CN113121636B - Complex for enriching n-tridecanol conjugate and preparation method and application thereof - Google Patents
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- XFRVVPUIAFSTFO-UHFFFAOYSA-N 1-Tridecanol Chemical compound CCCCCCCCCCCCCO XFRVVPUIAFSTFO-UHFFFAOYSA-N 0.000 title claims abstract description 96
- 238000002360 preparation method Methods 0.000 title abstract description 13
- 230000005291 magnetic effect Effects 0.000 claims abstract description 74
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 60
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 59
- 235000018102 proteins Nutrition 0.000 claims abstract description 58
- 239000011324 bead Substances 0.000 claims abstract description 53
- 238000000034 method Methods 0.000 claims abstract description 38
- 239000000203 mixture Substances 0.000 claims abstract description 38
- 238000005886 esterification reaction Methods 0.000 claims abstract description 25
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 23
- 150000001875 compounds Chemical class 0.000 claims abstract description 19
- 108010064851 Plant Proteins Proteins 0.000 claims abstract description 15
- 235000021118 plant-derived protein Nutrition 0.000 claims abstract description 15
- 230000003993 interaction Effects 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 107
- 239000003446 ligand Substances 0.000 claims description 36
- 241000196324 Embryophyta Species 0.000 claims description 20
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 15
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 15
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 14
- 230000027455 binding Effects 0.000 claims description 14
- 238000002156 mixing Methods 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 239000007864 aqueous solution Substances 0.000 claims description 11
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000007983 Tris buffer Substances 0.000 claims description 8
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 claims description 8
- 239000003480 eluent Substances 0.000 claims description 7
- 239000011780 sodium chloride Substances 0.000 claims description 7
- 239000002904 solvent Substances 0.000 claims description 7
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 238000000926 separation method Methods 0.000 claims description 6
- 239000004094 surface-active agent Substances 0.000 claims description 6
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 5
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 5
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 5
- 238000010828 elution Methods 0.000 claims description 5
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 4
- 239000012460 protein solution Substances 0.000 claims description 4
- 241000219000 Populus Species 0.000 claims description 3
- -1 hydrocarbon chain carboxylic acid Chemical class 0.000 claims description 3
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 230000005298 paramagnetic effect Effects 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 2
- 239000002131 composite material Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- REZQBEBOWJAQKS-UHFFFAOYSA-N triacontan-1-ol Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCCCCCCCO REZQBEBOWJAQKS-UHFFFAOYSA-N 0.000 abstract description 18
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 230000008827 biological function Effects 0.000 abstract 1
- 230000002068 genetic effect Effects 0.000 abstract 1
- 230000037361 pathway Effects 0.000 abstract 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 8
- 230000001737 promoting effect Effects 0.000 description 8
- BAQMYDQNMFBZNA-UHFFFAOYSA-N N-biotinyl-L-lysine Natural products N1C(=O)NC2C(CCCCC(=O)NCCCCC(N)C(O)=O)SCC21 BAQMYDQNMFBZNA-UHFFFAOYSA-N 0.000 description 4
- BAQMYDQNMFBZNA-MNXVOIDGSA-N biocytin Chemical group N1C(=O)N[C@@H]2[C@H](CCCCC(=O)NCCCC[C@H](N)C(O)=O)SC[C@@H]21 BAQMYDQNMFBZNA-MNXVOIDGSA-N 0.000 description 4
- 229960002685 biotin Drugs 0.000 description 4
- 235000020958 biotin Nutrition 0.000 description 4
- 239000011616 biotin Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 108010090804 Streptavidin Proteins 0.000 description 3
- 239000011230 binding agent Substances 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholiniumyl)ethanesulfonate Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 2
- 108090000288 Glycoproteins Proteins 0.000 description 2
- 102000003886 Glycoproteins Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 210000005069 ears Anatomy 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 230000029553 photosynthesis Effects 0.000 description 2
- 238000010672 photosynthesis Methods 0.000 description 2
- 239000012474 protein marker Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000003260 vortexing Methods 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 108091032955 Bacterial small RNA Proteins 0.000 description 1
- 241000499489 Castor canadensis Species 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 235000011779 Menyanthes trifoliata Nutrition 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000012271 agricultural production Methods 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 150000001615 biotins Chemical class 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000000111 isothermal titration calorimetry Methods 0.000 description 1
- 239000000696 magnetic material Substances 0.000 description 1
- 238000007885 magnetic separation Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000002366 mineral element Substances 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 108010051423 streptavidin-agarose Proteins 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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- 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
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N31/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic oxygen or sulfur compounds
- A01N31/02—Acyclic compounds
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/08—Magnoliopsida [dicotyledons]
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Agronomy & Crop Science (AREA)
- Organic Chemistry (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pest Control & Pesticides (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a compound for enriching n-tridecanol conjugate, which comprises the following components: a n-tridecanol moiety and a carboxylic magnetic bead, the carboxylic magnetic bead moiety being capable of being driven by a magnetic field to separate a complex or a complex containing the complex from a mixture system. The invention also discloses a preparation method of the compound, which comprises the following steps: the n-tridecanol and the carboxyl magnetic beads are subjected to esterification reaction to generate the magnetic beads with the n-tridecanol distributed on the surface. The invention also discloses a method for enriching the n-tridecanol interaction protein from the total plant protein by the compound. The method can enrich the n-triacontanol interaction protein, so as to further research the biological function and related genetic pathways of the n-triacontanol interaction protein and promote the improvement of the plant production performance.
