CN110294793A - SUMO fluorescence probe and preparation method thereof - Google Patents
SUMO fluorescence probe and preparation method thereof Download PDFInfo
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- CN110294793A CN110294793A CN201910550570.0A CN201910550570A CN110294793A CN 110294793 A CN110294793 A CN 110294793A CN 201910550570 A CN201910550570 A CN 201910550570A CN 110294793 A CN110294793 A CN 110294793A
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- 239000006166 lysate Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000011987 methylation Effects 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 210000003887 myelocyte Anatomy 0.000 description 1
- 230000006849 nucleocytoplasmic transport Effects 0.000 description 1
- 108010072637 phenylalanyl-arginyl-phenylalanine Proteins 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000029983 protein stabilization Effects 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 108010071207 serylmethionine Proteins 0.000 description 1
- 239000008925 shengjing Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000004885 white matter Anatomy 0.000 description 1
- 229920001285 xanthan gum Polymers 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G01N2333/4703—Regulators; Modulating activity
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- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
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Abstract
The present invention provides a kind of SUMO fluorescence probes and preparation method thereof.This method comprises: (1) prepares SUMO hydrazides SUMO2 (2-92)-NHNH based on the Intein N-S method migrated2；(2) it is based on SUMO hydrazides SUMO2 (the 2-92)-NHNH2, SUMO thioesters SUMO2 (2-92)-Mesna is obtained using hydrazides method；(3) it is reacted based on SUMO thioesters SUMO2 (the 2-92)-Mesna and fluorescein small molecule rhodamine, to obtain the SUMO fluorescence probe for having rhodamine.Preparation method provided by the present invention, which has, prepares yield height, and synthesis purity is high, operation is simple, the characteristics of can largely preparing, is suitble to the industrial application of SUMO fluorescence probe.
Description
Technical field
The present invention relates to protein synthesis technical fields, and in particular to a kind of SUMO fluorescence probe and preparation method thereof, especially
It is related to the preparation method of SUMO probe SUMO2-Rho110-Gly a kind of.
Background technique
Posttranslational modification (PTMs) be for many cell processes it is essential, be usually reversible.And have big
The posttranslational modification of amount is that (such as phosphorylation, methylation, acetylation and glycosylation) occurs in cell to live to adjust various life
It is dynamic.Its medium and small ubiquitin sample modification albumen (SUMO) change modification be one can be reversed by SUMO specific protease it is highly dynamic
Process, play an important role in various cell processes, such as adjust nucleo-cytoplasmic transport, protein stabilization, DNA damage repairs
It is multiple, Cycle Regulation, genetic transcription, differentiation and cellular localization etc..SUMO molecule by E1 activating enzymes, E2 desmoenzyme and
It is covalently bound under the participation of E3 ligase on substrate protein lysine residue, regulates and controls the structure and function of substrate protein, and
SUMO specific protease can be split from catalytic elimination SUMO on the target protein connecting with SUMO, and from its precursor protein
Solve SUMO, and then regulating cell function.For example, the progranulocyte leukemia egg of poly SUMOization (polySUMOylation)
White (PML) is preferentially identified by a kind of ubiquitin ligase RING Finger protein 4 (RNF4), then by uiquitin-protease
Enzyme body approach is degraded.This process is for most important with arsenic successful treatment acute promyelocytic leukemia.The phases such as cancer
Generation, the development of related disorders, are substantially Intracellular signals disorders, and the dynamic regulation of PTMs is unbalance, and the treatment of cancer relies on
In the explaination to these mechanism.In order to grasp effect of the SUMOization in related disease generation, need from gene to protein water
It is flat that comprehensive research is carried out to it.Therefore, by studying relevant SUMO fluorescence probe and Go to SUMOization enzyme capture probe,
Solve the dynamic change of SUMOization level in disease generating process in human body, and for tumour and other related diseases diagnosis and control
Treating and providing new method, technology and tool is current vital research direction.
However the preparation process of SUMO fluorescence probe also requires further improvement.
Summary of the invention
The present invention is directed to solve at least some of the technical problems in related technologies.For this purpose, of the invention
One purpose is to propose a kind of preparation method of SUMO fluorescence probe.The preparation side of SUMO fluorescence probe provided by the present invention
Method simple process is controllable, and at low cost, is suitble to the industrial production and application of SUMO fluorescence probe.Using provided by the present invention
Preparation method is particularly suitable for preparation SUMO2 probe SUMO2-Rho110-Gly, can be used as Go to SUMOization enzymatic activity screening
The important SUMO fluorescence probe with one kind of the screening of inhibitor.
