A short peptide binding to tissue-specific E3 ubiquitin ligase TRIM9 and its screening method

Abstract

The present invention discloses a short peptide binding to tissue-specific E3 ubiquitin ligase TRIM9 and a screening method thereof, and relates to the field of biopharmaceutical technology. The present invention obtains a short peptide binding to tissue-specific E3 ubiquitin ligase TRIM9 by screening through phage display technology, and uses the short peptide as a probe to interact with the full-length TRIM9 protein overexpressed in the cell, so that the polypeptide can be prepared into a drug, diagnostic reagent or research tool for regulating or intervening in a TRIM9-mediated biological process.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a short peptide binding to tissue-specific E3 ubiquitin ligase TRIM9 and a screening method thereof. Background Art

[0002] Protein-targeted degradation chimeras (PROTACs) are composed of a target protein ligand and an E3 ubiquitin ligase ligand connected by a linker. They induce ubiquitination of the target protein by shortening the distance between the target protein and the E3 ubiquitin ligase, thereby interfering with the function of the target protein by allowing the target protein to be recognized and degraded by the proteasome. Since the mechanism of action of PROTACs is completely different from that of small molecule inhibitors that directly inhibit the activity of substrate proteins, proteins that were originally difficult to be intervened by small molecule inhibitors (such as KRAS, BRAF, etc.) are expected to become drug targets that can be intervened.

[0003] The action of PROTAC depends on the recruitment of E3 ubiquitin ligases. Cereblon (CRBN) and Von Hippel-Lindau (VHL) are the E3 ubiquitin ligases currently recruited by most PROTACs, which are widely expressed in normal tissues. On the one hand, since target proteins such as transcription factor protein Myc, signal transducers and activators of transcription 3 (STAT3), and epidermal growth factor receptor (EGFR) are also expressed in normal tissues, PROTACs designed with E3 ubiquitin ligase ligands that are widely expressed throughout the body are prone to cause damage to normal tissues when degrading these non-tumor-specific expressed proteins; in addition, although mutant proteins of KRAS protein and BRAF protein have high tumor specificity, the widespread expression of E3 ubiquitin ligases also requires these target proteins to have highly specific recognition with their ligands in order to effectively control the risk of off-target, which greatly increases the difficulty of PROTAC research and development. Existing studies have shown that some of the more than 600 E3 ubiquitin ligases in the human body are only expressed in specific tissues or tumors. For example, the E3 ubiquitin ligase TRIM9 (Tripartite Motif Containing 9) is specifically and highly expressed in brain tissues, and is highly expressed in low-grade gliomas and glioblastoma multiforme. It has high tissue / tumor specificity, but there is currently a lack of TRIM9 ligands.

[0004] At present, researchers have limited knowledge of the structure of tissue / tumor-specific E3 ubiquitin ligases, making it difficult to develop small molecule ligands based on the structure. The recognition of E3 ubiquitin ligases and substrates is often achieved through certain specific peptide motifs. For example, the VHL ligand commonly used in PROTAC is optimized and modified based on a peptide sequence in the interaction region between VHL and its substrate HIF-1α. Protein interaction research based on organism display technology does not rely on the structure of the protein, and has the advantages of high throughput and controllable costs. Moreover, searching for natural peptide ligands for E3 ubiquitin ligases through organism display can avoid the dilemma of insufficient structural information. Among them, phage display technology is a technology that uses phages as carriers and can display hundreds of millions of exogenous peptides. It has extremely high library diversity and a short experimental cycle. It is the most commonly used and successful technology in organism display systems. At the same time, phage display has the advantage of unifying phenotype and genotype. The peptide sequence displayed on the phage surface can be obtained through gene sequencing. It is a convenient and efficient method for screening high-throughput peptide ligands. Therefore, short peptide ligands for tissue-specific E3 ubiquitin ligase TRIM9 based on phage display technology need to be developed. Summary of the invention

[0005] Based on the deficiencies in the prior art, the present invention provides a short peptide binding to tissue-specific E3 ubiquitin ligase TRIM9 and a screening method thereof.

[0006] The specific technical scheme of the present invention is as follows:

[0007] The present invention provides a short peptide binding to tissue-specific E3 ubiquitin ligase TRIM9, wherein the amino acid sequence of the short peptide is selected from any one of the following sequences: RNLMF, LTRQS, CLVAA, TVALR, SNMYY, SDFPH, FKWVV, CPMLY, RTRQS, and SCGPE.