Description
Technical Field
The invention belongs to the field of biochemistry, relates to a compound for enriching a n-tridecanol conjugate and a preparation method thereof, and particularly relates to a compound for enriching a n-tridecanol interaction protein based on magnetic separation and a preparation method and application thereof.
Background
The effects of the n-triacontanol (also called triacontanol, 1-triacontanol) are mainly embodied in promoting the plant to grow roots, promoting the plant to absorb mineral elements, promoting the photosynthesis of leaves, promoting the germination of flower buds and the like. In agricultural production, the application of n-tridecanol can increase the number of small ears and grains per ear of crops.
There is no report on the n-tridecanol interaction protein, and the main reason is that there is no effective method for finding the protein interacting with n-tridecanol.
It is possible that n-tridecanol functions together with its interacting protein, but the detailed mechanism thereof has not been reported at present.
If the components such as protein and the like interacting with the n-tridecanol in the plant tissues can be found, the further research on the composition and the properties of the components is facilitated, and further, the synthesis and decomposition routes of the n-tridecanol or the potential signal path initiated or participated by the n-tridecanol are intervened manually, so that the production performance of crops is improved.
Disclosure of Invention
In order to solve the problems of the prior art, the first aspect of the present invention provides a complex for enriching a n-tridecanol conjugate, the complex comprising a n-tridecanol moiety and a ligand moiety, the ligand moiety being capable of being driven to separate the complex or a complex containing the complex from a mixture system.
In some embodiments, the n-tridecanol conjugate is an n-tridecanol interacting protein.
In some embodiments, the n-tridecanol moiety and the ligand moiety are linked by a covalent bond.
In some embodiments, the n-tridecanol moiety and the ligand moiety are directly linked.
In some embodiments, the ligand moiety is capable of specifically binding to a ligand binder such that the complex or a complex containing the complex is separated from a mixture system by the ligand binder.
In some embodiments, the material of the ligand moiety is a modified biotin molecule capable of binding to n-tridecanol and the ligand conjugate is immobilized streptavidin.
In some embodiments, the material of the ligand moiety is biotin with a terminal carboxyl linker arm attached.
In some embodiments, the material of the ligand moiety is biocytin.
In some embodiments, the ligand binder is streptavidin sepharose.
In some embodiments, the n-tridecanol moiety and the ligand moiety are indirectly linked.
In some embodiments, the ligand moiety is a magnetic material, which can be actuated by a magnetic field to separate the complex or a complex containing the complex from a mixture system.
In some embodiments, the ligand moiety is a magnetic bead, the surface of which is modified with an active arm having a terminal group capable of reacting with an alcoholic hydroxyl group.
In some embodiments, the magnetic bead is a paramagnetic bead.
In some embodiments, the magnetic beads have a particle size of 30 to 100 microns (e.g., 40 microns, 50 microns, 60 microns, 70 microns, 80 microns, 90 microns).
In some embodiments, the magnetic beads are carboxyl magnetic beads.
In some embodiments, the active arm terminates with a carboxyl group.