In the research process of SUMO fluorescence probe, some small molecule fluorescent groups can always have fluorescence, and this is special
Property often by information valuable in biological experiment to masking, to hinder the fluorescent marker of imaging of biomolecules.
And rhodamine is as common coloring agent, since it has high absorption coefficient and high fluorescence in the visible light region of electromagnetic spectrum,
And have the characteristics that high-fluorescence quantum yield and photostability are strong and be widely used as fluorescence probe.Most as rhodamine B
The rhodamine 110 (Rho110) of final product, it is a kind of xanthan alkene dyestuff, is similar to fluorescein and eosin, conjugated pi abundant
Key makes itself have lasting and strong photoluminescent property.Therefore, Rhodamine 110 (Rho110) is now by as a kind of normal
The fluorescence probe and coloring agent seen have a wide range of applications in biomedical and industrial circle；And since its excellent light is steady
Qualitative and Photophysical Behaviors have potential application in the diagnosis of cancer in vivo.And fluorescence rhodamine 110 with ammonia
Base acid, polypeptide and small molecule combine, and are widely used in the bio-imaging of detecting cancer cell.Therefore, by Rho110
Have great importance with SUMO preparation SUMO fluorescence probe for the screening of Go to SUMOization enzymatic activity and the screening of inhibitor etc..
Specifically, the present invention provides the following technical scheme that
According to an aspect of the present invention, the present invention provides a kind of preparation methods of SUMO fluorescence probe, comprising: (1)
Method based on intein N-S migration prepares SUMO hydrazides SUMO2 (2-92)-NHNH2；(2) it is based on the SUMO hydrazides
SUMO2(2-92)-NHNH2, hydrazides method acquisition SUMO thioesters SUMO2 (2-92)-Mesna；(3) it is based on the SUMO thioesters
SUMO2 (2-92)-Mesna and fluorescein small molecule reaction, to obtain the SUMO fluorescence probe.
During preparing SUMO fluorescence probe, first with the strategy preparation SUMO hydrazides of Intein method N-S migration
SUMO2(2-92)-NHNH2, and SUMO thioesters SUMO2 (2-92)-Mesna is further converted to based on hydrazides method, in turn
By itself and fluorescein small molecule reaction, SUMO fluorescence probe is obtained.Method and step provided by the present invention is simple, and used
Each cost of material it is low, be suitble to large scale preparation obtain SUMO fluorescence probe.It is especially suitable for utilizing method provided by the present invention
SUMO probe SUMO2-Rho110-Gly is prepared, can be used for the screening etc. of the screening of Go to SUMOization enzymatic activity and inhibitor.
According to an embodiment of the invention, the preparation method of the above SUMO fluorescence probe may further include following skill
Art feature:
In some embodiments of the invention, the fluorescein small molecule rhodamine is Gly-Rho110-Gly, the band
The SUMO fluorescence probe for having rhodamine is SUMO2-Rho110-Gly.
In some embodiments of the invention, step (1) further comprises: (1-1) using containing be loaded with Intein and
The bacterial strain of the carrier of SUMO2 is cultivated, cracking, and separation obtains supernatant, is contained on the carrier for being loaded with Intein and SUMO2
There is Intein and SUMO2 coded sequence；The supernatant is carried out affinity chromatography by (1-2), obtains affinity chromatography product；(1-3)
Based on the affinity chromatography product, cutting process is carried out using the cutting buffer containing hydrazine hydrate, to obtain the SUMO
Hydrazides SUMO2 (2-92)-NHNH2.Hydrazine hydrate can be used as the N-terminal of nucleopilic reagent attack intein (Intein), to pass through
Nitrogen-sulphur (N-S) is migrated intein and target protein (SUMO2 (2-92)-NHNH2) separation, obtain the target that C-terminal is hydrazides
Protein S UMO (2-92)-NHNH2。
In some embodiments of the invention, the affinity chromatography is carried out using chitin pearl in step (1-2).
In some embodiments of the invention, cutting buffer described in step (1-3) contains: Hepes, NaCl, EDTA
With the hydrazine hydrate of 4%~6% volume.It is possible thereby to carry out fly-cutting to affinity chromatography product, SUMO hydrazides is obtained.