[0008] The above-mentioned method for screening short peptides that bind to tissue-specific E3 ubiquitin ligase TRIM9 comprises the following steps:

[0009] (1) constructing a phage display library in which a variety of short peptide sequences are displayed on the surface of the phage;

[0010] (2) performing affinity screening on the phage display library and the substrate recognition region of TRIM9, and recovering phages that bind to TRIM9 after affinity screening;

[0011] (3) Analyze the displayed short peptide sequence from the recovered phage;

[0012] (4) Verifying the binding activity of the short peptide sequence with TRIM9, thereby obtaining a short peptide that binds to the tissue-specific E3 ubiquitin ligase TRIM9.

[0013] In some embodiments of the present invention, the phage display library in step (1) is a pentapeptide library.

[0014] In some embodiments of the present invention, in step (2), the substrate recognition region of TRIM9 is a SPRY domain;

[0015] The affinity screening includes multiple rounds of panning, and the stringency of the screening is gradually increased in each round of panning to reduce non-specifically bound phages;

[0016] The increased stringency of screening is manifested in reducing the input amount of SPRY domain protein and increasing the number of panning times.

[0017] In some embodiments of the present invention, in step (3), the short peptide sequence displayed by the phage is analyzed by high-throughput sequencing technology.

[0018] The present invention also provides the use of the short peptide or the short peptide obtained by the screening method that binds to the tissue-specific E3 ubiquitin ligase TRIM9 in the preparation of drugs, diagnostic reagents or research tools, and the drugs, diagnostic reagents or research tools are used to regulate or intervene in TRIM9-mediated biological processes.

[0019] Specifically, the drug is a drug for treating a disease associated with abnormal TRIM9 activity;

[0020] The diseases associated with abnormal TRIM9 activity are brain tumors, such as low-grade gliomas and glioblastoma multiforme.

[0021] Specifically, the diagnostic reagent is used to detect the expression level or activity state of TRIM9, or to screen compounds that can regulate the activity of TRIM9.

[0022] Specifically, the research tool is used to study the biological function, regulatory mechanism or interaction of TRIM9 with other proteins.

[0023] The present invention also provides a pharmaceutical composition for treating diseases associated with abnormal TRIM9 activity, comprising the short peptide, or the short peptide binding to tissue-specific E3 ubiquitin ligase TRIM9 obtained by the screening method, and a pharmaceutically acceptable carrier or excipient.

[0024] Beneficial effects of the present invention:

[0025] The present invention obtains a short peptide that binds to the tissue-specific E3 ubiquitin ligase TRIM9 by screening through phage display technology. The short peptide is used as a probe to interact with the full-length TRIM9 protein overexpressed in the cell. Therefore, the polypeptide can be prepared into a drug, diagnostic reagent or research tool for regulating or intervening in TRIM9-mediated biological processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Figure 1 shows the analysis of PCR products and vector restriction enzyme digestion during phage library construction; A is the schematic diagram of the phage library plasmid after construction, with the inserted polypeptide gene, GGGGS flexible hinge, and E-tag (GAPVPYPDPLEPR) at the 5' to 3' ends, followed by the phage capsid protein PⅢ gene; B is the agarose gel image of the PCR product of the inserted polypeptide gene, with the main band located at around 750 bp; C is the double restriction enzyme digestion agarose gel image of the pCANTAB 5E plasmid.

[0027] Figure 2 ELISA verification of E-tag display on phage surface.

[0028] Figure 3 This is the sequence logo of the deep sequencing of the phage library.

[0029] Figure 4 Analysis and sequencing of the effect of Escherichia coli codon bias on enrichment; A is the relative usage of synonymous codons; B is the deep sequencing sequence logo of the effect of codon bias on library enrichment.

[0030] Figure 5 SDS-PAGE analysis of GST-TRIM9 protein.

[0031] Figure 6 This is the sequence logo of deep sequencing after panning of the phage pentapeptide library targeting GST-TRIM9 protein.

[0032] Figure 7 This is the result of affinity test of the peptide to TRIM9.