In some embodiments, the active arm is C10-15The hydrocarbon chain carboxylic acid of (1).
In a second aspect, the present invention provides a method for preparing the complex of the first aspect, the method comprising: chemically reacting the n-tridecanol and the ligand moiety starting materials to form the complex comprising the n-tridecanol moiety and the ligand moiety.
In some embodiments, the chemical reaction is an esterification reaction.
In some embodiments, the esterification reaction is an esterification reaction between n-tridecanol and biotin having a terminal carboxyl linker arm attached thereto.
In some embodiments, the esterification reaction is an esterification reaction between n-tridecanol and biocytin.
In some embodiments, the esterification reaction is an esterification reaction between n-tridecanol and a carboxylic magnetic bead.
In some embodiments, the esterification reaction is: and mixing the n-tridecanol solution with the carboxyl magnetic bead solution, and carrying out esterification reaction to obtain the crude compound.
In some embodiments, the n-tridecanol solution is an ethanolic n-tridecanol solution.
In some embodiments, the amount of n-tridecanol solution to ethanol is in a mass ratio of 2 to 6:1 (e.g., 3:1, 4:1, 5: 1).
In some embodiments, the n-tridecanol solution includes a surfactant.
In some embodiments, the surfactant is Tween-20.
In some embodiments, the n-tridecanol solution has a volume ratio of the surfactant to the ethanol of 0.5 to 2: 1.
In some embodiments, the solvent of the carboxylic magnetic bead solution is MES solution.
In some embodiments, the MES solution is 2- (N-morpholine) ethanesulfonic acid in an aqueous solution at a concentration of 10 to 30 g/L.
In some embodiments, the concentration of the solution of carboxylic magnetic beads is 30-70 mg/ml (e.g., 35 mg/ml, 40 mg/ml, 45 mg/ml, 50 mg/ml, 55 mg/ml, 60 mg/ml, 65 mg/ml).
In some embodiments, the carboxylic magnetic beads are used after washing.
In some embodiments, the carboxyl magnetic beads are washed with MES solution and used.
In some embodiments, the solvent of the carboxylic magnetic bead solution is MES-Mix solution.
In some embodiments, the MES-Mix solution is an aqueous solution containing 10 to 30 mg/ml 2- (N-morpholino) ethanesulfonic acid and 3 to 7 mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
In some embodiments, the concentration of the solution of carboxylic magnetic beads is 15-35 mg/ml (e.g., 18 mg/ml, 20 mg/ml, 22 mg/ml, 24 mg/ml, 26 mg/ml, 28 mg/ml, 30 mg/ml, 32 mg/ml).
In some embodiments, in the esterification reaction, the amount by mass ratio of the n-tridecanol to the carboxyl magnetic beads is: 1:20-40 (e.g., 1:22, 1:24, 1:26, 1:28, 1:30, 1:32, 1:34, 1:36, 1: 38).
In some embodiments, the esterification reaction temperature 20-30 ℃ (e.g., 22 ℃, 24 ℃, 26 ℃, 28 ℃).
In some embodiments, the esterification reaction time is 60-180min (e.g., 80min, 100min, 120min, 140min, 160 min).
In some embodiments, the blending occurs once every 3-10min during the esterification reaction.
In some embodiments, during the esterification reaction, the Mix solution is added to the reaction system once every 10 to 30 minutes, and the volume ratio of each addition amount to the reaction system is: 1:10-25.
In some embodiments, the Mix solution is an aqueous solution containing 10 to 30 mg/ml 2- (N-morpholino) ethanesulfonic acid and 30 to 90 mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
In some embodiments, the reaction system containing the crude complex is magnetically adsorbed, and the liquid components of the reaction system are discarded to obtain a purified complex.
In some embodiments, the purified complex is washed with a wash solution to obtain a refined complex.
In some embodiments, the wash solution is a PBST solution.
In some embodiments, the method of cleaning is: mixing the washing solution with the purified compound, magnetically adsorbing a reaction system containing the crude compound, and discarding liquid components in the reaction system to obtain a refined compound.
In some embodiments, the refined complex is dissolved in a TE-TW solution to provide a solution of the complex for use.
