Construction method and application of tiny binding protein of targeted vascular endothelial growth factor receptor 2

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a tiny binding protein of a targeted vascular endothelial growth factor receptor 2 (VEGFR2) as well as a construction method and application of the tiny binding protein. The amino acid sequence of the tiny binding protein is shown as SEQ ID NO. 5, and the tiny binding protein is of a triple helix structure. The tiny binding protein of the vascular endothelial growth factor receptor 2 (VEGFR2), provided by the invention, can be used as a tool protein to be combined with VEGFR2 protein and is used for regulating vascular endothelial growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a computer-aided design of a vascular endothelial growth factor receptor 2 minibinder (VEGFR2-mb) and its applications. Background Technology

[0002] Vascular endothelial growth factor (VEGF) stimulates endothelial cell growth and functions in multiple diseases. This growth factor primarily exerts its effects by binding to the VEGFR2 receptor. Angiogenesis plays a crucial role in various disease processes; for example, diabetic retinopathy is mainly characterized by pannus formation, leading to blindness. In tumorigenesis, angiogenesis is a core factor. Anti-VEGFR2 receptor action can inhibit tumor growth, and anti-VEGFR2 antibodies are already marketed anti-tumor drugs. Furthermore, in cardiovascular diseases, angiogenesis contributes to blood flow to ischemic tissues. VEGFR2 is a key target whose function has been thoroughly elucidated and confirmed. Given its well-defined mechanism and function, it is a mature drug target. Therefore, selecting novel drug targets around the VEGFR2 receptor will contribute to therapeutic effects against various angiogenesis-related diseases.

[0003] De novo protein synthesis is a crucial component of synthetic biology and medical research. Synthetic proteins are nanoparticles with specific properties, applicable to various aspects of biomedicine, including drug delivery, image diagnostics, basic experimental research, antibody preparation, and biopharmaceutical development. Due to their highly specific targets, these synthetic proteins offer significant advantages in treating some genetic and intractable diseases, providing novel treatment options. Minibinder protein probes targeting a specific epitope should target the protein rather than interfering with its function, localization, or expression. Because of their small molecular weight, these proteins can access proteins in the tiny corners of cells. Studies have shown that minibinders can fuse with other gene-encoded elements or proteins to monitor and interfere with cellular components and processes. Recent studies have shown that fluorescent proteins have been used in vivo to track proteins in small subcellular structures. They have also fused with protein degradation mechanisms to target proteins of interest for controlled degradation or scaffolding, and can also aggregate different molecular components and drive gene expression. Computer-aided design is one of the key methods for designing binding proteins in modern times. Traditional methods for empirically preparing binding proteins, such as antibodies, while mature, are cumbersome and have significant limitations. High R&D costs and time requirements further complicate this process. With the advancements in computer technology and algorithms in recent years, computer-aided design of binding proteins has become increasingly cost-effective and efficient. It also provides new solutions to problems that traditional methods cannot address. Among these, rifdock and motif graft algorithms, proposed in recent years, are capable of designing binding proteins based on specified binding sites, and are of great significance for designing novel molecular therapies and detecting related diseases. This patent will employ an AI-assisted minibinder design method to design active biological drugs that target and modulate the function of VEGFR2. These drugs will simultaneously possess dual-modal functions of in vivo angiography and angiogenesis inhibition. Summary of the Invention

[0004] This invention synthesizes a novel VEGFR2 small molecule binding protein, VEGFR2-mb, as a candidate drug for the treatment of vascular diseases based on the rifdock and motifgraft algorithms.

[0005] In a first aspect, the present invention provides a micro-binding protein targeting VEGFR2 protein, the amino acid sequence of which is shown in SEQ ID NO. 5, and the micro-binding protein has a triple helix structure.

[0006] In a second aspect, the present invention provides a method for constructing the micro-binding protein, comprising the following steps:

[0007] The amino acid sequence of the extracellular region of VEGFR2 protein, from 331 to 362, was selected as the binding target. Based on the rifdock and Motifg Raft algorithms, the top 5 micro-binding proteins (VEGFR2-mb) were designed.

