CN109824764B - Method for simply synthesizing ubiquitin probe Ub-Rho110-Gly - Google Patents
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Abstract
The invention discloses a method for simply synthesizing ubiquitin probe Ub-Rho110-Gly, which comprises the steps of firstly preparing ubiquitin hydrazide Ub (1-75) -NHNH by using an N-S acyl migration semisynthesis strategy 2 And further converting the ubiquitin thioester Ub (1-75) -Mesna based on a hydrazide method, and further obtaining the ubiquitin probe Ub-Rho110-Gly through a direct ammonolysis method. The method has high preparation yield and high synthesis purity (Ub (1-75) -NHNH can be obtained in 1L culture medium after freeze-drying 2 30-40 mg), simple operation and mass preparation.
Description
Technical Field
The invention relates to a method for simply synthesizing a ubiquitin probe Ub-Rho110-Gly, belonging to the technical field of protein synthesis.
Background
Ubiquitination is a common post-translational modification involved in regulating a number of basic physiological processes, such as protein degradation, transcription and DNA damage repair. Ubiquitination is generally achieved by the synergistic action of three enzymes (E1, E2, E3), i.e. the glycine at the C-terminus of ubiquitin is linked to the side chain amino group of the middle lysine of the substrate protein by an isopeptide bond. The ubiquitin molecule can be used as a substrate protein, and N-terminal Met or seven lysines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 or Lys63) of the ubiquitin molecule are utilized to form uniform or non-uniform polyubiquitin chains with different lengths, and the polyubiquitin chains are identified in cell lysates. In addition, Met 1 at the N-terminal of ubiquitin can also be used for intermolecular ligation to form a linear polyubiquitin chain. Ubiquitination is a reversible process, and deubiquitinase carries out reverse regulation on ubiquitin-related processes by hydrolyzing isopeptide bonds between ubiquitin-ubiquitin or between ubiquitin and substrate proteins. At present, more than 90 kinds of deubiquitinases are known to exist in human body, wherein functional disorder of a plurality of deubiquitinases is related to a plurality of important diseases, such as cancer, inflammation, neurodegenerative diseases and the like, and some deubiquitinases become important drug action targets. For example, USP10, USP11, etc. are highly expressed in malignant lymphoma cells, USP1 is associated with the initial formation of colorectal cancer, while USP2 is highly expressed in ovarian cancer and cervical cancer. Therefore, the deubiquitinase also becomes a potential drug target, and the research on the enzyme activity property of the deubiquitinase and the screening of a specific deubiquitinase small-molecule inhibitor have important significance for the regulation and control mechanism of the life process, the search of a new drug target, and the diagnosis and treatment of diseases.
Ub-Rho110-Gly as a ubiquitin probe is widely applied to monitoring the activity of deubiquitinase and high-throughput screening of deubiquitinase inhibitors and the like. Since conventional small molecule fluorophores always have fluorescence, this property often masks valuable information in biological experiments, thereby preventing fluorescent labeling for imaging biomolecules. Rhodamine has a wide application range as a common coloring agent, and has a plurality of derivatives, wherein rhodamine B is a typical representative, but has high toxicity, so the application of the rhodamine is greatly limited. According to many animal experiments, it was found that rhodamine B metabolite is produced by hepatic deacetylation, and rhodamine 110, a final metabolite, is a xanthogen dye, similar to fluorescein and eosin. But its toxicity is less than that of its parent molecule and its abundance of conjugated pi bonds lends itself to long-lasting and intense fluorescent properties. Therefore, Rhodamine 110 is now used as a common fluorescent probe and stain, and has wide applications in biomedical and industrial fields; and has potential application value in the diagnosis of in vivo cancer due to the excellent light stability and photophysical properties. In addition, the fluorescent rhodamine 110 has been combined with amino acids, polypeptides and small molecules, and has been widely used in biological imaging for detecting cancer cells. Therefore, it is necessary to obtain the important ubiquitin probe Ub-Rho110-Gly by a simple method.
A novel DUB detection method is reported in an article (Analytical biochemistry, 2007,371, 201-207.) and takes ubiquitin-rhodamine 110-glycine as a substrate to measure the fluorescence intensity of ubiquitinase. The ubiquitin-rhodamine 110-glycine substrate has the characteristics of excellent optical performance, wide dynamic range and the like, so that the ubiquitin-rhodamine 110-glycine substrate is widely applied to drug discovery research, compound analysis and kinetic experiments.