In some embodiments, the concentration of the complex in the complex solution is 4.0-8.0 mg/ml (e.g., 4.5 mg/ml, 5.0 mg/ml, 5.5 mg/ml, 6.0 mg/ml, 6.5 mg/ml, 7.0 mg/ml, 7.5 mg/ml).
In some embodiments, the TE-TW solution is an aqueous solution containing 0.5 to 1.5 v/v% Tris, 0.2 to 0.5g/L disodium EDTA, and 0.005 to 0.015 v/v% Tween-20.
In some embodiments, the complex solution is stored at a temperature of 1-6 deg.C (e.g., 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C).
In a third aspect, the present invention provides a method for enriching a n-tridecanol conjugate, which comprises: using the complex according to the first aspect of the invention, n-tridecanol conjugates are enriched in plant tissue.
In some embodiments, the plant is a poplar.
In some embodiments, the tissue is a root.
In some embodiments, the enrichment method comprises the steps of:
s1, mixing the plant total protein solution with the compound solution, and incubating to obtain a first mixture;
s2, driving the ligand moiety in the first mixture, and separating the complex or a complex containing the complex from the first mixture to obtain a separation product;
s3, eluting the separation product to obtain the n-tridecanol conjugate.
In some embodiments, the solvent is a protein binding solution, and the concentration of the total plant protein in the solution is 5-15 μ g/mL (e.g., 6 μ g/mL, 7 μ g/mL, 8 μ g/mL, 9 μ g/mL, 10 μ g/mL, 11 μ g/mL, 12 μ g/mL, 13 μ g/mL, 14 μ g/mL).
In some embodiments, the protein binding solution comprises 0.1-0.3M Tris,0.4-0.6M NaCl, 15-25mM MgCl20.8-1.2 v/v% Tween-20 in water, pH 7.5.
In some embodiments, in the solution of total plant protein, the total plant protein is total protein in the plant tissue as a dry powder.
In some embodiments, the concentration of the complex in the complex solution is 4.0-8.0 mg/ml (e.g., 4.5 mg/ml, 5.0 mg/ml, 5.5 mg/ml, 6.0 mg/ml, 6.5 mg/ml, 7.0 mg/ml, 7.5 mg/ml).
In some embodiments, the complex solution is a TE-TW solution.
In some embodiments, the TE-TW solution is an aqueous solution containing 0.5 to 1.5 v/v% Tris, 0.2 to 0.5g/L disodium EDTA, and 0.005 to 0.015 v/v% Tween-20.
In some embodiments, the amount by mass ratio of the total plant protein to the complex is: 1:30-100 (e.g., 1:40, 1:50, 1:60, 1:70, 1:80, 1: 90).
In some embodiments, in step S2, the incubation time is 2-10min (e.g., 3min, 4min, 5min, 6min, 7min, 8min, 9 min).
In some embodiments, in step S2, the incubation condition is on ice.
In some embodiments, in step S2, the complex or a complex containing the complex is separated from the first mixture by magnetically attracting the ligand moiety to obtain the separation product.
In some embodiments, in step S3, the ligand moiety is magnetically attracted and the separation product is eluted with a protein eluent comprising the n-tridecanol conjugate.
In some embodiments, in step S3, the elution time is 2-10min (e.g., 3min, 4min, 5min, 6min, 7min, 8min, 9 min).
In some embodiments, in step S3, the elution condition is on ice.
In some embodiments, the eluent composition is 10-30mM Tris,5-15mM NaCl,0.08-0.12 v/v% Tween-20 in water, pH 7.5.
In a fourth aspect, the present invention provides a n-tridecanol conjugate obtained by using the enrichment method according to the third aspect of the present invention.
The fifth aspect of the invention provides the use of a complex according to the first aspect of the invention, a method of preparing a complex according to the second aspect of the invention, an enrichment method according to the third aspect of the invention or a combination of n-tridecanol according to the fourth aspect of the invention in the preparation of a formulation for any one of promoting rooting of a plant, promoting mineral uptake by a plant, promoting photosynthesis in leaves, promoting germination of flower buds, increasing the number of ears of a crop and increasing the number of grains per ear of a crop.
Compared with the prior art, the invention has the advantages that: the method is more convenient to find the interacting protein of a certain substance, and is simpler and more practical and less in cost compared with the prior yeast single hybridization, isothermal titration calorimetry, nuclear magnetic resonance and other technologies. Through an immunization method, a method for researching triacontanol interaction protein is found, and a new thought is provided for researching interaction protein of other substances.