In some embodiments of the invention, step (2) further comprises: (2-1) is by the SUMO hydrazides SUMO2 (2-
92)-NHNH2It is dissolved in the buffer containing guanidine hydrochloride, carries out first with sodium sulfite aqueous solution and react；(2-2) is by step (2-
1) reaction solution and mesna carries out the second reaction, and purifying obtains SUMO thioesters SUMO2 (the 2-92)-Mesna.
In some embodiments of the invention, the temperature of first reaction is subzero 20 degrees Celsius, second reaction
Temperature be 25 degrees Celsius.
In some embodiments of the invention, step (3) further comprises: (3-1) is by the SUMO thioesters SUMO2 (2-
92)-Mesna and Boc2O acid anhydrides carries out third reaction, to obtain Boc-SUMO2 (2-92)-Mesna；(3-2) will be described
Boc-SUMO2 (2-92)-Mesna and the fluorescein small molecule carry out the 4th reaction, small to obtain Boc-SUMO- fluorescein
Molecule rhodamine；(3-3) is small to the Boc-SUMO- fluorescein using the cutting liquid containing trifluoroacetic acid and tri isopropyl silane
Molecule rhodamine is cut, and purifying obtains the SUMO fluorescence probe.
In some embodiments of the invention, the body of trifluoroacetic acid described in the cutting liquid, tri isopropyl silane and water
Product is than being 90~95:2.5~5:2.5~5.
In some embodiments of the invention, SUMO thioesters SUMO2 (the 2-92)-Mesna is dissolved in dimethyl sulfoxide
In and Boc2O acid anhydrides, n,N-diisopropylethylamine mixing, carry out the third reaction, under 20~30 degrees Celsius to obtain
Boc-SUMO2(2-92)-Mesna。
In some embodiments of the invention, the Boc-SUMO2 (2-92)-Mesna is dissolved in dimethyl sulfoxide and benzene
Thiophenol, fluorescein small molecule Gly-Rho110-Gly, n,N-diisopropylethylamine mixing, under 20~30 degrees Celsius described in progress
4th reaction, to obtain Boc-SUMO-Rho110-Gly.Using DMSO as organic solvent, by SUMO thioesters SUMO2 (2-
92)-Mesna is SUMO2-Rho110-Gly with the direct ammonolysis of Gly-Rho-Gly, has reaction high conversion efficiency, synthesis step
Simply, the low feature of synthesis cost.
According to the second aspect of the invention, the present invention provides a kind of SUMO fluorescence probe, the SUMO fluorescence probe roots
It is prepared according to method described in first aspect present invention any embodiment.
It is obtained by the present invention to have the beneficial effect that the present invention prepares SUMO acyl by the strategy migrated with Intein method N-S
Hydrazine SUMO2 (2-92)-NHNH2, having and prepares yield height, synthesis purity is high, operation is simple, the characteristics of can largely preparing, freeze-drying
SUMO2 (2-92)-NHNH of 12mg about can be obtained in every 1L culture medium afterwards2.By SUMO thioesters SUMO2 (2-92)-Mesna and fluorescence
Plain small molecule rhodamine reaction generates fluorescence probe, reacts high conversion efficiency, and synthesis step is simple, and synthesis cost is low, is suitble to
The industrial production and preparation of SUMO probe.
Detailed description of the invention
Fig. 1 is SUMO hydrazides SUMO2 (2-92)-NHNH that embodiment according to the present invention provides2High performance liquid chromatography
Figure.
Fig. 2 is SUMO hydrazides SUMO2 (2-92)-NHNH that embodiment according to the present invention provides2Mass spectrogram.
Fig. 3 is the high performance liquid chromatography for SUMO thioesters SUMO2 (2-92)-Mesna that embodiment according to the present invention provides
Figure.
Fig. 4 is the mass spectrogram for SUMO thioesters SUMO2 (2-92)-Mesna that embodiment according to the present invention provides.
Fig. 5 is the high performance liquid chromatography for the SUMO probe SUMO2-Rho110-Gly that embodiment according to the present invention provides
Figure.
Fig. 6 is the mass spectrogram for the SUMO probe SUMO2-Rho110-Gly that embodiment according to the present invention provides.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, it is intended to is used to explain the present invention, and is not considered as limiting the invention.
Herein when indicating amino acid, can use form of presentation generally in the art, that is, use three letters or
Person is stated with a letter, such as Gly or G can be used to indicate glycine.
