Method for synthesizing ubiquitin protein probe through enzyme mediation

Abstract

The invention discloses a method for synthesizing a ubiquitin protein probe by enzyme mediation, which comprises the following steps: carrying out recombinant expression on ubiquitin protein HA-Ub (1-73)-NSL-His6 containing an OaAEP1 ligase recognition sequence, catalyzing by using OaAEP1-C247A ligase, efficiently loading a chemical warhead carrying a single glycine or double glycine motif to a ubiquitin protein substrate at a fixed point, and carrying out enzyme mediation to synthesize the ubiquitin protein probe. And purifying to obtain the high-purity ubiquitin probe. Compared with a traditional method, the complex chemical coupling step is omitted, and the method has the advantages of being easy and convenient to operate, high in yield, high in specificity, mild in reaction condition and the like and is suitable for large-scale preparation. The obtained probe can be widely applied to the fields of ubiquitination modification mechanism research, deubiquitination enzyme (DUBs) function analysis, drug development and the like, and an efficient tool is provided for analysis of a ubiquitination regulation and control network and disease treatment target research.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry and precise protein modification technology, specifically relating to an enzyme-mediated method for synthesizing ubiquitin protein probes. Background Technology

[0002] Ubiquitination is a crucial post-translational modification of proteins in cellular physiological processes, comprehensively participating in the regulation of basic cellular life activities, including core aspects such as protein targeted degradation, gene transcription regulation, cell cycle progression, and DNA damage repair. This modification requires the coordinated action of E1, E2, and E3 enzymes. First, under ATP-powered energy, the E1 enzyme activates ubiquitin by forming a thioester bond with the C-terminal glycine residue. The activated ubiquitin then transfers to the active cysteine ​​residue of the E2 enzyme, generating the E2-ubiquitin thioester intermediate. Finally, the E3 enzyme recognizes and catalyzes the formation of an isopeptide bond between the C-terminal glycine of ubiquitin and the lysine side chain amino group of the substrate protein, completing the modification. Ubiquitin can modify other proteins and can also act as a substrate for polymerization. Through the N-terminal methionine and seven lysine residues, ubiquitin can construct various polyubiquitin chains.

[0003] Leveraging cutting-edge technologies, polyubiquitin chains with different linkage mechanisms have been identified. For example, the Lys48 linkage often mediates protein-targeted 26S proteasome degradation, while the Lys63 linkage is primarily involved in signal transduction and DNA damage repair. Ubiquitination is inversely regulated by deubiquitinating enzymes (DUBs). Over 90 DUBs have been identified in the human body, which hydrolyze the isopeptide bonds between ubiquitin and substrates, and within ubiquitin molecules, maintaining a dynamic balance of ubiquitination modifications. DUB dysfunction is associated with various diseases. In cancer, USP10 and USP11 are highly expressed in malignant lymphoma cells, interfering with the degradation of tumor suppressor factors and promoting tumor cell proliferation. In neurodegenerative diseases, UCHL1 dysregulation is associated with Parkinson's disease, leading to the accumulation of misfolded proteins and enhanced neurotoxicity. Given the crucial role of DUBs in disease, they have become a focus of drug development. By utilizing cutting-edge techniques such as structural drug design and high-throughput screening, we can conduct in-depth research on the enzymatic properties of DUBs and screen for highly efficient and specific small molecule inhibitors. This research is of great value for elucidating life regulatory mechanisms, exploring drug targets, and diagnosing and treating diseases.

