is performed to remove soluble proteins, to remove periplasm proteins, to extract proteins and to separate undissolved proteins and/or impurities. Further, the incubation temperature is 3-5 ℃, and the centrifugation temperature is 3-5 ℃. The purpose of the incubation is to lyse the cells and thereby aid in the purification effect.
According to some embodiments of the invention, the performing protein purification comprises:
(1) Using buffer A and buffer B to lyse cells and remove soluble proteins and peripheral membrane proteins;
(2) Extracting membrane protein by using an extract C;
wherein the buffer solution A comprises 20-30mmol/L Hepes, 8-15mmol/L magnesium chloride, 18-23mmol/L potassium chloride, 0.4-0.7mmol/L tricarboxyethyl phosphine, 750-850nmol/L aprotinin, 0.3-0.5 mmol/L4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mug/ml leupeptin;
the buffer solution B comprises 20-30mmol/L of hydroxyethyl piperazine ethanesulfonic acid, 8-15mmol/L of magnesium chloride, 0.8-1.2mol/L of NaCl, 0.4-0.7mmol/L of tricarboxyethyl phosphine, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride and 4-6 mu g/ml of leupeptin;
the extract C comprises: 20-30mmol/L of hydroxyethyl piperazine ethylsulfanilic acid, 1.5-2.5mmol/L of magnesium chloride, 0.08-0.12mol/L of NaCl, 0.3-0.7mmol/L of tricarboxyethyl phosphine, 9-12% of glycerol, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mu g/ml of leupeptin, 0.8-1.2% of dodecyl-BETA-D-maltoside, and 0.2-0.4% of cholesterol succinic acid monoester; the pH value of the hydroxyethyl piperazine ethylsulfanilic acid is 7-7.5.
According to some embodiments of the invention, the affinity chromatography comprises:
(1) Incubating in a sugar starch column for 30-50min;
(2) Washing at least 4 column volumes with a first washing liquid comprising: 20-30mmol/L of hydroxyethyl piperazine ethylsulfanilic acid, 0.08-0.12mol/L of NaCl, 1.5-2.5mmol/L of magnesium chloride, 0.4-0.7mmol/L of tricarboxyethyl phosphine, 4% -6% of glycerol, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mu g/ml of leupeptin, 0.04% -0.06% of dodecyl-beta-D-maltoside, 0.008% -0.012% of cholesterol succinic acid monoester;
(3) Washing at least 4 column volumes with a second washing liquid comprising: 20-30mmol/L of hydroxyethyl piperazine ethylsulfanilic acid, 0.08-0.12mol/L of NaCl, 1.5-2.5mmol/L of magnesium chloride, 0.4-0.7mmol/L of tricarboxyethyl phosphine, 1.8-2.2% of glycerol, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mu g/ml of leupeptin, 0.02% -0.04% of dodecyl-beta-D-maltoside and 0.006% -0.008% of cholesterol succinic monoester.
According to some embodiments of the invention, the directly performing enzyme digestion on the column overnight, removing the 2MBP tag to obtain the target protein specifically comprises:
(1) Directly adding at least 1 column volume of second cleaning solution, adding a proper amount of TEV protease, and enzyme-cutting at 0-10deg.C overnight to remove 2MBP tag;
(2) Collecting through a gravity column, collecting with at least 2 column volumes of a second cleaning solution;
(3) Mixing the collected proteins, and concentrating to 1.8-2.2mL by using a 100KD truncated tube;
(4) The target protein is obtained by molecular gel exclusion chromatography.