The article (J.Agric.food chem.2017,65,7797-7804) reported the development and validation of a rapid, stable, sensitive method for quantifying rhodamine 110 by high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) to evaluate its pharmacokinetics and its distribution in rat organs. The rhodamine 110 has excellent photostability and photophysical properties due to rich conjugated pi bonds, so that the rhodamine has potential application value in-vivo cancer diagnosis.
Disclosure of Invention
The invention aims to provide a method for simply synthesizing a ubiquitin probe Ub-Rho110-Gly, and the ubiquitin hydrazide Ub (1-75) -NHNH is prepared by an N-S acyl migration semisynthesis strategy 2 Has high preparation yield and high synthesis purity (Ub (1-75) -NHNH can be obtained in each 1L culture medium after freeze-drying 2 30-40 mg), simple operation and mass preparation.
The invention relates to a method for simply synthesizing ubiquitin probe Ub-Rho110-Gly, which firstly adopts an N-S acyl migration semi-synthesis strategy to prepare ubiquitin hydrazide Ub (1-75) -NHNH 2 And further converting the ubiquitin thioester Ub (1-75) -Mesna based on a hydrazide method, and further obtaining the ubiquitin probe Ub-Rho110-Gly through a direct ammonolysis method.
The invention relates to a method for simply synthesizing an ubiquitin probe Ub-Rho110-Gly, which specifically comprises the following steps:
step 1: preparation of ubiquitylenes by N-S acyl migration semi-synthesis strategyHydrazine Ub (1-75) -NHNH 2
1a, selecting a monoclonal colony of a ubiquitin mutant Ub77C to 10mL LB culture medium containing ampicillin resistance, and culturing for 14-16h at 37 ℃ and 220 rpm;
the formula of the LB culture medium containing ampicillin resistance comprises the following components: 2g tryptone, 2g NaCl, 1g yeast powder, 200mL deionized water, 200 μ L of 50mg/mL Amp.
1b, amplifying the bacterial liquid cultured in the step 1a to 1L LB culture medium containing ampicillin resistance according to the volume ratio of 1:100, continuing to culture at 37 ℃ at 220rpm, adding 400 mu L of 1.0M IPTG to induce the bacterial liquid when the light absorption value of the bacterial liquid OD 600 reaches 0.8, and continuing to culture at 37 ℃ at 200rpm for 6 hours;
1c, collecting the bacterial solution (25 ℃, 8000rpm, 10min) cultured in 1b by centrifugation, discarding the supernatant, fully suspending the obtained bacterial cells with lysis buffer (50mM Tris-HCl, 150mM NaCl, pH 7.4) (the bacterial cells obtained in each 1L of LB medium are suspended in 20mL of lysis buffer), and lysing the bacterial cells by using an ultrasonicator;
1d, centrifuging the cell disruption solution obtained in the step 1c by using a high-speed refrigerated centrifuge (13000rpm, 30min, 4-8 ℃), collecting supernatant, adding perchloric acid into the supernatant according to the volume ratio of 200:1, precipitating hybrid protein, centrifuging at high speed (13000rpm, 30min, 4-8 ℃), and collecting supernatant;
1e, transferring the supernatant collected in 1d to a dialysis bag with 3000KDa, placing the dialysis bag in a dialysis buffer (50mM Tris-HCl, 150mM NaCl, pH 7.4) for dialysis for 12h, replacing the dialysis buffer, and repeating dialysis once;
1f, concentrating the protein solution dialyzed by the 1e until the final concentration of the protein is 20mg/mL to obtain a protein concentrated solution;
1g, adding mesna, TCEP and hydrazine hydrochloride into the protein concentrated solution obtained in the step 1f (100 mg of mesna, 5mg of TCEP and 50mg of hydrazine hydrochloride are added into each milliliter of the protein concentrated solution), uniformly mixing, adjusting the pH value of the solution to be 7.0, then placing the solution in a shaking table, shaking at 50 ℃ for 40 hours, after the reaction is finished, purifying the dialyzate by using semi-preparative high performance liquid chromatography (firstly washing with 20% acetonitrile for 2min, then washing with 70% acetonitrile for 32min), collecting the purified solution, and freeze-drying the solution to obtain the freeze-dried dialyzateTo obtain ubiquitin hydrazide Ub (1-75) -NHNH 2 。
And 2, step: preparation of ubiquitin thioester Ub (1-75) -Mesna by protein hydrazide method