Herein, the amino acid sequence of the 1-93 of SUMO2 albumen is MADEKPKEGV KTENNDHINLKVAGQDGSVV
QFKIKRHTPLSKLMKAYCER QGLSMRQIRF RFDGQPINET DTPAQLEMEDEDTIDVFQQQ TGG(SEQ ID
NO:1)。
Herein, in term " SUMO2 (2-92)-NHNH2 " 2-92 represent the amino acid sequence site of SUMO from the 2nd to
92 ADEKPKEGV KTENNDHINLKVAGQDGSVV QFKIKRHTPL SKLMKAYCER QGLSMRQIRF
RFDGQPINET DTPAQLEMEDEDTIDVFQQQ TG。
Herein, term " SUMO2-Rho110-Gly " represents SUMO2 (2-93)-Rho110-G, is by SUMO2 (2-
92) there are two the fluorescein small molecule Gly-Rho110-Gly of glycine modification (can also be abbreviated as G- for-Mesna and band
Rho110-G) condensation coupling obtains.Fluorescein small molecule G-Rho110-G is just a lot of after being combined with SUMO2 (2-92)-Mesna
One Gly, and the 93rd amino acid sequence of SUMO2 also just arrives as G, so final product obtained is with rhodamine
SUMO2 fluorescence probe (i.e. SUMO2-Rho110-Gly) is SUMO2 (2-93)-Rho110-G.
According to an aspect of the present invention, the present invention provides a kind of facile syntheesis SUMO probe SUMO2-Rho110-Gly
Method, specifically comprise the following steps:
Step 1: with strategy preparation SUMO hydrazides SUMO2 (2-92)-NHNH of Intein method N-S migration2
1a, the plasmid vector containing Intein and SUMO2 coded sequence is obtained, then converts Escherichia coli, picking contains
The monoclonal colonies of the plasmid vector contain to 4mL in the LB culture medium of ammonia benzyl resistance, and 37 DEG C, 220rpm overnight incubation make bacterium
Liquid is sufficiently muddy；
The formula of the LB culture medium containing ammonia benzyl resistance constitutes as follows: 2g tryptone, 2g NaCl, 1g yeast powder,
200mL deionized water, the Amp of 200 μ L 50mg/mL.
1b, taking the bacterium solution cultivated in 1a, 1:100 is amplified in the 1L LB culture medium containing ammonia benzyl resistance by volume, and 37
DEG C, 220rpm continue to cultivate, when 600 light absorption value of bacterium solution OD reaches 0.6~0.8, be added 500 μ L 1.0M IPTG to bacterium solution
Induced, 37 DEG C, 220rpm continue 5~6h of culture；
1c, the bacterium solution that 1b culture is completed is collected by centrifugation, discard supernatant after liquid with lysis buffer (20mM HEPES, 300~
500mM NaCl, 1mMEDTA, pH7.4) gained thallus is sufficiently resuspended (thallus obtained by every 1L LB culture medium is resuspended in 20~
In 30mL lysis buffer), thallus is cracked using Ultrasonic Cell Disruptor；
In general, the time of ultrasonication and the biomass direct proportionality for needing to be crushed, every 1L LB culture medium institute
It obtains thallus normal need and is crushed 1~2h.Thallus after being resuspended with lysate when unbroken at milkiness liquid, can be by after to be broken
Gradual change must clarify, bright.
1d, the clasmatosis liquid centrifugation (13000rpm, 30min, 4~8 DEG C) for being obtained 1c using high speed freezing centrifuge
After collect supernatant, with 0.22 μm of filtering with microporous membrane；
1e, balance chitin pearl: taking the liquid chitin pearl of about 10mL volume to be placed in Peptide systhesis pipe, will by gravity
Liquid is flow to end.Chitin pearl is rinsed with the lysis buffer of 10 times of bed volumes, by gained egg in step 1d after sufficiently balancing
Yu Zhuzhong is slowly added dropwise in white matter supernatant, mixes well it with chitin pearl, stands 0.5h at room temperature；
Due to can gradually be settled during chitin pearl and albumen supernatant mixing, can place it on shaking table
It slowly shakes to guarantee to mix well.
1f, the lower mouth for decontroling composite tube flow to end protein supernatant slowly by gravity, and (coutroi velocity is in 1~2mL/
Min), chitin pearl is rinsed to remove heteroproteins with the lysis buffer of 20 times of volumes, flow down solution slowly by gravity；
By the step, amalgamation and expression has the target protein of CBD structural domain that can specifically bind with chitin pearl, and miscellaneous egg
It is white to be removed, tentatively achieve the purpose that protein purification.