According to some embodiments of the invention, the method for purifying a protein having phospho-invertase activity comprises the steps of:
(S1) providing an original protein, and ligating a TEV cleavage site and 2MBP tags into the gene C segment of the original protein to form a recombinant protein gene;
(S2) transferring the recombinant protein gene to a pFastBacDual expression vector;
(S3) transferring the pFastBacDual expression vector to Sf9 insect cells;
(S4) culturing the Sf9 insect cell under conditions suitable for expression of the protein to obtain a recombinant cell; the temperature suitable for expression is 25-28 ℃, and the time suitable for expression is 48-52 hours;
(S5) incubating the recombinant cells with buffer A at 4℃for 30 minutes; the buffer solution A comprises 20-30mmol/L Hepes, 8-15mmol/L magnesium chloride, 18-23mmol/L potassium chloride, 0.4-0.7mmol/L tricarboxyethyl phosphine, 750-850nmol/L aprotinin, 0.3-0.5 mmol/L4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mug/ml leupeptin; the pH value of the hydroxyethyl piperazine ethylsulfanilic acid is 7-7.5;
(S6) centrifuging the incubated product at 4 ℃ for 20-40min, and discarding the upper layer;
(S7) mixing with the buffer B, homogenizing the precipitate by a homogenizer until no particles exist, and then incubating for 30min at 4 ℃; the buffer solution B comprises 20-30mmol/L of hydroxyethyl piperazine ethanesulfonic acid, 8-15mmol/L of magnesium chloride, 0.8-1.2mol/L of NaCl, 0.4-0.7mmol/L of tricarboxyethyl phosphine, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride and 4-6 mu g/ml of leupeptin; the pH value of the hydroxyethyl piperazine ethylsulfanilic acid is 7-7.5;
(S8) centrifuging the incubated product at 4 ℃ for 20-40min, and discarding the upper layer;
(S9) mixing with the extract C, homogenizing the precipitate until no particles exist, adding 1% dodecyl-BETA-D-maltoside and 0.2% cholesterol succinic monoester, and incubating at 4deg.C for 2 hr; the extract C comprises: 20-30mmol/L of hydroxyethyl piperazine ethylsulfanilic acid, 1.5-2.5mmol/L of magnesium chloride, 0.08-0.12mol/L of NaCl, 0.3-0.7mmol/L of tricarboxyethyl phosphine, 9-12% of glycerol, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mu g/ml of leupeptin, 0.8-1.2% of dodecyl-BETA-D-maltoside, and 0.2-0.4% of cholesterol succinic acid monoester; the pH value of the hydroxyethyl piperazine ethylsulfanilic acid is 7-7.5;
(S10) continuously centrifuging the incubated product at 4 ℃ for 20-30min;
(S11) taking supernatant, and incubating the supernatant for 30-50min at the temperature of 4 ℃ in a sugar starch column;
(S12) washing 5 column volumes with a first washing liquid;
(S13) clearing 5 column volumes with a second cleaning solution;
(S14) adding 1 column volume of the second cleaning solution, adding an appropriate amount of TEV, mixing, and performing enzyme digestion at 4 ℃ overnight to cut off the MBP label;
(S15) collecting through a gravity column, washing 2 column volumes with a second washing liquid and collecting;
(S16) mixing the collected proteins and concentrating the mixture to 1.8-2.2mL by using a 100KD truncated tube;
(S17) obtaining the target protein by molecular gel exclusion chromatography.
According to some embodiments of the invention, the protein of interest has phospholipidinvertase activity.
To achieve the above object, a second aspect of the present invention discloses a protein having phospholipidinvertase activity, which is produced by any of the purification methods described above.
Therefore, the beneficial effects of the invention include:
according to the method for purifying the protein with the phospholipide invertase activity, the 2MBP label is selected and added, and the 2MBP label is directly removed after protein purification and affinity chromatography are carried out, so that the protein with the phospholipide invertase activity with larger yield is obtained, and conditions can be provided for in vitro functional activity macromolecular machine recombination;
the purification method is simple and is easy to realize industrialization.