1-fold amount of Ub (1-75) -NHNH 2 Dissolving in PBS buffer solution containing 6.0M guanidine hydrochloride at pH 3.0, adding 10 times of sodium nitrite aqueous solution, and reacting at-20 deg.C for 20 min; after the reaction is finished, adding 40 times of mesna, adjusting the pH value to 5.0, and reacting for 20min at room temperature; after the reaction is finished, purifying by using semi-preparative high performance liquid chromatography (washing with 30% acetonitrile for 2min, then washing with 70% acetonitrile for 32min), collecting a purified solution, and freeze-drying to obtain Ub (1-75) -Mesna;
and step 3: preparation of ubiquitin probe Ub-Rho110-Gly by directly aminolysis of ubiquitin thioester
Dissolving the Ub (1-75) -Mesna protein dry powder prepared in the step 2 by using an organic solvent, adding 2% (volume fraction) of thiophenol according to the volume of the solution, adding 50eq of Gly-Rho110-Gly and 100eq of DIEA (N, N-diisopropylethylamine), stirring and dissolving, reacting for 2 hours at room temperature, precipitating the reaction solution by using ether after the reaction is completed, purifying by using semi-preparative high performance liquid chromatography (firstly washing for 5 minutes by using 20% of acetonitrile, then washing for 35 minutes by using 70% of acetonitrile), collecting the purified solution, and freeze-drying to obtain the target ubiquitin probe Ub-Rho 110-Gly.
In step 1a, the gene sequence of the single clone of ubiquitin mutant Ub77C is as follows:
MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGC。
in step 3, the organic solvent is DMSO (dimethyl sulfoxide).
The invention has the following beneficial effects:
the invention prepares ubiquitin hydrazide Ub (1-75) -NHNH by using an N-S acyl migration semisynthesis strategy 2 Has the characteristics of high preparation yield, high synthesis purity, simple operation and mass preparation, and Ub (1-75) -NHNH can be obtained in 1L of culture medium after freeze-drying 2 30-40 mg. Direct amination of ubiquitin thioester Ub (1-75) -Mesna with Gly-Rho-Gly by optimizing the reaction solvent using pure DMSO as organic solventThe product is Ub-Rho110-Gly and has the characteristics of high reaction conversion efficiency, simple synthesis steps and low synthesis cost.
Drawings
FIG. 1 is ubiquitin hydrazide Ub (1-75) -NHNH 2 The high performance liquid chromatogram of (1).
FIG. 2 is ubiquitin hydrazide Ub (1-75) -NHNH 2 Mass spectrum of (2).
FIG. 3 is a high performance liquid chromatogram of the ubiquitin thioester Ub (1-75) -Mesna.
FIG. 4 is a mass spectrum of ubiquitin thioester Ub (1-75) -Mesna.
FIG. 5 is the HPLC chromatogram of ubiquitin probe Ub-Rho 110-Gly.
FIG. 6 is a mass spectrum of ubiquitin probe Ub-Rho 110-Gly.
Detailed Description
For the purpose of facilitating an understanding of the present invention, the following description is made in conjunction with specific embodiments thereof, and the description is made only for the purpose of illustrating the features and advantages of the present invention and not for the purpose of limiting the appended claims.
Example 1:
single colonies of ubiquitin mutant Ub77C were picked to 40mL LB medium containing ampicillin resistance and cultured at 37 ℃ and 220rpm for 14 h.
Amplifying the bacterial liquid cultured in the steps to 4L LB culture medium containing ampicillin resistance according to the volume ratio of 1:100, continuing culturing at 37 ℃ and 220rpm, adding 1.0M IPTG stock solution until the final concentration of IPTG is 0.4mM when the OD 600 light absorption value of the bacterial liquid reaches 0.8, inducing the bacterial liquid, and continuing culturing at 37 ℃ and 200rpm for 6 hours.
The cultured cell suspension was collected by centrifugation (25 ℃, 8000rpm, 10min), the supernatant was discarded, and the obtained cells were thoroughly resuspended in 80mL of lysis buffer (50mM Tris-HCl, 150mM NaCl, pH 7.4), and lysed using an ultrasonication apparatus. The cell disruption solution was centrifuged (13000rpm, 30min, 4-8 ℃) by using a high-speed refrigerated centrifuge, and then the supernatant was collected, 800uL of perchloric acid was added to the supernatant to precipitate impure protein, and after separation by high-speed centrifugation (13000rpm, 30min, 4-8 ℃), the supernatant was collected.