[0004] Existing technology reports a diubiquitin-based fluorescence resonance energy transfer (FRET) probe for quantitative analysis of the specificity of deubiquitinating enzymes (DUBs) for different ubiquitin chain linkage types. This probe, using diubiquitin as a substrate, was prepared via solid-phase peptide synthesis (SPPS) and natural chemical linking (NCL) techniques, and equipped with rhodamine 110 and tetramethylrhodamine as the FRET pair. Due to its excellent optical properties and wide dynamic range, this probe has been widely used in drug discovery, compound analysis, and kinetic experiments (ChemBioChem. 2016, 17, 816-820). Furthermore, existing technology also reports an active site-directed probe for studying protease function. This probe, based on a previously considered non-reactive warhead molecule, achieves selective reaction with cysteine ​​residues at the active site of deubiquitinating enzymes (DUBs) by replacing the C-terminal carboxylic acid of ubiquitin with an alkyne functional group. Studies have shown that this alkynyl-modified ubiquitin (Ub-PA) can react with various DUBs to form stable covalent bonds, and this reaction exhibits high selectivity (J. Am. Chem. Soc. 2013, 135, 2867-2870). Nevertheless, previously reported methods for preparing ubiquitin probes still suffer from drawbacks such as numerous operational steps and complex chemical synthesis. Therefore, there is an urgent need to develop novel, simpler, and more efficient methods for preparing ubiquitin probes.

[0005] In recent years, with the deepening research on ubiquitination modification, scientists' demand for ubiquitination tools has been increasing. Developing new research tools and methods is of great significance for a deeper understanding of the mechanisms of ubiquitination modification. OaAEP1-C247A, as a novel ligase, provides an ideal tool for site-directed modification of ubiquitin proteins. It has advantages such as high catalytic efficiency, broad substrate compatibility, and mild reaction conditions, effectively solving the problems of cumbersome chemical coupling steps, poor site specificity, and low reaction yields in existing ubiquitination tool synthesis methods. Summary of the Invention

[0006] To address the above problems, the present invention aims to provide a method for enzyme-mediated synthesis of ubiquitin protein probes. The specific technical solution is as follows:

[0007] A method for enzyme-mediated synthesis of ubiquitin protein probes, comprising the following steps:

[0008] 1) Select single colonies of the ubiquitin mutant HA-Ub(1-73)-NSL-His6, whose amino acid sequence is shown in SEQ ID NO.1, and culture them in LB medium containing ampicillin resistance.

[0009] 2) Take the bacterial culture from step 1) and scale it up to 1L of LB medium containing ampicillin resistance at a volume ratio of 1:100. Continue culturing until the OD600 absorbance of the bacterial culture reaches 0.8. Then add IPTG to induce the bacterial culture and continue culturing.

[0010] 3) Centrifuge to collect the bacterial culture from step 2), discard the supernatant, resuspend the obtained bacterial cells thoroughly with lysis buffer, and lyse the bacterial cells using an ultrasonic disruptor;

[0011] 4) Collect the supernatant using a centrifuge;

[0012] 5) The collected supernatant was purified using Ni-NTABeads via gravity column method, and the protein eluent was concentrated using ultrafiltration tubes.

[0013] 6) After adding the protein solution from step 5) to the buffer solution, add OaAEP1-C247A, a glycyl compound, and incubate at 37°C for 2-12 hours. Purify the protein solution using Ni-NTA Beads via gravity column method, collect the flow-through, remove small molecules using ultrafiltration tube, and concentrate the protein solution to obtain the ubiquitin probe.

[0014] Furthermore, the LB medium containing ampicillin resistance in step 1) consists of 2g tryptone, 2g NaCl, 1g yeast extract, 200mL deionized water, and 200μL 50mg / mL Amp.

[0015] Furthermore, the culture conditions in step 1) are 37℃ and 220rpm for 8-12h; the culture conditions in step 2) are 37℃ and 220rpm.

[0016] Furthermore, in step 3), the conditions for centrifuging and collecting the cultured bacterial solution are 4℃, 8000rpm, 10min; the lysis buffer is 50mM Tris-HCl, 150mM NaCl, pH=7.4; and the resuspension conditions are that the bacterial solution obtained from 1LLB medium is resuspended in 20mL of lysis buffer.

[0017] Furthermore, in step 4), the conditions for collecting the supernatant in the centrifuge are 4°C, 8000 rpm, and 1 h.