1g, cutting buffer (20mM HEPES, the 500mM NaCl, 1mM that about 2 times of volumes are added into chitin pearl
EDTA, 4%~6% hydrazine hydrate, pH7.4), 2~3h is stood at room temperature；
By the step, hydrazine hydrate as nucleopilic reagent can attack Intein N-terminal, by nitrogen-sulfur transfer by Intein
It is separated with target protein, obtains target protein SUMO2 (2-92)-NHNH that C-terminal is hydrazides2。
1h, chitin column is rinsed using the lysis buffer of 3 times of volumes, collects efflux.Gained contains SUMO2 (2-
92)-NHNH2Solution isolated and purified using RP-HPLC.Every 1L LB culture medium can about obtain the SUMO2 of 12mg after freeze-drying
(2-92)-NHNH2。
Step 2: protein hydrazides method prepares SUMO thioesters SUMO2 (2-92)-Mesna
SUMO2 (2-92)-NHNH that 1 times is measured2It is dissolved in the PBS buffer solution containing 6.0M guanidine hydrochloride of pH=3.0, and
The sodium nitrite in aqueous solution of 10 times of amounts is added, reacts 20min under the conditions of -20 DEG C；After reaction, the U.S. department of 40 times of amounts is added
Sodium, and pH to 5.0 is adjusted, react at room temperature 20min；It is purified, is received using semipreparative high performance liquid chromatography after the reaction was completed
The solution of collection after purification, SUMO2 (2-92)-Mesna of the about available 6~8mg of every 1L LB culture medium after freeze-drying；
Step 3: passing through Boc2O acid anhydrides protects SUMO thioesters SUMO2 (2-92)-Mesna to form Boc-SUMO2 (2-92)-
Mesna obtains SUMO probe SUMO2-Rho110- followed by direct ammonolysis Boc-SUMO2 (2-92)-Mesna
Gly。
3a, SUMO2 (the 2-92)-Mesna protein dry powder organic solvent DMSO (dimethyl sulfoxide) for preparing step 2 are molten
Solution, adduction enter Boc2O acid anhydrides (di-tert-butyl dicarbonate) and DIEA (n,N-diisopropylethylamine) use ice after stirring 1h at room temperature
Ether is settled out albumen, and naturally dry obtains Boc-SUMO2 (2-92)-Mesna；
3b, the albumen being precipitated out in 3a is dissolved in DMSO, the benzenethiol of 2% (v/v) is added according to liquor capacity, and
After small molecule Gly-Rho110-Gly is completely dissolved the DIEA of 10eq is added, in room in the Gly-Rho110-Gly that 5eq is added
After 1.5~2h of the lower reaction of temperature, reaction solution ice ether is precipitated, and then naturally dry, obtains Boc-SUMO2-Rho110-Gly；
3c, suitable cutting liquid (TFA (trifluoroacetic acid): TIS (triisopropyl silicon is added in the albumen being precipitated out in 3b
Alkane): H2O=95:2.5:2.5), addition ice ether is settled out albumen after stirring 30min at room temperature, then adds after natural drying
Enter after solvent dissolved, is purified using semipreparative high performance liquid chromatography, collect solution after purification, every 1L after freeze-drying
The target product SUMO probe SUMO2-Rho110-Gly of the about available 2~3mg of LB culture medium.
In step 3, the organic solvent is DMSO (dimethyl sulfoxide).
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available.
Embodiment 1
Embodiment 1 prepares SUMO hydrazides SUMO2 (2-92)-NHNH using Intein method N-S migration strategy2.It is based on quotient
Bacterial strain containing Intein connects SUMO2 albumen in the N-terminal of Intein, in its C based on routine techniques by means of carrier
End connection chitin binding domain, converts Escherichia coli, to obtain the bacterial strain that can express Intein and SUMO2 albumen.Wherein,
Bacterial strain containing Intein is commercialized BL21, is purchased from Quan Shi King Company, and the carrier cloned in the bacterial strain is PTXB1,
Restriction enzyme site is NdeI and SapI.
By the strain culturing that can express Intein and SUMO2, cracking obtains supernatant, then utilizes chitin pearl
Affinity chromatography is carried out, the chitin-binding protein in chitin pearl and supernatant combines, chitin-binding protein and intein phase
Even, the other end of intein is connected with SUMO2 target protein, thus SUMO2 target protein is fixed on chitin pearl.Then
It is cracked using hydrazine hydrate, obtains SUMO hydrazides SUMO2 (2-92)-NHNH2。
Include the following steps:
In the LB culture medium that the monoclonal colonies that picking can express Intein and SUMO2 contain ammonia benzyl resistance to 4mL, 37
DEG C, 220rpm overnight incubation.Wherein, the formula of the LB culture medium containing ammonia benzyl resistance constitutes as follows: 2g tryptone, 2g
NaCl, 1g yeast powder, 200mL deionized water, the Amp of 200 μ L 50mg/mL.
