Cell indicator as well as preparation method and application thereof

Abstract

The invention relates to a cell indicator system as well as a preparation method and application thereof, and belongs to the technical field of biomacromolecule detection. According to the cell indicator system, cells comprise at least one of a carrier A, a carrier B and a carrier C; the carrier A contains a TetR protein coding gene; the carrier B contains a biologically acceptable peptide and a coding gene of ubiquitin; and the vector C contains coding genes of targeted E3 ligase and biotin ligase. According to the application, the cell line capable of expressing the TetR protein, the biological accepting peptide, the ubiquitin, the targeted E3 ligase and the biotin ligase is constructed, the targeted E3 ligase can be combined with the substrate through the induced expression of the doxycycline, so that the substrate is ubiquitinated and biotinylated, and the known and unknown ubiquitinated substrates can be rapidly detected and identified.

Description

Technical Field

[0001] This application relates to the technical field of biomacromolecule detection, and particularly relates to a cell indicator system, a preparation method thereof, and an application thereof. Background Art

[0002] Biotinylation-based identification of interacting proteins (BioID) is a proximity-dependent biotin identification technology that can be used to study the interactions between proteins, proteins and RNAs, and proteins and DNAs. The use of the BioID method is limited to the interaction between two known proteins. For example, protein A is linked to BirA (biotin ligase). When protein B is close to or binds to A-BirA, protein B will be biotinylated. However, there are still great limitations in applying it to the ubiquitination of substrate proteins by E3 ligases.

[0003] Proteolysis-targeting chimeras (PROTAC) is a drug development technology that degrades target proteins using the ubiquitin-proteasome system (UPS). With the rapid development of PROTAC technology, there is an urgent need to improve and innovate the existing BioID technology to facilitate the application of BioID to PROTAC technology. Summary of the Invention

[0004] The purpose of this application is to overcome the deficiencies of the prior art and provide a cell indicator system that can target E3 ubiquitin ligase to induce ubiquitination and biotinylation of substrate proteins, a preparation method thereof, and an application thereof.

[0005] To achieve the above purpose, the technical solution adopted in this application is as follows:

[0006] In the first aspect, this application provides a cell indicator system, which includes at least one of vector A, vector B, and vector C in the cell;

[0007] The vector A contains the TetR protein coding gene;

[0008] The vector B contains the coding genes of a biotin acceptor peptide and ubiquitin;

[0009] The vector C contains the coding genes of a target E3 ligase and a biotin ligase.

[0010] This application constructs a cell line capable of expressing TetR protein, biotin acceptor peptide, ubiquitin, targeted E3 ligase, and biotin ligase. Through induction by doxycycline, it can target the binding of E3 ligase to the substrate, ubiquitinate and biotinylate the substrate, and can quickly detect known and identify unknown ubiquitinated substrates.

[0011] The cells in the cell indicator of this application can be common model cells in the laboratory, or tumor cells, pancreatic tissue cells, mouse liver cells, etc. can be selected as carrier cells to construct the cell indicator according to the actual situation.

[0012] As a preferred embodiment of the cell indicator system described in this application, it includes at least one of the following (Ⅰ)-(Ⅱ):

[0013] (Ⅰ) The vector A is a lentiviral expression vector, and the coding gene sequence of the TetR protein is as shown in SEQ ID NO.1;

[0014] (Ⅱ) The vector B also contains a TetO element and a FLAG tag;

[0015] The coding gene sequence of the biotin acceptor peptide is as shown in SEQ ID NO.2;

[0016] The coding gene sequence of ubiquitin is as shown in SEQ ID NO.3;

[0017] The nucleotide sequence of the TetO element is as shown in SEQ ID NO.4;

[0018] The nucleotide sequence of the FLAG tag is as shown in SEQ ID NO.5.

[0019] The addition of the Tet-On system can control the expression of the biotin acceptor peptide and ubiquitin using doxycycline, restricting the combination of substrate biotinylation and ubiquitination within a controlled time window. The biotin acceptor peptide (BAP) is a specific receptor for biotin and can bind to biotin. Ubiquitin (UB) is a small molecule protein that can label proteins to be degraded. FLAG is a small peptide tag that is beneficial for subsequent judgment of whether BAP-UB can be expressed.