The supernatant was transferred to a 3000kDa dialysis bag and dialyzed in a dialysis buffer (50mM Tris-HCl, 150mM NaCl, pH 7.4)) for 12 hours, the dialysis buffer was changed, and the dialysis was repeated once.
And concentrating the dialyzed protein solution until the final concentration of the protein is 20mg/mL to obtain about 20mL of protein concentrate. 2g of mesna, 100mg of TCEP and 1g of hydrazine hydrochloride are added into the protein concentrated solution, and after uniform mixing, the pH value of the solution is adjusted to 7.0. Placing the reaction solution in a shaking table, shaking at 50 ℃ for 40h, after the reaction is finished, purifying the dialysate by using semi-preparative high performance liquid chromatography, collecting the purified solution, and freeze-drying to obtain the ubiquitin hydrazide Ub (1-75) -NHNH 2 About 100 mg.
Example 2:
50mg of Ub (1-75) -NHNH 2 Dissolving in 6mL of PBS buffer solution containing 6M guanidine hydrochloride and 0.2M disodium hydrogen phosphate at pH 3.0, adding 60uL of 1.0M sodium nitrite aqueous solution, and reacting at-20 deg.C for 20 min; after the reaction is finished, 39mg of mesna is added, the pH value is adjusted to 5.0, and the reaction is carried out for 20min at room temperature; after the reaction was completed, the above reaction solution was purified using semi-preparative high performance liquid chromatography, and the purified solution was collected and lyophilized to obtain 40mg of ubiquitin thioester Ub (1-75) -Mesna.
Example 3:
weighing 50mg of ubiquitin thioester Ub (1-75) -Mesna protein dry powder, dissolving in 5mL of pure DMSO, adding 2% (volume fraction) of thiophenol according to the volume of the solution, adding 129mg of Gly-Rho110-Gly, adding 96mL of DIEA, stirring and dissolving, reacting for 2h at room temperature after stirring and dissolving, after complete reaction and complete reaction, precipitating the reaction solution with 20mL of diethyl ether, purifying by using semi-preparative high performance liquid chromatography, collecting the purified solution, and freeze-drying to obtain the target ubiquitin probe Ub-Rho 110-Gly.
In conclusion, the invention provides a synthetic method for simply obtaining the ubiquitin probe Ub-Rho 110-Gly.
While the invention has been described with reference to specific preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> university of fertilizer industry
<120> simple method for synthesizing ubiquitin probe Ub-Rho110-Gly
<130> 2019.03.06
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 76
<212> PRT
<213> Artificial Synthesis
<400> 1
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu
1 5 10 15
Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp
20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
35 40 45
Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu
50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Cys
65 70 75
Claims (4)
1. A method for simply synthesizing an ubiquitin probe Ub-Rho110-Gly is characterized by comprising the following steps:
firstly, the semi-synthesis strategy of N-S acyl migration is applied to prepare the ubiquitin hydrazide Ub (1-75) -NHNH 2 Further converting the ubiquitin thioester Ub (1-75) -Mesna into a ubiquitin thioester Ub (1-75) -Mesna based on a hydrazide method, and further obtaining a ubiquitin probe Ub-Rho110-Gly through a direct ammonolysis method; the method comprises the following steps:
step 1: preparation of ubiquitin hydrazide Ub (1-75) -NHNH by N-S acyl migration semisynthesis strategy 2
1a, selecting a monoclonal colony of a ubiquitin mutant Ub77C to 10mL LB culture medium containing ampicillin resistance, and culturing for 14-16h at 37 ℃ and 220 rpm; the amino acid sequence of Ub77C in the single clone of the ubiquitin mutant Ub77C is shown in SEQ ID No: 1 is shown in the specification;
1b, amplifying the bacterial liquid cultured in the step 1a to 1L LB culture medium containing ampicillin resistance according to the volume ratio of 1:100, continuing to culture at 37 ℃ at 220rpm, adding 400 mu L of 1.0M IPTG to induce the bacterial liquid when the light absorption value of the bacterial liquid OD 600 reaches 0.8, and continuing to culture at 37 ℃ at 200rpm for 6 hours;