Taking the bacterium solution of above-mentioned steps culture, 1:100 is amplified in the 1L LB culture medium containing ammonia benzyl resistance by volume, and 37
DEG C, 220rpm continue to cultivate, when 600 light absorption value of bacterium solution OD reaches 0.6~0.8, be added 500 μ L 1.0M IPTG to bacterium solution
Induced, 37 DEG C, 220rpm continue 5~6h of culture.
The bacterium solution that culture is completed is collected by centrifugation, uses lysis buffer (20mM HEPES, 300~500mM after discarding supernatant liquid
NaCl, 1mM EDTA, pH7.4) gained thallus is sufficiently resuspended to (thallus obtained by every 1L LB culture medium is resuspended in 20~30mL and splits
Solve in buffer), thallus is cracked using Ultrasonic Cell Disruptor；Then the clasmatosis liquid obtained 1c using high speed freezing centrifuge
Supernatant is collected in centrifugation (13000rpm, 30min, 4~8 degree Celsius) afterwards, with 0.22 μm of filtering with microporous membrane.
Balance chitin pearl: taking the liquid chitin pearl of about 10mL volume to be placed in Peptide systhesis pipe, by gravity by liquid
Body is flow to end.Chitin pearl is rinsed with the lysis buffer of 10 times of bed volumes, by gained albumen in step 1d after sufficiently balancing
Yu Zhuzhong is slowly added dropwise in matter supernatant, mixes well it with chitin pearl, stands 0.5h at room temperature.
Decontrol composite tube lower mouth make protein supernatant slowly flow to end by gravity (coutroi velocity in 1~2mL/min,
Chitin pearl is rinsed to remove heteroproteins with the lysis buffer of 20 times of volumes, leaves solution slowly by gravity).
Be added into chitin pearl about 2 times of volumes cutting buffer (cutting buffer contains 20mM HEPES,
500mM NaCl, 1mM EDTA, 4%~6% hydrazine hydrate, pH7.4), 2~3h is stood at room temperature.
Chitin column is rinsed using the lysis buffer of 3 times of volumes, collects efflux.Gained contains SUMO2 (2-92)-
The solution of NHNH2 is isolated and purified using RP-HPLC, as shown in Figure 1.Every 1L LB culture medium can about obtain 12mg's after freeze-drying
SUMO2(2-92)-NHNH2。
Fig. 1 is SUMO2 (2-92)-NHNH2High-efficient liquid phase chromatogram, wherein abscissa be the time, expression be with height
It is the length of time used that different material, which is eluted, when effect liquid phase chromatogram isolates and purifies, and ordinate indicates absorption value, by table 1
It can reflect out the expression effect of the albumen.
Sample corresponding to the peak occurred when purifying simultaneously to high performance liquid chromatography separation carries out Mass Spectrometer Method, obtained
The molecular weight of the albumen is obtained by calculation as shown in Fig. 2, wherein abscissa represents mass-to-charge ratio in Fig. 2 in mass spectral results, then with
The theoretical molecular weight of the albumen is compared, so that whether detect obtained albumen correct.Calculation formula are as follows: lotus obtained
Matter ratio (m/z) * n-n=molecular weight of albumen.Wherein, n represents charge number.It is computed, and compared with theoretical molecular weight, confirmation obtains
The albumen needed.
Embodiment 2
Embodiment 2 prepares SUMO thioesters SUMO2 (2-92)-Mesna using protein hydrazides method, includes the following steps:
By SUMO2 (2-92)-NHNH of 50mg2It is dissolved in the 6M guanidine hydrochloride of 6mL pH=3.0,0.2M disodium hydrogen phosphate
In PBS buffer solution, and 60uL 1.0M sodium nitrite in aqueous solution is added, reacts 20min under the conditions of -20 DEG C；After reaction,
The mesna of 39mg is added, and adjusts pH to 5.0, reacts at room temperature 20min；Semi-preparative High Performance liquid phase color is used after the reaction was completed
Spectrum, as shown in figure 3, above-mentioned reaction solution is purified, collects solution after purification, every 1L LB culture medium Yue Ke get after freeze-drying
To SUMO thioesters SUMO2 (2-92)-Mesna of 6~8mg.