Drawings
FIG. 1 shows the electrophoresis of protein, wherein lane 1 represents protein marker, the molecular weight from top to bottom is: 250kDa, 150kDa, 100kDa, 70kDa, 50kDa, 40kDa, 30kDa, 25kDa, 20kDa, 15kDa, lane 2 represents total plant protein and lane 3 represents n-tridecanol-interacting protein.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be described in detail with reference to the accompanying drawings.
The invention provides a complex for enriching n-tridecanol and an enrichment method thereof, and the main technical concept is as follows:
binding of n-tridecanol to magnetic beads to form magnetic bead-n-tridecanol complex, incubating the complex with total plant protein to bind protein to the n-tridecanol moiety in the complex to form magnetic bead-n-tridecanol-protein complex. The magnetic beads are magnetically adsorbed by a magnetic frame so as to fix the magnetic bead-n-tridecanol-protein complex. And then incubating the complex and protein eluent together, and eluting the protein by changing the binding environment to obtain the protein interacted with the n-tridecanol.
The principle of the combination of magnetic beads and n-tridecanol is as follows: the carboxyl group of the magnetic beads and the hydroxyl group of the n-tridecanol are subjected to esterification reaction, and the n-tridecanol is tightly bonded to the magnetic beads through an ester bond.
Definition of
(1) N-tridecanol conjugate:
a biomolecule or biomolecule complex capable of specific binding to n-tridecanol through a non-covalent bond pattern, including but not limited to: proteins, glycoproteins, protein complexes, glycoprotein complexes, protein-glycoprotein complexes, and complexes of the foregoing with RNA.
When the n-tridecanol conjugate is a protein capable of specifically binding to n-tridecanol by a non-covalent bond pattern, the n-tridecanol conjugate can be referred to as a n-tridecanol interacting protein.
(2) N-tridecanol moiety:
also referred to as the n-tridecanol residue, refers to the moiety from n-tridecanol in the complex.
(3) Ligand moiety:
a ligand moiety refers to the moiety from a ligand in a complex, and when the ligand is a small molecule, the ligand moiety may also be referred to as a ligand residue.
The aforementioned n-tridecanol moiety is stably associated with the aforementioned ligand moiety, and in some cases, the two are associated by covalent bonding to form a complex for enriching the aforementioned n-tridecanol conjugate.
(4) Complex-containing complex:
in some cases, refers to a complex formed by combining a complex with an n-tridecanol conjugate.
(5) Is driven to separate the complex or complexes containing the complex from the mixture system:
including, but not limited to, 1) driving a ligand moiety (e.g., a magnetic bead, sometimes with a linker arm residue on its surface for binding to triacontanol) and driving the complex or complexes containing the complex to separate from the mixture system (which may contain the complex, potential triacontanol conjugates and other components) by a non-contact means (e.g., a magnetic field); 2) the complex or complex-containing complex is separated from the mixture system (which may contain a complex, a mixture of potential n-tridecanol conjugates and other components) by affinity driving of the ligand moiety using a solid phase (e.g., a support capable of immobilizing streptavidin) (e.g., an avidin biotin moiety), e.g., by binding the solid phase support to the ligand moiety of the complex or complex-containing complex, thereby removing the other components of the mixture system.
Example 1: preparation of the composite
Materials (I) and (II)
Carboxyl magnetic beads: commercially available from beaver heads, with a particle size of 30-100 microns, paramagnetic, and the surface of the magnetic beads is modified with a group of twelve carbon carboxylic acids.
Secondly, a solution preparation method comprises the following steps:
(1) MES solution preparation method:
19.5g of 2- (N-morpholine) ethanesulfonic acid was weighed into a 1L flask and made up to 1L with ultrapure water to form a MES solution.
(2) The Mix solution preparation method:
1 ml of MES solution was added to a 2 ml centrifuge tube, 60 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide was added thereto, and the mixture was sufficiently inverted to dissolve 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to form a Mix solution.
(3) The preparation method of the MES-Mix solution comprises the following steps:
add 58.4. mu.l of MES solution to 5.1. mu.l of Mix solution to form a MES-Mix solution.