[0033] Figure 8 This is an immunoprecipitation experiment of TRIM9 full-length protein using RNLMF probe; wherein, A is the overexpression of TRIM9 full-length protein in HEK-293T cells; B is the result of immunoprecipitation experiment of TRIM9 protein using RNLMF polypeptide probe. DETAILED DESCRIPTION

[0034] Example 1 Construction of phage pentapeptide display library

[0035] The (NNK) 5 primers (including S I restriction site, NNK represents a degenerate codon, N represents the possibility of ATCG appearing at this base position, each with a 25% probability, and K represents the possibility of TG appearing at this base position, each with a 50% probability. The NNK degenerate codon can cover the coding of all 20 natural amino acids) as a forward primer to synthesize the carboxyl terminus of bacteriophage PIII protein (containing Bam HI restriction site) as the reverse primer, the gene encoding the bacteriophage PIII protein (Gene ID: 927334) as the template for PCR, and the DNA fragment library was obtained by agarose gel recovery method (purchased from Novezan, FastPureGel DNA Extraction Mini Kit, catalog number: DC301-01). The theoretical fragment length was 750 bp ( Figure 1 B). The phagemid plasmid pCANTAB 5E was double-digested in a 37°C metal bath, and the restriction site was S Ⅰ and Bam HⅠ, DNA gel recovery after 2 hours of restriction digestion. The obtained DNA fragment library was homologously recombined with the phagemid plasmid pCANTAB5E after double restriction digestion (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., Hieff Clone Universal OneStep Cloning Kit, catalog number: 10922ES20) ( Figure 1 C in the figure). The reaction solution after homologous recombination was dialyzed through a semipermeable membrane (purchased from Merck Life Sciences, MF-Millipore, catalog number: VSWP09025) to remove salts, and then electroporated into SS320 Escherichia coli (purchased from Jinweizhi Biotechnology Co., Ltd., catalog number: GWEC101). The bacterial solution was evenly spread on a 2YT-Tet-Amp solid plate (containing tetracycline and ampicillin resistance) and placed upside down in a 37°C incubator for overnight culture. The next day, an appropriate volume of 2YT liquid culture medium was added to the plate, and the bacterial film was gently scraped off with a disposable coating stick, and the eluted bacterial solution (which contained the phage library plasmid after construction, as shown in the schematic diagram) was collected. Figure 1 A), and glycerol was added to a final concentration of 15% and stored at -80°C. At the same time, the library capacity was estimated based on the dilution point plate results. A total of 1.2×10 8 Take the E. coli library liquid and resuspend it in 2YT-Tet-Amp-Dextrose medium. Resuscitate to its OD 600The value was about 0.7, and sufficient helper phage M13KO7 (purchased from Thermo Fisher Scientific, catalog number: 18311019) was added for infection. After 1 hour, the cells were centrifuged and resuspended in 2YT-Amp-Kan liquid medium and cultured overnight. The next day, the progeny phage library was extracted by polyethylene glycol precipitation, and the phage precipitate was resuspended in 1× PBS, which was the phage pentapeptide library. Glycerol was added to a final glycerol concentration of 10%, and the cells were stored at -80°C after aliquoting.

[0036] Example 2 Surface display verification of phage pentapeptide library

[0037] The phage prepared in Example 1 was diluted with the coating solution and coated on an ELISA adsorption plate (purchased from Corning, Costar Assay Plate, 96 Well) at a ratio of 1:9, 1:27, and 1:81, with a volume of 100 μL added to each well, and coated overnight in a refrigerator at 4°C. The next day, the coating solution in the plate was discarded, washed twice with 0.05% PBST, and blocked with 5% skim milk, with an addition volume of 100 μL per well, and blocked at 37°C for 1 hour. After blocking, the skim milk was discarded, washed twice with 0.05% PBST, and then the primary antibody prepared with skim milk (purchased from Biotech, E-tag Mouse Monoclonal Antibody, Catalog No.: AF0135, diluted 1:1000 when used) was added, and incubated in an incubator at 37°C for 1 hour. The primary antibody was discarded, and the plate was washed 4 times with 0.05% PBST. The secondary antibody labeled with HRP (purchased from Biotech, horseradish peroxidase-labeled goat anti-mouse IgG (H+L), catalog number: A0350, diluted 1:5000 when used) was added. The plate was incubated at room temperature for 30 minutes. After the incubation, the plate was discarded, and the plate was washed 8 times with 0.05% PBST. The color developing solution (purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number: E661007) was prepared. The same volume of color developing solution was quickly added to each well, and the plate was placed in an incubator at 37°C. When the color was obvious, the plate was taken out, 1 M sulfuric acid was added to stop the color development, and the OD was detected. 450 Numeric value.