[0018] Further, in step 6), the buffer is a PBS buffer with pH = 6.0, and the molar ratio of OaAEP1-C247A: ubiquitin protein in the protein solution: glycyl compound is 1:10-100:1000-10000.

[0019] The glycyl compound is a monoglycine warhead or a diglycine warhead. The monoglycine warhead can be G-PA, and its structural formula is shown in (Ⅰ) below. The diglycine warhead can be GG-PA or GG-TZ, and its structural formula is shown in (Ⅱ) or (Ⅲ).

[0020]

[0021] The beneficial effects of this invention are as follows:

[0022] Compared with traditional methods, this invention omits cumbersome chemical coupling steps and has advantages such as simple operation, high yield, strong specificity and mild reaction conditions, making it suitable for large-scale preparation. The obtained probes can be widely used in the study of ubiquitination modification mechanisms, functional analysis of deubiquitinating enzymes (DUBs) and drug development, providing an efficient tool for elucidating ubiquitination regulatory networks and studying disease therapeutic targets. Attached Figure Description

[0023] Figure 1 The high-performance liquid chromatogram of ubiquitin protein HA-Ub(1-73)-NSL-His6;

[0024] Figure 2 The high-performance liquid chromatogram of the ubiquitin probe HA-Ub(1-75,R74N)-PA;

[0025] Figure 3 The high-performance liquid chromatogram of the ubiquitin probe HA-Ub(1-76,R74N)-PA;

[0026] Figure 4 The high-performance liquid chromatogram of the ubiquitin probe HA-Ub(1-76,R74N)-TZ;

[0027] Figure 5 Western blot image of cell lysate labeled with ubiquitin probe HA-Ub(1-76,R74N)-PA in vitro;

[0028] Figure 6 This is a Western blot image of cell lysates labeled with the ubiquitin probe HA-Ub(1-76,R74N)-TZ in vitro. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments. Although ubiquitin is used as a specific embodiment, the scope of protection of the present invention is not limited thereto. This method is also applicable to the preparation of other ubiquitin protein probes with similar sequences, including ubiquitin-like proteins such as Nedd8, Fub1, SUMO1, ISG15, and FAT10.

[0030] Example 1: Preparation of HA-Ub(1-75,R74N)-PA ubiquitin probe (probe 1)

[0031] 1) Pick a single colony of the ubiquitin mutant HA-Ub(1-73)-NSL-His6, whose amino acid sequence is shown in SEQ ID NO.1, and add it to 10 mL of LB medium containing ampicillin resistance. The high-performance liquid chromatogram of the ubiquitin mutant HA-Ub(1-73)-NSL-His6 is shown in Figure 1. Figure 1 As shown, the culture was carried out at 37℃ and 220rpm for 8h. The LB medium containing ampicillin resistance was formulated as follows: 2g tryptone, 2g NaCl, 1g yeast extract, 200mL deionized water, and 200μL 50mg / mL ampicillin (Amp).

[0032] 2) Take the bacterial culture from step 1) and scale it up to 1L of LB medium containing ampicillin resistance at a volume ratio of 1:100. Continue to culture at 37℃ and 220rpm. When the OD600 absorbance of the bacterial culture reaches 0.8, add 400μL of 1.0M IPTG to induce the bacterial culture. Continue to culture at 37℃ and 200rpm for 6h.

[0033] 3) Centrifuge to collect the bacterial culture from step 2) (4℃, 8000rpm, 10min), discard the supernatant, and then fully resuspend the obtained bacterial cells in lysis buffer (50mM Tris-HCl, 150mM NaCl, pH=7.4) (the bacterial cells obtained from 1L LB medium are resuspended in 20mL lysis buffer), and the bacterial cells are lysed using an ultrasonic disruptor;

[0034] 4) Collect the supernatant using a centrifuge (4℃, 8000rpm, 1h);

[0035] 5) The collected supernatant was purified using Ni-NTABeads via gravity column method, and the protein eluent was concentrated using ultrafiltration tubes.