[ description of the drawings ]
FIG. 1 is a method for purifying a protein having phospholipidinvertase activity in an embodiment of the present invention;
FIG. 2 is a graph showing the comparison of the purification effect of the protein of the present invention in example 1 and comparative example 1;
FIG. 3 is a size exclusion chromatogram of the Opsin protein after cleavage of the 2Strep-Flag tag in comparative example 1;
FIG. 4 SDS-PAGE of Opsin proteins after cleavage of the 2Strep-Flag tag in comparative example 1;
FIG. 5 is a size exclusion chromatogram of the Opsin protein after cleavage of the 2MBP tag in example 1;
FIG. 6 is a SDS-PAGE of the Opsin protein after cleavage of the 2MBP tag in example 1;
FIG. 7 is a diagram of unlabeled Opsin protein from comparative example 1, after eluting the 2 MBP-tagged Opsin protein from the column overnight, removing the 2MBP tag by affinity chromatography, and then by size exclusion chromatography;
FIG. 8 is a SDS-PAGE of unlabeled Opsin protein from comparative example 1 with 2MBP tag after elution from the column overnight and then by size exclusion chromatography after removal of 2MBP tag by affinity chromatography;
FIG. 9 is a diagram of unlabeled Opsin protein from example 1 with a 2MBP tag directly after overnight cleavage on column material by size exclusion chromatography;
FIG. 10 is a SDS-PAGE of unlabeled Opsin protein from example 1 of FIG. 9 after direct digestion on the column overnight by size exclusion chromatography.
[ detailed description ] of the invention
For the purpose of making the technical solution and advantages of the present invention more apparent, the present invention will be further described in detail below with reference to the accompanying drawings and examples of implementation. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Phospholipases are a molecular machine capable of transporting macromolecules such as lipids across membranes. Phospholipases are generally derived from the purification of natural membrane proteins. However, the existing protein purification method has the defects of low content, low yield, complex living purification process and the like. Therefore, the embodiment of the invention provides a purification system with higher protein yield and protein activity, thereby laying a good foundation for deeply exploring the physiological function of the purification system and taking the purification system as a drug target to carry out small molecule drug screening and treating related diseases, and providing a reliable tool for application research.
The following describes the technical scheme provided by the embodiment of the invention with reference to the attached drawings.
Example 1
As shown in fig. 1, a method for purifying a protein having phospholipidinvertase activity, comprising: constructing a 2MBP tag protein expression vector of the target protein to form recombinant protein, expressing the recombinant protein, purifying the protein, separating by utilizing affinity chromatography to obtain semi-finished product protein, and directly removing the 2MBP tag on the semi-finished product protein to obtain the target protein.
Wherein the protein of interest: including but not limited to membrane proteins such as TMEM41B, VMP 1. In particular, the TMEM41B membrane protein is a phospholipid invertase which is long sought in the art.
Specifically, the purification method comprises the following steps:
(1) Providing original protein, and connecting a TEV enzyme cutting site and 2MBP labels in a gene C section of the original protein to form a recombinant protein gene.
(2) Transferring the recombinant protein gene to a pFastBacDual expression vector;
(3) Transferring the pFastBacDual expression vector to Sf9 insect cells;
(4) Culturing the Sf9 insect cell under conditions suitable for expression of the protein to obtain a recombinant cell;
specifically, recombinant protein was expressed at 27 ℃, the Sf9 insect cell density was 2.5×106 (please provide units of this density) for 48h; centrifuging the expressed solution at 4deg.C for 10min, and collecting cell precipitate to obtain recombinant cell.
(5) Incubating the cell pellet with buffer a to lyse the cells, in particular, incubating in buffer a comprising: 25mmol/L Hepes, 10 mmol/L magnesium chloride, 20 mmol/L potassium chloride, 0.5mmol/L tricarboxyethyl phosphine, 800 nmol/L aprotinin, 0.4 mmol/L4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 5 μg/ml leupeptin;
the incubation temperature was 4℃and the incubation time was 30min. By this incubation, the recombinant cells were lysed and centrifuged for 30min. After centrifugation, the supernatant was discarded.
(6) Re-suspending the precipitate with buffer B, homogenizing the precipitate with a homogenizer until no granule exists, and incubating at 4deg.C for 30min; centrifuging at 4deg.C for 30min after incubation, and removing supernatant after centrifugation;
the buffer B comprises 25mmol/L of hydroxyethyl piperazine ethanesulfonic acid, 10 mmol/L of magnesium chloride, 1 mol/L of NaCl, 0.5mmol/L of tricarboxyethyl phosphine, 800 nmol/L of aprotinin, 0.4mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride and 5 mug/ml of leupeptin.