[0020] As a preferred embodiment of the cell indicator system described in this application, the vector C also contains an HA tag; the vector C is a lentiviral expression vector;

[0021] The coding gene sequence of the targeted E3 ubiquitin ligase is as shown in SEQ ID NO.6;

[0022] The coding gene sequence of the biotin ligase is as shown in SEQ ID NO.7;

[0023] The nucleotide sequence of the HA tag is as shown in SEQ ID NO.8.

[0024] A targeting E3 ligase is an enzyme that can specifically ubiquitinate substrates, recognize target proteins and ubiquitinate them. Biotin ligase (BirA) is an enzyme that catalyzes the activity of biotin and the binding of biotin to biotin receptors.

[0025] As a preferred embodiment of the cell indicator system described in this application, it includes at least one of the following (I)-(II):

[0026] (I) The biotin acceptor peptide in vector B is located at the N-terminus of ubiquitin;

[0027] (II) The biotin ligase in vector C is located at the N-terminus or C-terminus of the targeting E3 ligase.

[0028] In a second aspect, this application provides a method for preparing the above cell indicator system, including the following steps:

[0029] S1. Package lentiviruses of vector A and vector C in vector cells to obtain lentivirus A containing vector A and lentivirus C containing vector C;

[0030] S2. Infect vector cells with the lentivirus A obtained in step S1 to obtain cell A containing vector A;

[0031] S3. Transfect the above vector B into cell A obtained in step S2 to obtain cell B containing vector A and vector B;

[0032] S4. Infect cell B obtained in step S3 with the lentivirus C obtained in step S1 to obtain cell C containing vector A, vector B and vector C, and the obtained cell C is the cell indicator system.

[0033] In a third aspect, this application provides the use of the above cell indicator system in detecting and / or identifying ubiquitinated substrates.

[0034] In this application, by detecting whether a sample can bind to streptavidin agarose beads, form a precipitate in an immunoprecipitation experiment, and using WB or mass spectrometry to detect whether it is biotinylated, it can be determined whether the substrate is an ubiquitinated substrate. This method is fast, convenient, does not require complex instrument equipment, and has high economic benefits.

[0035] In a fourth aspect, this application provides the use of the above cell indicator system in detecting the process of the formation of ternary complexes between PROTAC molecules and ligands and / or the degree of ubiquitination.

[0036] By detecting the degree of biotinylation, the present application can evaluate the process of the formation of ternary complexes by PROTAC molecules and ligands, and monitor the degree of ubiquitination of ternary complexes, enabling rapid detection and monitoring of the ubiquitination process of ternary complexes.

[0037] In a fifth aspect, the present application provides a kit for detecting and / or identifying ubiquitinated substrates, including the above-mentioned cell indicator system, doxycycline, biotin, and streptavidin agarose beads.

[0038] As a preferred embodiment of the kit of the present application, the kit further includes a PROTAC molecule.

[0039] In a sixth aspect, the present application provides a method for detecting and / or identifying ubiquitinated substrates, including the following steps:

[0040] (1) Co-culture the above-mentioned cell indicator system, add doxycycline for induction to obtain a biotin acceptor peptide-ubiquitin conjugate and an E3 ubiquitin ligase-biotin ligase conjugate;

[0041] (2) Mix the biotin acceptor peptide-ubiquitin conjugate and the E3 ubiquitin ligase-biotin ligase conjugate obtained in step (1) with the sample to be tested, add biotin and / or a PROTAC molecule to obtain cells containing the ubiquitinated and biotinylated sample to be tested;

[0042] (3) Lyse the cells containing the ubiquitinated and biotinylated sample to be tested obtained in step (2) to obtain a lysate supernatant, perform immunoprecipitation of the obtained lysate supernatant with streptavidin agarose beads, and then detect and / or identify the ubiquitination and biotinylation of the sample to be tested by immunoblotting and / or mass spectrometry techniques.

[0043] The present application can confirm whether the substrate can be ubiquitinated by the binding of streptavidin agarose beads to biotin and through immunoprecipitation techniques and WB or mass spectrometry techniques, and can detect both known and unknown ubiquitinated substrates.

[0044] Compared with the prior art, the beneficial effects of the present application are as follows:

[0045] (1) By constructing a cell line capable of expressing TetR protein, biotin acceptor peptide, ubiquitin, target E3 ligase, and biotin ligase, and inducing expression with doxycycline, it can target the binding of E3 ligase to the substrate, ubiquitinate and biotinylate the substrate, and can rapidly detect known and identify unknown ubiquitinated substrates.