(4) The preparation method of the n-tridecanol solution comprises the following steps:
weighing 2 mg of n-tridecanol, adding the n-tridecanol into a 2 ml centrifuge tube, adding 500 microliters of 60 ℃ absolute ethyl alcohol, fully inverting to dissolve the n-tridecanol, and adding 500 microliters of Tween-20 to form a n-tridecanol solution.
(5) The PBST solution formulation method:
8g of sodium chloride, 0.2g of potassium chloride, 1.44g of disodium hydrogen phosphate and 0.24g of potassium dihydrogen phosphate were dissolved in 800ml of deionized water, the pH was adjusted to 7.4 with hydrochloric acid, and 200. mu.l of Tween-20 was added to obtain a PBST solution.
(6) The preparation method of the TE-TW solution comprises the following steps:
adding 10 ml of tris aqueous solution (pH 8.0,1 mol/L) into a 1L conical flask, adding 0.336 g of disodium ethylene diamine tetraacetate, fixing the volume to 1L, and adding 100 microliters of Tween-20 to obtain a TE-TW solution. Storing at 4 ℃.
Thirdly, a complex synthesis process:
1. adding 30 microliters of carboxyl magnetic beads (50 mg/ml, wherein the solvent is MES solution) into a 1.5 ml centrifuge tube, adding 200 microliters of MES solution, reversing and mixing uniformly, adsorbing the carboxyl magnetic beads by using a magnetic frame, and adsorbing the MES solution; and adding 200 microliters of MES solution again, reversing and mixing uniformly, adsorbing the carboxyl magnetic beads by using a magnetic frame, and adsorbing the MES solution. The MES solution has the function of stabilizing the pH value and preventing the pH value of the solution from changing violently.
2. Add 60. mu.l MES-Mix solution to a 1.5 ml centrifuge tube and invert to resuspend the beads. The MES-Mix solution activates the magnetic beads.
3. 12.4. mu.l of magnetic beads were added to 200. mu.l centrifuge tubes, and 5. mu.l of n-tridecanol solution was added to each of the centrifuge tubes, for a total of five centrifuge tubes.
4. After blowing and mixing, the centrifuge tube was placed in a metal bath at 25 ℃ for 20 minutes and inverted every 5 minutes.
5. Then 1 microliter of Mix solution was added to each centrifuge tube. The Mix solution acts to activate the magnetic beads.
6. After vortexing, the tubes were placed in a 25 ℃ metal bath for 20 minutes, and inverted every 5 minutes.
7. An additional 1 microliter of Mix solution was added to each centrifuge tube.
8. After vortexing, the tubes were placed in a 25 ℃ metal bath for 80 minutes, and inverted every 5 minutes.
9. Adsorbing the carboxyl magnetic beads by using a magnetic frame, and adsorbing the solution in the centrifugal tube;
10. adding 200 microliters of PBST solution into the centrifugal tube, reversing and uniformly mixing, adsorbing carboxyl magnetic beads by using a magnetic frame, and adsorbing the PBST solution; adding 200 microliter PBST solution again, reversing and mixing evenly, adsorbing carboxyl magnetic beads by using a magnetic frame, and absorbing the PBST solution.
11. Adding 50 microliters of TE-TW solution into the centrifuge tube, reversing and uniformly mixing the solution and the resuspended magnetic beads to form a magnetic bead-n-tridecanol complex suspension, and storing at 4 ℃.
Example two: application of magnetic bead-n-tridecanol complex
1. Obtaining of Total plant protein
The 84K poplar root total protein dry powder was extracted according to the procedures described in the protocol of the plant protein extraction kit (C500053) of the Producer company.
2. Incubation
10 micrograms of dry plant total protein powder was dissolved in 1 ml of protein binding solution (0.2M Tris (pH 7.5),0.5M NaCl,20mM MgCl21 v/v% Tween-20 Detergent), 100. mu.l of a suspension of magnetic bead-n-tridecanol complex was added. On ice, bind for 5 minutes. Forming a magnetic bead-n-tridecanol-protein complex.
3. Magnetic treatment
The magnetic beads are magnetically adsorbed by a magnetic frame so as to fix the magnetic bead-n-tridecanol-protein complex.
The liquid component is removed.