Cells can be lysed and soluble proteins and peripheric membrane proteins removed via buffer A and buffer B.
(7) Extracting membrane protein by using an extract C;
specifically, the precipitate is resuspended with extract C, homogenized with a homogenizer until no more particles are present, then dodecyl-BETA-D-maltoside with a concentration of 1% and 0.2% cholesterol succinate monoester are added, and incubation is continued for 2 hours at 4deg.C; centrifuging at 4deg.C for 30min after incubation, and collecting supernatant;
the extract C comprises: 25mmol/L of hydroxyethylpiperazine ethylsulfanilic acid, 2mmol/L of magnesium chloride, 0.1 mol/L of NaCl, 0.5mmol/L of tricarboxyethyl phosphine, 10% of glycerol, 800 nmol/L of aprotinin, 0.4mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 5. Mu.g/ml of leupeptin;
(8) Centrifuging the membrane protein to separate undissolved protein;
specifically, the supernatant and the sugar starch column were incubated at 4 ℃ for 40 minutes;
(9) Affinity chromatography separation of the membrane protein;
specifically, the product after the incubation of step (8) is washed with a first washing liquid for 5 column volumes, and then with a second washing liquid for 5 column volumes.
Wherein the first cleaning liquid comprises: 25mmol/L of hydroxyethylpiperazine ethylsulfanilic acid, 0.1 mol/L of NaCl, 2mmol/L of magnesium chloride, 0.5mmol/L of tricarboxyethyl phosphine, 5% glycerol, 800 nmol/L of aprotinin, 0.4mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 5. Mu.g/ml of leupeptin, 0.05% of dodecyl-beta-D-maltoside, 0.01% of cholesterol succinic monoester;
the second cleaning liquid comprises: 25mmol/L of hydroxyethyl piperazine ethanesulfonic acid, 0.1 mol/L of NaCl, 2mmol/L of magnesium chloride, 0.5mmol/L of tricarboxyethyl phosphine, 2% of glycerol, 800 nmol/L of aprotinin, 0.4mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 5 mug/ml of leupeptin, 0.03% of dodecyl-BETA-D-maltoside and 0.006% of cholesterol succinic monoester.
(10) Cleaving the MBP tag in the membrane protein by TEV enzyme;
specifically, adding 1 column volume and a proper amount of TEV into the product after chromatographic separation in the step (6), uniformly mixing, and then carrying out enzyme digestion at 4 ℃ overnight;
(11) Collecting proteins after enzyme digestion overnight through a gravity column, and then washing 2 column volumes with a second washing liquid and collecting; the collected proteins were then mixed and concentrated to 2mL using a 100KD cutoff tube.
(12) Separating by size exclusion chromatography to obtain the target protein.
Comparative example 1
The difference compared to example 1 is that 2Strep tags are used instead of the MBP tag.
Comparative example 2
The difference compared to example 1 is that the Opsin protein with 2MBP tag was eluted from the column, digested overnight, and subjected to affinity chromatography to remove the 2MBP tag, followed by size exclusion chromatography to obtain the unlabeled Opsin protein.
Performance testing
For better testing, the purification effect of the protein with phosphoinvertase activity of the present invention was evaluated by SDS-PAGE patterns.
FIG. 2 shows a comparison of the purification effect of the protein of the invention in example 1 and comparative example 1.
The first column corresponding to "1" in the figure indicates the Opsin-TEV-2Strep-Flag protein after being combined with the column, the second column corresponding to "2" in the figure indicates the Opsin protein after 2Strep-Flag labels are cut off on the column, the 3 rd and 4 th columns corresponding to "3" and "4" in the figure indicate the Opsin protein without labels which is penetrated and eluted after enzyme digestion overnight on the column, the 5 th column corresponding to "5" in the figure indicates Marker, and the "6789" and "1,2,3,4" in the figure respectively indicate the Opsin-TEV-2MBP proteins.