[0046] (2) In this application, by detecting whether the sample can bind to streptavidin agarose beads and form a precipitate during the co-immunoprecipitation experiment, and using WB or mass spectrometry to detect whether it is biotinylated, it can be determined whether the substrate is a ubiquitinated substrate. This method is rapid, convenient, does not require complex instrument equipment, and has high economic benefits.

[0047] (3) In this application, by detecting the degree of biotinylation, the process of the PROTAC molecule forming a ternary complex with the ligand can be evaluated, and the ubiquitination degree of the ternary complex can be monitored, enabling rapid detection and monitoring of the ubiquitination process of the ternary complex. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 It is the plasmid map of vector A in Example 1 of this application;

[0049] Figure 2 It is the plasmid map of vector B in Example 1 of this application;

[0050] Figure 3 It is the plasmid map of vector C in Example 1 of this application;

[0051] Figure 4 It is the WB verification result of cell A in Example 1 of this application;

[0052] Figure 5 It is the WB verification result of cell B in Example 1 of this application;

[0053] Figure 6 It is the plasmid map of vector C in Example 2 of this application;

[0054] Figure 7 It is the WB verification result of cell C in Example 1 and Example 2 of this application. In the figure, enTEL represents the endogenous TEL protein;

[0055] Figure 8 It is the operation process for detecting and / or identifying ubiquitinated substrates in Example 4 of this application;

[0056] Figure 9 It is the WB verification result of the biotinylation and ubiquitination of the cell indicator system in Effect Example 1 of this application;

[0057] Figure 10 It is the WB detection result of the ubiquitination and biotinylation of POI B after adding PROTAC in Example 4 of this application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] To better illustrate the purpose, technical solution, and advantages of this application, the following will further illustrate this application in combination with specific embodiments.

[0059] Materials, reagents, etc. used in the examples, comparative examples, and experimental examples can be obtained from commercial sources unless otherwise specified.

[0060] Lipofectamine TM The 3000 transfection reagent is provided by Invitrogen, with the product number L3000075.

[0061] Doxycycline (DOX) is provided by MCE, with the product number HY-N0565.

[0062] The PLV-EF1a-IRES-Hygro vector has the product number 85134, the PLV-EF1a-IRES-Puro vector has the product number 85132, the psPAX2 plasmid has the product number 12260, and the pMD2.G plasmid has the product number 12259. All of the above are provided by addgene.

[0063] The 293T cells are provided by Saiku Biotechnology, with the product number CC4003.

[0064] The anti-TetR antibody has the product number Abcam, ab302642, the anti-FLAG antibody has the product number Proteintech, 20543-1-AP, the anti-TEL antibody has the product number CST, 14866, and the anti-biotin antibody has the product number Bethyl, A150-109A.

[0065] Biotin is provided by MCE, with the product number HY-B0511.

[0066] Streptavidin agarose beads are provided by Millipore, with the product number 16-126.

[0067] The homologous recombination technology is completed using a kit. The relevant operation steps are carried out according to the kit instructions. The kit is the ClonExpress Ultra One Step Cloning Kit V2, provided by Nanjing Novoprotein Scientific Co., Ltd., with the product number C116.

[0068] The PROTAC molecule can be a commercially available PROTAC molecule. The PROTAC molecules used in the following examples are designed and synthesized by the applicant.

[0069] The antibody against substrate A is provided by Proteintech, with the product number 10355-1-AP.

[0070] The cell lysate is CHPAS lysate.

[0071] The Western Blot (WB) was performed according to the steps described in the "Molecular Biology Experiment Manual". The secondary antibodies used were both goat anti-mouse and goat anti-Rabbit, provided by Transgen, with the product numbers HS201-01 and HS101-01.

[0072] The plasmid synthesis and sequencing used in the following examples were both completed by GenScript Biotech Corporation.

[0073] In the following examples and effect examples, unless otherwise specified, the cell culture conditions used were 37°C and 5% CO2, and the medium used was complete medium.

[0074] Example 1

[0075] This example provides a cell indicator system and a preparation method thereof. The preparation method includes the following steps:

[0076] I. Constructing vectors

[0077] 1.1 Construction of vector A

[0078] Using the lentiviral expression vector PLV-EF1a-IRES-Hygro, the gene fragment of the TetR protein was amplified by PCR using the tet-PLKO-sgRNA-pruo vector as a template. The TetR fragment was inserted into PLV-EF1a-IRES-Hygro by homologous recombination. The restriction enzyme sites used were BamH1 and EcoR1. Sequencing was performed to confirm that the vector contained the target fragment, and vector TetR, namely vector A, was obtained. The map of vector A is shown in Figure 1 , and the coding gene of the TetR protein is as shown in SEQ ID NO.1.