4. Elution is carried out
Add 500. mu.l protein eluent (20mM Tris (pH 7.5),10mM NaCl,0.1 v/v% Tween-20 Detergent).
Place on ice for 5 minutes. The protein is eluted by changing the solution in such a way that the binding of n-tridecanol to the protein is disrupted.
The magnetic beads are magnetically adsorbed by a magnetic frame.
And sucking the liquid to obtain the protein interacted with the n-tridecanol.
5. Electrophoresis
Using conventional SDS-PAGE, stained with Coomassie Brilliant blue, protein Marker was added in lane 1 at molecular weights of 250, 150, 100, 70 (red), 50, 40, 30, 25 (red), 20, 15kDa, respectively, in sequence from top to bottom, the total plant protein sample obtained in step 1 above was added in lane 2, and the n-tridecanol interacting protein sample in lane 3. Electropherograms are shown in FIG. 1.
As can be seen from FIG. 1, the electrophoresis result in lane 3 shows two proteins, protein 1, with a molecular weight of about 70kDa and protein 2, with a molecular weight of about 20 kDa.
It can be seen that, in plants, n-tridecanol can be combined with protein 1 and protein 2, and protein 1 and protein 2 may act as plant hormones of n-tridecanol by forming a complex with n-tridecanol and other possible components. Up-regulating the expression of protein 1 and protein 2 in plants (e.g., by transgenically transferring protein 1 and protein 2 genes to stronger promoters) may increase the sensitivity of plants to n-tridecanol and promote plant growth.
More triacontanol interacting proteins may also be found when working with plant total proteins of different species, different tissues, and different developmental stages.
When whole cell homogenate is used instead of total plant protein, the magnetic bead-triacontanol complex may also separate other components involved in the protein, such as small RNA.
Therefore, the magnetic bead-n-tridecanol complex of the present invention has many potential uses.
EXAMPLE III use of biocytin-n-tridecanol Complex
The carboxyl of lysine in biocytin and the hydroxyl of n-tridecanol are subjected to esterification reaction to form a biocytin-n-tridecanol compound.
The compound is combined with the n-tridecanol interacting protein after being incubated with the total plant protein to form a biocytin-n-tridecanol interacting protein compound.
And (3) binding streptavidin agarose gel with biotin part of biocytin to capture biocytin-n-tridecanol interaction protein complex, further eluting and releasing n-tridecanol interaction protein.
It will be appreciated by those skilled in the art that the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The embodiments disclosed above are therefore to be considered in all respects as illustrative and not restrictive. All changes which come within the scope of or equivalence to the invention are intended to be embraced therein.
Claims (18)
1. The compound for enriching the n-tridecanol conjugate consists of n-tridecanol and a ligand, wherein the n-tridecanol and the ligand are connected through an ester bond, the ligand is a magnetic bead, the magnetic bead is a carboxyl magnetic bead, the surface of the magnetic bead is modified with an active arm, the tail end of the active arm is a carboxyl group, the n-tridecanol conjugate is a n-tridecanol interaction protein, and the n-tridecanol interaction protein is a protein capable of being specifically combined with the n-tridecanol through a non-covalent bond mode.
2. The complex of claim 1, wherein the magnetic bead is a paramagnetic bead.
3. The composite of claim 1, wherein the magnetic beads have a particle size of 30 to 100 microns.
4. The complex of claim 1, wherein the active arm is C10-15The hydrocarbon chain carboxylic acid of (1).
5. A method of preparing a complex according to any one of claims 1 to 4, said method comprising: and carrying out esterification reaction on the n-tridecanol and the carboxyl magnetic beads to form the complex comprising the n-tridecanol and the ligand.
6. The method of claim 5, wherein the esterification reaction is: mixing a n-tridecanol solution and a carboxyl magnetic bead solution, and carrying out esterification reaction to obtain a crude compound;
the n-tridecanol solution is an ethanol solution of n-tridecanol;
in the n-tridecanol solution, the mass ratio of the n-tridecanol to the ethanol is 2-6: 1;
the n-tridecanol solution contains a surfactant;
the surfactant is Tween-20;
in the n-tridecanol solution, the volume ratio of the dosage of the surfactant to the dosage of the ethanol is 0.5-2: 1;
the solvent of the carboxyl magnetic bead solution is MES-Mix solution;
the MES-Mix solution is an aqueous solution containing 10-30 mg/ml of 2- (N-morpholine) ethanesulfonic acid and 3-7 mg/ml of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide;
the concentration of the carboxyl magnetic bead solution is 15-35 mg/ml;
in the esterification reaction, the mass ratio of the n-tridecanol to the carboxyl magnetic beads is as follows: 1:20-40.