FIGS. 3 to 6 show graphs comparing the purification effect of the protein of example 1 of the present invention with that of comparative example 1:
specifically, FIG. 3 shows a size exclusion chromatogram of the Opsin protein after cleavage of the 2Strep-Flag tag in comparative example 1; FIG. 4 SDS-PAGE of Opsin proteins after cleavage of the 2Strep-Flag tag in comparative example 1; FIG. 5 is a size exclusion chromatogram of the Opsin protein after cleavage of the 2MBP tag in example 1; FIG. 6 is a SDS-PAGE of Opsin protein after cleavage of the 2MBP tag in example 1.
Based on the results shown in FIGS. 3 to 6, it can be seen that the same protein has a higher ultraviolet absorption value in FIG. 5 than in FIG. 3, and the amount of the target protein (the depth of the band) in FIG. 6 is greater than in FIG. 4. The number of bands of the target protein on the SDS-PAGE gel shown in FIGS. 4 and 6 corresponds to the amount of protein produced by the purification method having phospholipidinvertase activity.
According to the protein purification method provided by the invention, the added MBP label can increase the expression level and solubility of the target protein, and the stability and correct folding of the protein are enhanced. The 2MBP label is added with one MBP label on the basis of the MBP label, so that the expression level and the solubility of the protein are higher.
FIGS. 7 to 10 show graphs comparing the purification effect of the proteins of example 1 of the present invention with that of comparative example 2:
specifically, fig. 7 shows that the Opsin protein with the 2MBP tag is eluted from the column, digested overnight, subjected to affinity chromatography to remove the 2MBP tag, and subjected to size exclusion chromatography to obtain the unlabeled Opsin protein. FIG. 8 is a SDS-PAGE of FIG. 7. FIG. 9 is a graph of unlabeled Opsin protein from size exclusion chromatography after digestion of 2 MBP-tagged Opsin protein directly on a column overnight. FIG. 10 is a SDS-PAGE of FIG. 9.
By comparing FIG. 7 with FIG. 9, FIG. 8 and FIG. 10, it can be seen that the target protein content obtained after the direct digestion on the column overnight is higher, wherein the higher the peak height of the UV absorption of FIG. 7 and FIG. 9, and the darker the color of the band, the higher the content of the band on the SDS-PAGE gel of FIG. 8 and FIG. 10.
From the above experimental comparison, it is clear that the protein yield of the phosphoinvertase obtained by purifying 2 MBPs is more than 3 times of the corresponding protein yield by purifying 2 streps. The invention carries out purification treatment by carrying 2MBP labels on the protein C end of the phosphoinvertase, which is beneficial to improving the yield of phosphoinvertase protein.
Finally, it should be understood that the foregoing embodiments are merely illustrative of the technical solutions of the present invention, and that although the present invention has been described in detail with reference to the above preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solutions of the present invention.
It should be appreciated that reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Those skilled in the art will also appreciate that the embodiments described in the specification are alternative embodiments and that the acts and modules referred to are not necessarily required for the present invention.
In various embodiments of the present invention, it should be understood that the sequence numbers of the foregoing processes do not imply that the execution sequences of the processes should be determined by the functions and internal logic of the processes, and should not be construed as limiting the implementation of the embodiments of the present invention.
The above description of the protein with phospholipidosis invertase activity and its purification method disclosed in the present examples is provided for the purpose of illustrating the principles and embodiments of the present invention by applying specific examples, and the above examples are only for the purpose of helping to understand the method and core idea of the present invention; meanwhile, as for those skilled in the art, according to the idea of the present invention, there are changes in the specific embodiments and the application scope, and in summary, the present disclosure should not be construed as limiting the present invention, and any modifications, equivalent substitutions and improvements made within the principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A method for purifying a protein having phospholipidinvertase activity, comprising:
constructing a 2MBP tag protein expression vector of the target protein to form recombinant protein;
carrying out protein purification after expressing recombinant proteins;
directly performing enzyme digestion on the column material overnight after affinity chromatography, and removing the 2MBP tag to obtain target protein;
the forming of the recombinant protein comprises:
providing original protein, and connecting a TEV enzyme cutting site and 2MBP labels in a gene C section of the original protein to form a recombinant protein gene; transferring the recombinant protein gene to a pFastBacDual expression vector,
transferring the pFastBacDual expression vector to Sf9 insect cells;
the original protein is an Opsin protein.