[0079] 1.2 Construction of vector B

[0080] On the gene editing software, the base sequences of the TetO element, FLAG tag, BAP, and UB were integrated. BAP was located at the N-terminus of UB. It was commissioned to a biological company to synthesize it into fragment 1. Fragment 1 was ligated to the pCMV-Zeo vector by restriction enzyme digestion and ligation. Sequencing was performed to confirm that the vector contained the target fragment, and vector TetO-FLAG-BAP-UB, namely vector B, was obtained. The map of vector B is shown in Figure 2 , the coding gene sequence of BAP is as shown in SEQ ID NO.2, the coding gene sequence of UB is as shown in SEQ ID NO.3, the nucleotide sequence of the TetO element is as shown in SEQ ID NO.4, and the nucleotide sequence of the FLAG tag is as shown in SEQ ID NO.5.

[0081] 1.3 Construction of vector C

[0082] Integrate the TEL base sequence with HA tag, commissioned a biological company to synthesize it to obtain the TEL fragment with HA tag; amplify the BirA fragment by PCR, and use homologous recombination technology to ligate the TEL fragment and the BirA fragment into the lentiviral expression vector PLV-EF1a-IRES-Puro. The restriction enzyme sites are BamH1 and EcoR1. The BirA fragment is located at the C-terminus of the TEL fragment. Sequencing is performed to confirm that the vector contains the target fragment, and vector TEL-C-BirA, namely vector C, is obtained. The map of vector C is shown in Figure 3 ; The TEL coding gene sequence is as shown in SEQ ID NO.6, the BirA coding gene sequence is as shown in SEQ ID NO.7, and the nucleotide sequence of the HA tag is as shown in SEQ ID NO.8.

[0083] II. Construction of the cell indicator system

[0084] 2.1 Co-transfect the exponentially growing 293T cells with the auxiliary plasmids psPAX2 and pMD2.G and vector A obtained in step 1.1 or vector C obtained in steps 1 and 3, and concentrate the lentivirus by the PEG8000 concentration method to obtain lentivirus A or lentivirus C;

[0085] 2.2 Select 293T cells in the logarithmic growth phase, aspirate the culture medium, wash twice with PBS, replace with fresh medium, add lentivirus A obtained in step 2.1, and the volume ratio of lentivirus to cells is lentivirus: cells = 1:4. After mixing, culture for 48 h, aspirate the culture medium, add fresh medium containing hygromycin, culture for 7 days, collect some cells to extract protein for WB, and use the anti-TetR antibody to detect whether TetR can be stably expressed. The WB detection results are shown in Figure 4 , as Figure 4 shown, the anti-TetR antibody can bind to the cell protein, indicating that the obtained cells can stably express TetR, that is, cells A containing vector A are obtained;

[0086] 2.3 Transiently transfect vector B obtained in step 1.2 into cells A obtained in step 2.2 according to the Lipofectamine TM 3000 transfection reagent instruction manual. After culturing for 24 h, add bleomycin and screen for 7 days. Pick out the cloned cells and maintain them in a bleomycin-resistant environment. Add 0, 10, 100, 1000 ng / mL of DOX and treat for 24 h. Collect some cells to extract protein for WB, and use the anti-FLAG antibody to detect the expression of BAP-UB. The WB results are shown in Figure 5 , as Figure 5The anti-FLAG antibody shown can bind to FLAG, indicating that the obtained cells can express BAP-UB under DOX induction, that is, cells B containing vector A and vector B are obtained; in addition, the induction expression effect is better at 100 ng / mL DOX, and 100 ng / mL DOX is used for induction expression subsequently.

[0087] 2.4 Select cells B in the logarithmic growth phase obtained in step 2.3, aspirate the culture medium, wash twice with PBS, replace with fresh medium, add lentivirus C obtained in step 2.1, and the volume ratio of lentivirus to cells is lentivirus: cells = 1:4. After mixing, culture for 48 h, aspirate the culture medium, add fresh medium containing puromycin, culture for 7 days, pick clone cells and maintain them in a puromycin-resistant environment. Add 100 ng / mL DOX and treat for 24 h. Collect some cells to extract proteins for WB, and use anti-FLAG antibody and anti-TEL antibody to detect whether BAP-UB and TEL-C-BirA can be stably expressed. The WB detection results are shown in Figure 6 , as Figure 6 shown, the anti-FLAG antibody and anti-TEL antibody can bind to the endogenous TEL protein in the cells, indicating that the obtained cells can express BAP-UB and TEL-C-BirA, that is, cells C containing vector A, vector B, and vector C are obtained. The obtained cells C are the cell indicator system.