7. The method according to claim 6, wherein the carboxyl magnetic beads are washed with MES solution and used;
the MES solution is a 2- (N-morpholine) ethanesulfonic acid aqueous solution with the concentration of 10-30 g/L.
8. The method of claim 6, wherein the esterification reaction is carried out at a temperature of 20 to 30 ℃;
the esterification reaction time is 60-180 min;
uniformly mixing once every 3-10min in the esterification reaction process;
in the esterification reaction process, adding a Mix solution into a reaction system once at intervals of 10-30 minutes, wherein the volume ratio of each addition amount to the reaction system is as follows: 1: 10-25;
the Mix solution is an aqueous solution containing 10-30 mg/ml 2- (N-morpholine) ethanesulfonic acid and 30-90 mg/ml 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide.
9. The method according to claim 6, wherein the reaction system containing the crude complex is magnetically adsorbed, and a liquid component in the reaction system is discarded to obtain a purified complex.
10. The method according to claim 9, wherein the purified complex is washed with a washing solution to obtain a purified complex;
the washing solution is PBST solution;
the cleaning method comprises the following steps: mixing the washing solution with the purified compound, magnetically adsorbing a reaction system containing the crude compound, and discarding liquid components in the reaction system to obtain a refined compound.
11. The method according to claim 10, wherein the purified complex is dissolved in a TE-TW solution to obtain the complex solution for use;
in the complex solution, the concentration of the complex is 4.0-8.0 mg/ml;
the TE-TW solution is an aqueous solution containing 0.5-1.5 v/v% of tris (hydroxymethyl) aminomethane, 0.2-0.5g/L of disodium ethylenediaminetetraacetate and 0.005-0.015 v/v% of Tween-20.
12. The method according to claim 11, wherein the complex solution is stored at a temperature of 1 to 6 ℃.
13. An enrichment method of n-tridecanol conjugate, the enrichment method comprising the following steps: enriching n-tridecanol conjugates in plant tissue using the complex of any one of claims 1-4;
the plant is a poplar;
the tissue is a root;
the enrichment method comprises the following steps:
s1, mixing the plant total protein solution with the compound solution, and incubating to obtain a first mixture;
in the plant total protein solution, a solvent is a protein binding solution, and the concentration of the plant total protein is 5-15 mu g/mL;
the protein binding solution contains 0.1-0.3MTris,0.4-0.6M NaCl，15-25mM MgCl20.8-1.2 v/v% Tween-20 in water, pH 7.5;
in the complex solution, the concentration of the complex is 4.0-8.0 mg/ml;
in the complex solution, a solvent is TE-TW solution;
the TE-TW solution is an aqueous solution containing 0.5-1.5 v/v% of tris (hydroxymethyl) aminomethane, 0.2-0.5g/L of disodium ethylene diamine tetraacetate and 0.005-0.015 v/v% of Tween-20;
the dosage mass ratio of the total plant protein to the compound is as follows: 1: 30-100;
s2, attracting the ligand by magnetic force, and separating the complex or the complex containing the complex from the first mixture to obtain the separation product;
s3, attracting the ligand by magnetic force, eluting the separation product by protein eluent, wherein the eluent contains the n-tridecanol conjugate; the eluent comprises 10-30mM Tris,5-15mM NaCl,0.08-0.12 v/v% Tween-20 in water solution, and has pH of 7.5.
14. The enrichment method according to claim 13, wherein the total plant protein in the total plant protein solution is the dry powder of the total protein in the plant tissue.
15. The enrichment method of claim 13, wherein the incubation time is 2-10min in step S1.
16. The enrichment method of claim 13, wherein in step S1, the incubation condition is on ice.
17. The enrichment method of claim 13, wherein the elution time is 2-10min in step S3.
18. The enrichment method of claim 13, wherein the elution condition is on ice in step S3.
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