Fig. 3 is the high-efficient liquid phase chromatogram of SUMO thioesters SUMO2 (2-92)-Mesna, and abscissa represents time, ordinate
Represent absorption value.Sample corresponding to the peak occurred when purifying simultaneously to high performance liquid chromatography separation carries out Mass Spectrometer Method, is obtained
Result as shown in figure 4, referring to embodiment 1 calculation method, albumen required for confirmed acquisition.
Embodiment 3
Embodiment 3 provides a kind of utilization SUMO thioesters SUMO2 (2-92)-Mesna generation SUMO probe SUMO2-
The method of Rho110-Gly comprising: pass through Boc first2O acid anhydrides protection SUMO thioesters SUMO2 (2-92)-Mesna is formed
Boc-SUMO2 (2-92)-Mesna obtains SUMO probe followed by direct ammonolysis Boc-SUMO2 (2-92)-Mesna
SUMO2-Rho110-Gly。
Specifically comprise the following steps:
3a, SUMO2 (2-92)-Mesna protein dry powder 1.5mL DMSO (dimethyl sulfoxide) dissolution for weighing 43mg, and add
It is incorporated to the Boc of 18.5 μ L2The DIEA (n,N-diisopropylethylamine) of O acid anhydrides (di-tert-butyl dicarbonate) and 8 μ L, is stirred at room temperature
It is settled out albumen with ice ether after 1h, naturally dry obtains Boc-SUMO2 (2-92)-Mesna；
3b, the albumen being precipitated out in 3a is dissolved in 2.5mL DMSO, and the benzenethiol of 10 μ L is added, be then added
After small molecule Gly-Rho110-Gly is completely dissolved 8.5 μ L DIEA are added, in room temperature in the Gly-Rho110-Gly of 11.5mg
After 1.5~2h of lower reaction, reaction solution ice ether is precipitated, and then naturally dry, obtains Boc-SUMO2-Rho110-Gly；
3c, suitable cutting liquid (TFA (trifluoroacetic acid): TIS (triisopropyl silicon is added in the albumen being precipitated out in 3b
Alkane): H2O=95:2.5:2.5), addition ice ether is settled out albumen after stirring 30min at room temperature, then adds after natural drying
Enter after solvent dissolved, is purified using semipreparative high performance liquid chromatography, as shown in Figure 5.Solution after purification is collected,
Freeze-drying, obtains target product ubiquitin probe SUMO2-Rho110-Gly.
Fig. 5 is the high-efficient liquid phase chromatogram of probe SUMO2-Rho110-Gly, and wherein abscissa represents time, ordinate generation
Table absorption value.
Sample corresponding to the peak occurred when purifying simultaneously to high performance liquid chromatography separation carries out Mass Spectrometer Method, and result is such as
Shown in Fig. 6.Referring to the method for embodiment 1, the fluorescence probe of fluorescein small molecule rhodamine is had required for confirmed acquisition.
Herein, term " first ", " second " are used for description purposes only, and are not understood to indicate or imply relatively important
Property or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be expressed or
Person implicitly includes at least one this feature.In the description of the present invention, the meaning of " plurality " is at least two, such as two,
Three etc., unless otherwise specifically defined.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
It can be combined in any suitable manner in a or multiple embodiment or examples.In addition, without conflicting with each other, the technology of this field
The feature of different embodiments or examples described in this specification and different embodiments or examples can be combined by personnel
And combination.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
SEQUENCE LISTING
<110>Hefei Ke Shengjing peptide Biotechnology Co., Ltd
<120>SUMO fluorescence probe and preparation method thereof
<130> PIDC7190023
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 93
<212> PRT
<213> Artificial Sequence
<220>
<223> SUMO2
<400> 1
Met Ala Asp Glu Lys Pro Lys Glu Gly Val Lys Thr Glu Asn Asn Asp
1 5 10 15
His Ile Asn Leu Lys Val Ala Gly Gln Asp Gly Ser Val Val Gln Phe
20 25 30
Lys Ile Lys Arg His Thr Pro Leu Ser Lys Leu Met Lys Ala Tyr Cys
35 40 45
Glu Arg Gln Gly Leu Ser Met Arg Gln Ile Arg Phe Arg Phe Asp Gly
50 55 60
Gln Pro Ile Asn Glu Thr Asp Thr Pro Ala Gln Leu Glu Met Glu Asp
65 70 75 80
Glu Asp Thr Ile Asp Val Phe Gln Gln Gln Thr Gly Gly
85 90
Claims (10)
1. a kind of preparation method of SUMO fluorescence probe characterized by comprising
(1) method based on Intein N-S migration prepares SUMO hydrazides SUMO2 (2-92)-NHNH2；
(2) it is based on SUMO hydrazides SUMO2 (the 2-92)-NHNH2, SUMO thioesters SUMO2 (2-92)-is obtained using hydrazides method
Mesna；
(3) it is reacted based on SUMO thioesters SUMO2 (the 2-92)-Mesna and fluorescein small molecule rhodamine, to be had
The SUMO fluorescence probe of rhodamine.