1c, centrifugally collecting the bacterial liquid cultured in the step 1b, removing supernatant, fully suspending the obtained thalli by using a lysis buffer solution, and lysing the thalli by using an ultrasonic crusher;
1d, centrifuging the cell disruption solution obtained in the step 1c by using a high-speed refrigerated centrifuge, collecting supernate, adding perchloric acid into the supernate according to the volume ratio of 200:1, precipitating hybrid protein, and collecting supernate after high-speed centrifugation;
1e, transferring the supernatant collected in the step 1d to a 3000KDa dialysis bag, placing the bag in a dialysis buffer solution for dialysis for 12 hours, replacing the dialysis buffer solution, and repeating dialysis once;
1f, concentrating the protein solution dialyzed by the 1e until the final concentration of the protein is 20mg/mL to obtain a protein concentrated solution;
1g, adding mesna, TCEP and hydrazine hydrochloride into the protein concentrated solution obtained in the step 1f, uniformly mixing, adjusting the pH =7.0 of the solution, then placing the solution in a shaking table, shaking at 50 ℃ for 40h, after the reaction is finished, purifying the dialyzate by using semi-preparative high performance liquid chromatography, collecting the purified solution, and freeze-drying to obtain the ubiquitin hydrazide Ub (1-75) -NHNH 2 ；
Step 2: preparation of ubiquitin thioester Ub (1-75) -Mesna by protein hydrazide method
1-fold amount of Ub (1-75) -NHNH 2 Dissolving in PBS buffer solution containing 6.0M guanidine hydrochloride with pH =3.0, adding 10 times of sodium nitrite aqueous solution, and reacting at-20 deg.C for 20 min; after the reaction is finished, 40 times of mesna is addedAdjusting the pH value to 5.0, and reacting for 20min at room temperature; after the reaction is finished, purifying by using semi-preparative high performance liquid chromatography, collecting the purified solution, and freeze-drying to obtain Ub (1-75) -Mesna;
and step 3: preparation of ubiquitin probe Ub-Rho110-Gly by directly aminolysis of ubiquitin thioester
Dissolving the Ub (1-75) -Mesna protein dry powder prepared in the step 2 by using an organic solvent, adding 2vt% of thiophenol according to the volume of the solution, adding 50eq of Gly-Rho110-Gly and 100eq of N, N-diisopropylethylamine, stirring and dissolving, reacting for 2 hours at room temperature, precipitating the reaction liquid by using ether after the reaction is completed, purifying by using semi-preparative high performance liquid chromatography, collecting the purified solution, and freeze-drying to obtain a target product ubiquitin probe Ub-Rho 110-Gly;
in step 3, the organic solvent is dimethyl sulfoxide.
2. The method of claim 1, wherein:
in step 1a, the formulation of the LB medium containing ampicillin resistance was as follows: 2g tryptone, 2g NaCl, 1g yeast powder, 200mL deionized water, 200 μ L of 50mg/mL Amp.
3. The method of claim 1, wherein:
in step 1c, the resulting cells were resuspended in 20mL of lysis buffer per 1L of LB medium.
4. The method of claim 1, wherein:
in step 1g, 100mg of mesna, 5mg of TCEP and 50mg of hydrazine hydrochloride are added into each milliliter of protein concentrated solution.
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Non-Patent Citations (5)
| Title |
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| A sensitive fluorescence intensity assay for deubiquitinating proteases using ubiquitin-rhodamine110-glycine as substrate;Hassiepen U等;《Analytical biochemistry》;20071215;第371卷(第2期);第202页图1，第203页左栏最后一段 * |
| Chemical Synthesis of Natural Polyubiquitin Chains through Auxiliary-Mediated Ligation of an Expressed Ubiquitin Isomer;Xu L等;《Journal of the American Chemical Society》;20171226(第20期);第329-332页 * |
| Efficient semi-synthesis of ubiquitin-7-amino-4-methylcoumarin;Chu G等;《Tetrahedron》;20180712;第74卷(第28期);第3932页左栏第2段，Scheme 1，第3934页第4.4-4.5小节 * |
| Expedient Total Synthesis of Small to Medium-Sized Membrane Proteins via Fmoc Chemistry;Zheng J等;《Journal of the American Chemical Society》;20140221(第136期);第3702页右栏第2段 * |
| PDB: 1YX5_B;National Center for Biotechnology Information;《National Center for Biotechnology Information》;20180520;第1页 * |
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