[0088] Example 2

[0089] This example provides a cell indicator system and its preparation method. The preparation method is similar to that of Example 1, except that BirA in step 1.3 is located at the N-terminus of TEL, and the obtained vector is TEL-N-BirA. The vector map is shown in Figure 7 , and the remaining steps involving vector C are all replaced with TEL-N-BirA vectors; in step 2.4, the WB detection results are shown in Figure 6 , as Figure 6 shown, the anti-FLAG antibody and anti-TEL antibody can bind to the cell proteins, indicating that the obtained cells can express BAP-UB and TEL-N-BirA, that is, cells C containing vector A, vector B, and vector C are obtained. The obtained cells C are the cell indicator system; the remaining steps and their parameter conditions remain unchanged.

[0090] Comparative Example 1

[0091] This comparative example provides a cell indicator system and its preparation method. The preparation method is similar to that of Example 1, except that step 2.3 is not carried out, and lentivirus C is directly transferred into cells A obtained in step 2.2 in step 2.4, and the remaining steps and parameter conditions remain unchanged.

[0092] Comparative Example 2

[0093] This comparative example provides a cell indicator system and a preparation method thereof. The preparation method is similar to that of Example 2, except that step 2.3 is not performed, and lentivirus C is directly transferred into cell A obtained in step 2.2 in step 2.4, and the remaining steps and parameter conditions remain unchanged.

[0094] Effect Example 1

[0095] To detect whether the obtained cell indicator system can biotinylate and ubiquitinate a sample to be tested, WB was used to verify cells C obtained in Example 1 and Example 2. The specific scheme is as follows:

[0096] Cell C induced by 100 ng / mL DOX was mixed with 1 μg / mL biotin. After incubation at 37 °C for 6 h, the cells were washed, and cell proteins were extracted for WB detection. The antibodies used were anti-biotin antibody and anti-FLAG antibody. The results are shown in Figure 7 .

[0097] As Figure 7 shown, the cell indicator systems of Example 1 and Example 2 can both bind to the anti-biotin antibody and the anti-FLAG antibody, indicating that the cell indicator system of the present application can ubiquitinate the sample to be tested and bind to biotin to achieve biotinylation.

[0098] Example 3

[0099] This example provides a kit for detecting and / or identifying ubiquitinated substrates, including the cell indicator system of Example 1 or Example 2, doxycycline, biotin, and streptavidin agarose beads.

[0100] Example 4

[0101] This example provides a method for detecting and / or identifying ubiquitinated substrates. Substrate A (it is known that substrate A is a TEL substrate) is used as a sample to be tested for detection. The detection process is shown in Figure 8 , and the steps are as follows:

[0102] 1. Add 100 ng / mL of DOX to the cell indicator systems of Example 1, Example 2, Comparative Example 1, and Comparative Example 2, and cell B obtained in Example 1 respectively for induction for 12 h to express BAP-UB and TEL-C-BirA / TEL-N-BirA, so as to obtain BAP-UB and TEL-C-BirA / EL-N-BirA;

[0103] 2. To detect the ubiquitination of substrate A, add 1 μg / mL of biotin and incubate at 37 °C for 6 h to obtain cells containing ubiquitinated and biotinylated substrate A;

[0104] 3. Follow the instructions of the lysis buffer to add the lysis buffer to lyse the cells containing the ubiquitinated and biotinylated test samples obtained in step 2, centrifuge to obtain the lysis supernatant, and perform immunoprecipitation with the streptavidin agarose beads according to the instructions of the streptavidin agarose beads. Then use WB technology to detect the ubiquitination and biotinylation of substrate A. The antibody used in WB is anti-biotin antibody. In order to verify the expression of TEL and substrate A, TEL antibody and substrate A antibody were also added during the WB process. The WB results are shown in Figure 9 .

[0105] like Figure 9 As shown, only the ubiquitinated and biotinylated substrate A can bind to the streptavidin agarose beads to achieve immunoprecipitation and thus be detected by WB. It can be seen that the cell indicator system of the present application can simultaneously achieve ubiquitination and biotinylation of the sample to be tested.