2. The method of purification according to claim 1, wherein the expression of recombinant protein comprises:
culturing the Sf9 insect cell under conditions suitable for expression of the protein;
the temperature suitable for expression is 25-28 ℃, and the time suitable for expression is 48-52 hours.
3. The method of claim 1, wherein the performing protein purification comprises:
at least one incubation of the expressed recombinant protein, centrifugation, removal of soluble proteins, removal of periplasm proteins, extraction of proteins and separation of undissolved proteins and/or impurities;
the incubation temperature is 3-5 ℃, and the centrifugation temperature is 3-5 ℃.
4. A method of purifying according to claim 3, wherein the performing protein purification comprises:
using buffer A and buffer B to lyse cells and remove soluble proteins and peripheral membrane proteins;
wherein the buffer solution A comprises 20-30mmol/L Hepes, 8-15mmol/L magnesium chloride, 18-23mmol/L potassium chloride, 0.4-0.7mmol/L tricarboxyethyl phosphine, 750-850nmol/L aprotinin, 0.3-0.5 mmol/L4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mug/ml leupeptin;
the buffer solution B comprises 20-30mmol/L of hydroxyethyl piperazine ethanesulfonic acid, 8-15mmol/L of magnesium chloride, 0.8-1.2mol/L of NaCl, 0.4-0.7mmol/L of tricarboxyethyl phosphine, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride and 4-6 mu g/ml of leupeptin.
5. The method according to claim 4, further comprising, after removing the soluble protein and the peripheral membrane protein:
extracting membrane protein by using an extract C;
the extract C comprises: 20-30mmol/L of hydroxyethyl piperazine ethylsulfanilic acid, 1.5-2.5mmol/L of magnesium chloride, 0.08-0.12mol/L of NaCl, 0.3-0.7mmol/L of tricarboxyethyl phosphine, 9-12% of glycerol, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mu g/ml of leupeptin, 0.8-1.2% of dodecyl-BETA-D-maltoside, and 0.2-0.4% of cholesterol succinic acid monoester; the pH value of the hydroxyethyl piperazine ethylsulfanilic acid is 7-7.5.
6. The purification method of claim 1, wherein the affinity chromatography separation comprises:
incubating in a sugar starch column for 30-50min;
washing at least 4 column volumes with a first washing liquid comprising: 20-30mmol/L of hydroxyethyl piperazine ethylsulfanilic acid, 0.08-0.12mol/L of NaCl, 1.5-2.5mmol/L of magnesium chloride, 0.4-0.7mmol/L of tricarboxyethyl phosphine, 4% -6% of glycerol, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mu g/ml of leupeptin, 0.04% -0.06% of dodecyl-beta-D-maltoside, 0.008% -0.012% of cholesterol succinic acid monoester;
washing at least 4 column volumes with a second washing liquid comprising: 20-30mmol/L of hydroxyethyl piperazine ethylsulfanilic acid, 0.08-0.12mol/L of NaCl, 1.5-2.5mmol/L of magnesium chloride, 0.4-0.7mmol/L of tricarboxyethyl phosphine, 1.8-2.2% of glycerol, 750-850nmol/L of aprotinin, 0.3-0.5mmol/L of 4- (2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 4-6 mu g/ml of leupeptin, 0.02% -0.04% of dodecyl-B-D-maltoside and 0.006% -0.008% of cholesterol succinic monoester.
7. The method of claim 6, wherein the directly cleaving the column overnight to remove the 2MBP tag to obtain the protein of interest further comprises:
directly adding at least 1 column volume of second cleaning solution, adding a proper amount of TEV protease, and enzyme-cutting at 0-10deg.C overnight to remove 2MBP tag;
collecting through a gravity column, collecting with at least 2 column volumes of a second cleaning solution;
mixing the collected proteins, and concentrating to 1.8-2.2mL by using a 100KD truncated tube;
the target protein is obtained by molecular gel exclusion chromatography.
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