2. the method according to claim 1, wherein the fluorescein small molecule rhodamine is Gly-Rho110-
Gly, the SUMO fluorescence probe with rhodamine are SUMO2-Rho110-Gly.
3. the method according to claim 1, wherein step (1) further comprises:
(1-1) is cultivated using the bacterial strain containing the carrier for being loaded with Intein and SUMO2, is cracked, and separation obtains supernatant, institute
It states and contains Intein and SUMO2 coded sequence on the carrier for being loaded with Intein and SUMO2；
The supernatant is carried out affinity chromatography by (1-2), obtains affinity chromatography product；
(1-3) is based on the affinity chromatography product, cutting process is carried out using the cutting buffer containing hydrazine hydrate, to obtain
SUMO hydrazides SUMO2 (the 2-92)-NHNH2；
Optionally, the affinity chromatography is carried out using chitin pearl in step (1-2).
4. according to the method described in claim 3, it is characterized in that, cutting buffer described in step (1-3) contains: Hepes,
The hydrazine hydrate of NaCl, EDTA and 4%~6% volume.
5. the method according to claim 1, wherein step (2) further comprises:
(2-1) is by SUMO hydrazides SUMO2 (the 2-92)-NHNH2It is dissolved in the buffer containing guanidine hydrochloride, with sodium sulfite water
Solution carries out the first reaction；
The reaction solution of step (2-1) and mesna are carried out the second reaction by (2-2), and purifying obtains the SUMO thioesters SUMO2
(2-92)-Mesna；
Optionally, the temperature of first reaction is subzero 20 degrees Celsius, and the temperature of second reaction is 25 degrees Celsius.
6. the method according to claim 1, wherein step (3) further comprises:
(3-1) is by SUMO thioesters SUMO2 (the 2-92)-Mesna and Boc2O acid anhydrides carries out third reaction, to obtain Boc-
SUMO2(2-92)-Mesna；
The Boc-SUMO2 (2-92)-Mesna and the fluorescein small molecule rhodamine are carried out the 4th reaction by (3-2), so as to
Obtain Boc-SUMO- fluorescein small molecule rhodamine；
(3-3) is using the cutting liquid containing trifluoroacetic acid and tri isopropyl silane to Boc-SUMO- fluorescein small molecule sieve
Pellet is bright to be cut, and purifying obtains the SUMO fluorescence probe.
7. according to the method described in claim 6, it is characterized in that, trifluoroacetic acid described in the cutting liquid, triisopropyl silicon
The volume ratio of alkane and water is 90~95:2.5~5:2.5~5.
8. according to the method described in claim 6, it is characterized in that, SUMO thioesters SUMO2 (the 2-92)-Mesna is dissolved in
In dimethyl sulfoxide and Boc2O acid anhydrides, n,N-diisopropylethylamine mixing, it is anti-to carry out the third under 20~30 degrees Celsius
It answers, to obtain Boc-SUMO2 (2-92)-Mesna.
9. according to the method described in claim 6, it is characterized in that, the Boc-SUMO2 (2-92)-Mesna is dissolved in diformazan
Base sulfoxide and benzenethiol, fluorescein small molecule Gly-Rho110-Gly, n,N-diisopropylethylamine mixing, it is Celsius 20~30
Degree is lower to carry out the 4th reaction, to obtain Boc-SUMO-Rho110-Gly.
10. a kind of SUMO fluorescence probe, which is characterized in that SUMO fluorescence probe institute according to claim 1~any one of 9
The method stated is prepared.
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| CN112500465A (en) * | 2020-12-01 | 2021-03-16 | 大连理工大学 | Synthetic method and application of SUMO modified capture probe |
| CN114507278A (en) * | 2022-01-27 | 2022-05-17 | 苏州大学 | Usu probe UFM1-Lys-TAMRA and synthetic method thereof |
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