[0036] 6) Add 300 μM of the protein solution from step 5) to a buffer solution (PBS, pH 6.0), add 0.01 eq (molar equivalent to the protein substrate) of OaAEP1-C247A, add 100 eq of the G-PA probe, incubate at 37°C for 2 h, purify the protein using a gravity column method with Ni-NTABeads, collect the flow-through, remove small molecules using ultrafiltration and concentrate the protein solution to obtain approximately 270 μM HA-Ub(1-75,R74N)-PA ubiquitin probe. The high-performance liquid chromatography (HPLC) chromatogram is shown below. Figure 2 As shown, most of the raw materials (t) R1 =21.84min) is converted into product (t) R2=22.56min), with a yield of approximately 90%.

[0037] Example 2: Preparation of HA-Ub(1-76,R74N)-PA ubiquitin probe (probe 2)

[0038] Add 300 μM of the protein solution prepared in step 5) of Example 1 to a buffer solution (PBS, pH 6.0), add 0.1 eq (molar equivalent relative to the protein substrate) of OaAEP1-C247A, add 10 eq of GG-PA probe, and incubate at 37°C for 4 h. Purify the protein using a gravity column method with Ni-NTABeads, collect the flow-through, remove small molecules using ultrafiltration, and concentrate the protein solution to obtain approximately 255 μM HA-Ub(1-76,R74N)-PA ubiquitin probe. Its high-performance liquid chromatography (HPLC) chromatogram is shown below. Figure 3 As shown, most of the raw materials (t) R1 =21.84min) is converted into product (t) R3 =23.11min), with a yield of approximately 85%.

[0039] Example 3: Preparation of HA-Ub(1-76,R74N)-TZ ubiquitin probe (probe 3)

[0040] Add 300 μM of the protein solution prepared in step 5) of Example 1 to a buffer solution (PBS, pH 6.0), add 0.1 eq (molar equivalent relative to the protein substrate) of OaAEP1-C247A, add 10 eq of GG-TZ probe, and incubate at 37°C for 2-12 h. Purify the protein using a gravity column method with Ni-NTABeads, collect the flow-through, remove small molecules using ultrafiltration, and concentrate the protein solution to obtain approximately 250 μM HA-Ub(1-76,R74N)-TZ ubiquitin probe. Its high-performance liquid chromatography (HPLC) chromatogram is shown below. Figure 4 As shown, most of the raw materials (t) R1 =21.84min) is converted into product (t) R2 =22.31min), with a yield of approximately 83%.

[0041] Example 4: Using HeLa and SW620 cell lysates to investigate the effectiveness of the probe.

[0042] Mature HeLa and SW620 cell lines were collected by centrifugation, washed with PBS (pH 7.4), centrifuged again to collect cells, added an appropriate amount of cell lysis buffer for lysis, and gently pipetted to mix thoroughly. The cells were then placed on ice for 10 min for lysis. After complete lysis, the cells were centrifuged at 12000g for 4 min and the supernatant was collected to obtain the cell lysate.

[0043] Prepare the reaction systems according to Table 1 and Table 2 respectively.

[0044] Table 1. Experimental reaction system for in vitro labeling of cell lysates with ubiquitin probe HA-Ub(1-76,R74N)-PA.

[0045]

[0046] For the ubiquitin probe HA-Ub(1-76,R74N)-PA (probe 2), different experimental and control groups were designed according to Table 1. Groups 1, 2, and 4 were control groups, and groups 3 and 5 were experimental groups. After preparation, the mixture was incubated at 37℃ for 1 h. After the reaction, 10 μL of the reaction solution was taken and subjected to SDS-PAGE electrophoresis using an 8% protein gel. Subsequently, Western blotting experiments were performed. Figure 5 As shown, no antibody-binding bands appeared in the two control groups where only cell lysates were added. However, a series of antibody-binding bands appeared in the two experimental groups, indicating that probe 2 successfully labeled multiple deubiquitinated proteases present in the cells, thus confirming the effectiveness of probe 2 in complex cellular environments. "+" indicates the addition of the corresponding substrate, and "-" indicates the opposite.