[0038] The results are as follows Figure 2 As shown, the presence of E-tag was detected on the surface of the library phage, and the absorbance value of the library phage had a dose-effect relationship with the phage coating amount. The color intensity decreased with the decrease of the phage coating amount, indicating that the color was specific, indicating that the polypeptide was successfully displayed on the phage surface.

[0039] Example 3 Verification of the diversity of the phage pentapeptide library

[0040] The phage library was subjected to high-throughput sequencing. Sequencing primers were designed with the library sequence insertion region as the center and the extension of about 100 bp before and after. A small amount of library phage was taken and used as a template for PCR. The band at 250 bp was recovered, and the sample was then sent to Beijing Novogene Technology Co., Ltd. for high-throughput sequencing. The sequencing platform selected was Novasek-PE150. After sequencing, the sequencing data was analyzed using the Perl language. 36,950 sequences were sequenced. From the overall polypeptide sequence, the most frequent polypeptide sequence only appeared 38 times, and the most abundant polypeptide accounted for 0.1% of the sequencing library. No nonspecific enrichment occurred in the overall polypeptide sequence, indicating that the errors introduced by humans in the early PCR and recombination and operation process were very low, and the overall sequence diversity of the library was well maintained.

[0041] Then, we analyzed whether there was any single-site amino acid enrichment. We used R language to analyze these peptide sequences and drew sequence logos.

[0042] The results are as follows Figure 3 As shown in the figure, the more times an amino acid appears at a certain site, the larger the letter of the amino acid on the sequence logo will be, which can reflect whether there is an amino acid ratio enrichment at a certain site in the polypeptide sequence. Due to the degeneracy of codons, the same amino acid is encoded by different numbers of codons, among which arginine (Arginine, R), leucine (Leucine, L) and serine (Serine, S) are each encoded by 6 codons, and glycine (Glycine, G), proline (Proline, P), valine (Valine, V), threonine (Threonine, T) and alanine (Alanine, A) are each encoded by 4 codons, so the probability of these amino acids appearing is relatively large compared to other amino acids, and it is also relatively obvious in the sequence logo. Overall, the distribution of the enrichment of amino acids at a single site reflected by the sequence logo is consistent with the law of codon degeneracy, indicating that there is no non-specific enrichment of a certain amino acid at a certain site in the library, and the amino acid distribution of the overall polypeptide sequence and the unit site of the polypeptide sequence is relatively balanced, indicating that the original library is unbiased and the library quality is good.

[0043] Example 4 Analysis of the influence of Escherichia coli codon bias on sequence enrichment

[0044] There is a bias in codon usage in Escherichia coli, so we investigated whether there is codon bias in the subsequent phage screening process, which leads to non-antigen-specific enrichment of certain sequences at the polypeptide translation level.

[0045] In 2YT-Dextrose liquid medium, inoculate ER2738 strain (purchased from Lucigen, catalog number 60522-1) and culture until OD 600 The value is about 0.6, and the library phage is added for infection, and then the Escherichia coli infected by the library phage is screened using ampicillin resistance plates, and the phagemid vector in the infected Escherichia coli is packaged using helper phage M13KO7 to obtain phages after one round of infection-packaging. These phages are used to repeat the above infection-packaging steps, and a total of 3 rounds of infection and packaging are performed. Then PCR is performed on the phages produced in the last round, and high-throughput sequencing technology is used to investigate the effect of the preference of Escherichia coli codons on the enrichment of phage library polypeptide sequences in the absence of affinity screening. The relative usage of synonymous codons (RSCU) is introduced, which represents the ratio of the actual usage frequency of codons encoding an amino acid to the theoretical usage frequency. If the RSCU value is greater than 1, it means that the actual occurrence frequency of the codon is higher than the theoretical occurrence frequency.

[0046] The results are as follows Figure 4 As shown, the codon bias of the E. coli host during the continuous infection-packaging process does not cause a high level of background enrichment in affinity screening.