[0106] Example 5

[0107] This example provides a method for detecting and / or identifying ubiquitinated substrates, wherein substrate A (known to be a TEL substrate) is used as a test sample for detection, and the steps are similar to those of Example 4, except that in step 2, 5 μM PROTAC molecules are added after adding biotin, and the remaining steps and parameters remain unchanged. The obtained WB results are shown in FIG. Figure 10 .

[0108] like Figure 10 As shown, the method of detecting ubiquitinated substrates of the present application can detect the degree of ubiquitination of the ternary complex formed by the PROTAC molecule and the ligand, indicating that the cell indicator of the present application can be used to monitor the ubiquitination process of the ternary complex.

[0109] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A cell indicator system, characterized in that, The cell includes at least one of vector A, vector B, and vector C; Vector A contains a TetR protein coding gene; Vector B contains the coding genes of a biotin acceptor peptide and ubiquitin; Vector C contains the coding genes of a targeting E3 ligase and a biotin ligase.

2. The cell indicator system according to claim 1, wherein It includes at least one of the following (Ⅰ)-(Ⅱ): (Ⅰ) Vector A is a lentiviral expression vector, and the TetR protein coding gene sequence is as shown in SEQ ID NO.1; (Ⅱ) Vector B also contains a TetO element and a FLAG tag; The biotin acceptor peptide coding gene sequence is as shown in SEQ ID NO.2; The ubiquitin coding gene sequence is as shown in SEQ ID NO.3; The nucleotide sequence of the TetO element is as shown in SEQ ID NO.4; The nucleotide sequence of the FLAG tag is as shown in SEQ ID NO.

5.

3. The cell indicator system according to claim 1, characterized in that, Vector C also contains an HA tag; Vector C is a lentiviral expression vector; The targeting E3 ligase coding gene sequence is as shown in SEQ ID NO.6; The biotin ligase coding gene sequence is as shown in SEQ ID NO.7; The nucleotide sequence of the HA tag is as shown in SEQ ID NO.

8.

4. The indicator system according to claim 1, wherein It includes at least one of the following (Ⅰ)-(Ⅱ): (Ⅰ) The biotin acceptor peptide in vector B is located at the N-terminus of ubiquitin; (Ⅱ) The biotin ligase in vector C is located at the N-terminus or C-terminus of the targeting E3 ligase.

5. The preparation method of the cell indicator system according to any one of claims 1-4, characterized in that, It includes the following steps: S1. Package lentiviruses of vector A and vector C in a vector cell to obtain lentivirus A containing vector A and lentivirus C containing vector C; S2. Infect the vector cell with lentivirus A obtained in step S1 to obtain cell A containing vector A; S3. Transfect vector B as described in any one of claims 1-4 into cell A obtained in step S2 to obtain cell B containing vector A and vector B; S4. Infect cell B obtained in step S3 with lentivirus C obtained in step S1 to obtain cell C containing vector A, vector B, and vector C, and the obtained cell C is the cell indicator system.

6. The application of the cell indicator system as described in any one of claims 1-4 in detecting and / or identifying ubiquitinated substrates.

7. The application of the cell indicator system as described in any one of claims 1-4 in detecting the process of the formation of a ternary complex between a PROTAC molecule and a ligand and / or the degree of ubiquitination.

8. A kit for detecting and / or identifying ubiquitinated substrates, characterized in that, It includes the cell indicator system as described in any one of claims 1-4, doxycycline, biotin, and streptavidin agarose beads.

9. The kit according to claim 8, wherein The kit further includes a PROTAC molecule.

10. A method for detecting and / or identifying ubiquitinated substrates, characterized in that, It includes the following steps: (1) Add doxycycline to the cell indicator system as described in any one of claims 1-4 to induce expression to obtain a biotin acceptor peptide-ubiquitin conjugate and an E3 ubiquitin ligase-biotin ligase conjugate; (2) Mix the biotin acceptor peptide-ubiquitin conjugate and the E3 ubiquitin ligase-biotin ligase conjugate obtained in step (1) with the sample to be tested, and add biotin and / or PROTAC molecules to obtain cells containing the ubiquitinated and biotinylated sample to be tested. (3) Lyse the cells containing the ubiquitinated and biotinylated sample to be tested obtained in step (2) to obtain a lysate supernatant. Immunoprecipitate the obtained lysate supernatant with streptavidin agarose beads, and then detect and / or identify the ubiquitination and biotinylation of the sample to be tested by immunoblotting and / or mass spectrometry techniques.

Images