[0047] Table 2. Experimental reaction system for in vitro labeling of cell lysates with ubiquitin probe HA-Ub(1-76,R74N)-TZ.

[0048] For the ubiquitin probe HA-Ub(1-76,R74N)-TZ (probe 3), different experimental and control groups were designed according to Table 2, where groups 1-3, 5, and 6 were control groups, and groups 4 and 7 were experimental groups. After preparation, the experimental groups were treated with 365nm UV light for 20 min, and then incubated with the control groups at 37℃ in the dark for 1 h. After the reaction, 10 μL of the reaction solution was taken and subjected to SDS-PAGE electrophoresis using an 8% protein gel, followed by Western blotting experiments, such as... Figure 6 As shown, no antibody-binding bands appeared in either the cell lysate or the control group (without 365nm UV activation). However, a series of antibody-binding bands appeared in the experimental group treated with 365nm UV light for 20 min. This result indicates that probe 3 not only has activity targeting deubiquitinating enzymes but can also be activated on demand through UV irradiation.

Claims

1. A method for enzyme-mediated synthesis of ubiquitin protein probes, characterized in that, Includes the following steps: 1) Select ubiquitin mutants with amino acid sequences as shown in SEQ ID NO.

1. HA-Ub(1-73)-NSL-His6 monoclonal colonies were cultured in LB medium containing ampicillin resistance. 2) Take the bacterial culture from step 1) and scale it up to 1L of LB medium containing ampicillin resistance at a volume ratio of 1:

100. Continue culturing until the OD600 absorbance of the bacterial culture reaches 0.

8. Then add IPTG to induce the bacterial culture and continue culturing. 3) Centrifuge to collect the bacterial culture from step 2), discard the supernatant, resuspend the obtained bacterial cells thoroughly with lysis buffer, and lyse the bacterial cells using an ultrasonic disruptor; 4) Collect the supernatant using a centrifuge; 5) The collected supernatant was purified using Ni-NTA Beads via gravity column chromatography, and the protein eluent was concentrated using ultrafiltration tubes. 6) Add the protein solution from step 5) to the buffer solution, add OaAEP1-C247A, a glycyl compound, and incubate at 37°C for 2-12 hours. Purify the protein solution using Ni-NTABeads via gravity column method, collect the flow-through, remove small molecules using ultrafiltration tube and concentrate the protein solution to obtain the ubiquitin probe.

2. The method for enzyme-mediated synthesis of ubiquitin protein probes as described in claim 1, characterized in that, The LB medium containing ampicillin resistance in step 1) consists of 2g tryptone, 2g NaCl, 1g yeast extract, 200mL deionized water, and 200μL 50mg / mL Amp.

3. The method for enzyme-mediated synthesis of ubiquitin protein probes as described in claim 1, characterized in that, The culture conditions in step 1) are 37℃ and 220rpm for 8-12h; the culture conditions in step 2) are 37℃ and 220rpm.

4. The method for enzyme-mediated synthesis of ubiquitin protein probes as described in claim 1, characterized in that, In step 3), the conditions for centrifuging and collecting the cultured bacterial solution are 4℃, 8000rpm, 10min; the lysis buffer is 50mM Tris-HCl, 150mM NaCl, pH=7.4; the resuspending conditions are that the bacterial solution obtained from each 1L LB medium is resuspended in 20mL of lysis buffer.

5. The method for enzyme-mediated synthesis of ubiquitin protein probes as described in claim 1, characterized in that, In step 4), the conditions for collecting the supernatant in the centrifuge are 4°C, 8000 rpm, and 1 h.

6. The method for enzyme-mediated synthesis of ubiquitin protein probes as described in claim 1, characterized in that, In step 6), the buffer is a PBS buffer with pH = 6.

0. The molar ratio of ubiquitin protein in the protein solution to glycyl compound in OaAEP1-C247A is 1:10-100:1000-10000.

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