[0047] Example 5 Expression and purification of the SPRY domain protein of TRIM9

[0048] Beijing Qingke Biotechnology was commissioned to synthesize the substrate recognition region of TRIM9 (i.e., SPRY region (Gene ID: cd12889 in 114088), N-terminal fusion GST tag) sequence in pGEX-4T1, and transferred into BL21 (DE3) competent cells (purchased from Beijing Qingke Biotechnology, catalog number: TSC-E01). After amplification, isopropyl β-D-Thiogalactoside (IPTG) was added to induce expression, and Escherichia coli was disrupted by a high-pressure cell disruptor. Affinity purification was performed using an AKTA purifier and a GST tag affinity column (purchased from Yisheng Biotechnology (Shanghai) Co., Ltd., GSTSep Glutathione 4FFChromatography Column, catalog number: 20510ES08). The purified eluate was ultrafiltered and concentrated, and the protein storage buffer was replaced from the eluate containing glutathione to PBS buffer. SDS-PAGE analysis of GST-TRIM9 protein is as follows. Figure 5 As shown, the protein band is at the theoretical protein position, and the overall purity of the protein is >90%, so the next step of phage display panning experiment can be carried out.

[0049] Example 6 Affinity screening of phage pentapeptide library for E3 ubiquitin ligase TRIM9

[0050] The phages in this example were subjected to four rounds of panning. In the first round of panning, the original phage library prepared in Example 1 and the recombinant fusion protein obtained in Example 5 were directly used for incubation. The initial input amount of the original phage library was 10 11 The input amount of recombinant fusion protein was 6 μg. After GST affinity magnetic beads (purchased from Changzhou Tiandi Renhe Biotechnology Co., Ltd., Glutathione Magarose Beads, Catalog No.: SM002005) were washed (TBS-T with 0.05% Tween-20) and blocked (0.05% TBS-T with 0.5% BSA), phage and recombinant fusion protein were added and incubated at room temperature. The magnetic beads were then washed to remove non-specifically adsorbed phages. The first round of panning was washed 12 times, followed by the addition of 1 mL of elution buffer (200 mM glycine with 0.1% BSA, pH = 2.5), incubated at room temperature for 15 minutes, and then the magnetic beads were adsorbed with a magnetic stand, the supernatant was carefully aspirated, and finally neutralized with neutralizing solution (2 M Tris-Hcl, pH = 9.0) to a pH of about 7. The eluted phages were used to infect ER2738 Escherichia coli in the logarithmic growth phase, and the helper phage M13KO7 was used to package the progeny viruses, so that the genetic information of the recovered phages could be replicated and amplified in large quantities, producing a progeny phage library that had undergone a round of affinity screening.

[0051] Negative panning was introduced in the second round of panning, mainly for GST-tagged proteins. The phage library amplified in the first round was first incubated with GST protein and GST affinity magnetic beads, and then the supernatant was retained and incubated with the recombinant fusion protein. At the same time, the stringency of the panning was appropriately increased, the amount of recombinant fusion protein input in the second round was reduced to 3 μg, the number of washes was increased to 14 times, and the content of Tween-20 in TBS-T was increased to 0.1%, so that phages with higher affinity have an advantage in the competition, recover as many high-affinity phages as possible and reduce nonspecific adsorption. In the third and fourth rounds of panning, the amount of fusion protein input was reduced to 1 μg, and the number of washes was increased to 16 times. TBS-T with 0.1% Tween-20 was used. After the panning was completed, a small amount of phages that had completed 4 rounds of panning were taken and pre-treated in a 95°C metal bath for 15 minutes. High-throughput sequencing fragments were prepared using PCR and sent to Beijing Novogene Technology Co., Ltd. for high-throughput sequencing. The results are as follows: Figure 6 As shown, the pentapeptide library was enriched for GST-TRIM9, and a total of 25,200 valid data were obtained by high-throughput sequencing. The top two peptides were RNLMF and LTRQS, which appeared 4,556 and 4,128 times, respectively (Table 1).

[0052] Table 1 Enriched peptides from the pentapeptide library for TRIM9

[0053]

[0054] Example 7 Affinity verification of phage display enriched peptides for TRIM9 protein

[0055] The EGFP-tagged TRIM9-SPRY sequence was synthesized by Beijing Qingke Biotechnology in pET-28a, and EGFP-TRIM9 was expressed by Escherichia coli and diluted to 1.6 μM. The peptide to be detected (synthesized by Suzhou Modifu Biotechnology Co., Ltd.) was dissolved into a 20 mM concentration of the mother solution, diluted to 4 mM with 1 × TBS-T buffer (0.05% Tween-20), and 10 μL of gradient diluted peptide solution and 10 μL of EGFP-TRIM9 protein solution were added to each well of two columns of eight-tube strips. After repeated pipetting and thorough mixing, the mixture was incubated at room temperature for 25 minutes, and a microscale thermophoresis (MST) capillary (purchased from Nano Temper Technologies, catalog number: MO-K022) was added to absorb the incubation solution, and then the detection was performed by the MO control module, and the obtained data were analyzed using MO Affinity Analysis Software.

[0056] In this example, the affinity of the peptide with the highest enrichment and the peptide with relatively high enrichment was investigated to evaluate the relationship between enrichment and affinity. According to the results in Table 1, the enrichment of the first and second ranked peptides is similar. In this example, the top two ranked peptides: RNLMF (4556 / 25200), LTRQS (4218 / 25200) and the eighth, ninth and tenth ranked peptides CPMLY (182 / 25200), RTRQS (131 / 25200), SCGPE (121 / 25200) were synthesized as controls.

[0057] The affinity test results of the peptides are as follows Figure 7As shown. The affinity of the peptide with the highest enrichment is 25.6 μM, and the second one is 96 μM. Although the proportion of these two peptides in the enrichment is very close, the affinity for EGFP-TRIM9 is quite different. It can be considered that the reason for the similar enrichment is that the phage has affinity for two independent regions in GST-TRIM9 that do not compete with each other during the panning process, and the phage competition process mainly screens the peptide with the best relative affinity in the library. High relative affinity does not mean strong absolute affinity. Therefore, the two independent regions in the fusion protein are enriched with peptides with better relative affinity in the library. Among the three selected control peptides, the affinity of two peptides is above 900 μM, which is very low. The ninth-ranked RTRQS peptide differs from the second-ranked LTRQS peptide by only one amino acid. The affinity of the RTRQS peptide is 146.6 μM, which is slightly inferior to LTRQS, but its enrichment is quite different from LTRQS. It can be considered that the reason for the large difference in the enrichment of these two peptides is that they have the same affinity for the same site and there is a competition between them. LTRQS has a better relative affinity, so it has a greater advantage in competitive panning and thus has a higher enrichment, but in terms of absolute affinity, the difference between the two is not large.

[0058] Example 8 Affinity verification of RNLMF for TRIM9 full-length protein

[0059] The full-length TRIM9 protein gene with a Flag tag (Gene ID: 114088) was synthesized by Beijing Qingke Biotechnology in the pCDH plasmid and transfected using the jetPRIME transfection reagent. ® (Purchased from Polyplus, catalog number: 101000046) The plasmid was transfected into HEK-293T (Purchased from Shanghai Cell Bank, Chinese Academy of Sciences) cells. After 24 hours, the cells were harvested and lysed with 1% NP40 (Purchased from Biyuntian, catalog number: P0013F) and protease inhibitor (Purchased from TargetMol, Protease Inhibitor Cocktail, catalog number: C0001). The RNLMF-PEG2 (polyethylene glycol)-Biotin peptide probe (synthesized from Suzhou Modifu Biotechnology Co., Ltd.) was co-incubated with the cell lysate, and then the affinity of Streptavidin magnetic beads with Biotin was used for immunoprecipitation experiments to investigate whether the peptide probe can bind and immunoprecipitate the overexpressed protein in the cell lysate. In the immunoprecipitation experiment, four groups were set up, namely the control group (no probe was added) and the experimental group (the incubation amount of the peptide probe was 20 μM, 100 μM, and 500 μM, respectively).

[0060] The results are as follows Figure 8As shown in the figure, although there is a slight amount of nonspecific adsorption of Streptavidin magnetic beads, the experimental group incubated with the added peptide probe pulled down more TRIM9 protein, and it was positively correlated with the input amount of peptide probe, indicating that the RNLMF peptide can be used as a probe to interact with the full-length TRIM9 protein overexpressed in the cell.

Claims

1. A short peptide that binds to tissue-specific E3 ubiquitin ligase TRIM9, characterized in that: The amino acid sequence of the short peptide: RNLMF.

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