[0008] The binding of the five VEGFR2-mb molecules to VEGFR2 was evaluated, and VEGFR2-mb molecules were constructed.

[0009] Preferably, the binding status of several micro-binding proteins to VEGFR2 is evaluated using the alphafold2 algorithm.

[0010] Preferably, the minibinder that binds to the extracellular region of VEGFR2 needs to be purified, and the purification is carried out according to the following steps:

[0011] A his-tag was inserted at the N-terminus and an HA-tag was inserted at the C-terminus of the minibinder DNA sequence to obtain the VEGFR2-mbHis tag and HA tag fusion protein; then, the DNA sequence of the minibinder-tag fusion protein was constructed into a plasmid through two restriction enzyme sites, XhoⅠ and NcoⅠ, to obtain an expression plasmid carrying the DNA sequence of VEGFR2.

[0012] The plasmid carrying the DNA sequence of VEGFR2-minibinder was transformed into competent cells, cultured, induced to express, and purified to obtain the mini-binding protein.

[0013] Preferably, the competent cells are DH5α Escherichia coli competent cells, and the plasmid is pet28α(+) plasmid.

[0014] In a third aspect, the present invention provides a nucleotide sequence encoding the microbinding protein.

[0015] In a fourth aspect, the present invention provides an expression vector containing the nucleotide sequence said in the invention.

[0016] In a fifth aspect, the present invention provides the use of the microbinding protein or the expression vector in binding the VEGR2 protein.

[0017] Preferably, the microbinding protein or the expression vector is used to regulate vascular endothelial function, including vascular endothelial cell activity and angiogenesis.

[0018] Preferably, the microbinding protein or the expression vector is used for the expression detection of VEGFR2 in live cells.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] 1. This invention, based on the RIFDock and MotifGraft algorithms, is the first to design and synthesize a novel VEGFR2 small molecule binding protein (minibinder, mb) targeting the extracellular region of the VEGFR2 protein as a candidate biopharmaceutical for vascular diseases. This minibinder, with a length of 56-65 amino acids, forms a stable triple helix structure. Compared to other types of binding proteins, such as antibodies synthesized using traditional methods, it has advantages such as short sequence and high synthesis efficiency. Compared to small molecule drugs, it has advantages such as high affinity and good targeting. Since VEGFR2 is a cylindrical structure assembled from multiple α-helices, in the design process, to enable the minibinder to bind to the active site of VEGFR2 and thus intervene in VEGFR2 function, this invention selects amino acids from the extracellular region of VEGFR2 as the target, allowing the minibinder to interact with the amino acid residues in the extracellular region of VEGFR2, thereby intervening in VEGFR2 function.

[0021] 2. The micro-binding protein targeting VEGFR2 provided by this invention can interact with VEGFR2 and promote angiogenesis. Using this as a tool, in vivo angiogenesis capacity can also be evaluated.

[0022] 3. The interaction between the micro-binding protein and VEGFR2 provided by this invention can perform in vivo protein tracing function. Attached Figure Description

[0023] Figure 1 The docking result between VEGFR2-mb10913 and VEGFR2 is obtained after designing based on rifdock and motifgraft;

[0024] Figure 2 This is an SDS-PAGE image of the VEGFR2-mb10913 nickel column affinity chromatography and its target peak;

[0025] Figure 3 The results are from VEGFR2-mb10913 protein sequencing.

[0026] Figure 4 This is the result of VEGFR2-mb10913 in HUVEC cells undergoing Transwell migration.

[0027] Figure 5 It is the function of VEGFR2-mb10913 in regulating HUVEC cell angiogenesis. Detailed Implementation

[0028] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0029] Unless otherwise stated, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental methods not specifically described in the following examples are generally performed under conventional conditions as described in Cao Longxing et al., *Design of protein-binding proteins from the target structure alone* and Sambrook et al., *Molecular Cloning: A Laboratory Manual* (New York: Cold Spring Harbor Laboratory Press, 4th edition), or as recommended by the manufacturer.

[0030] Example 1: Obtaining VEGFR2-mb

[0031] VEGFR2-mb design

[0032] According to the method for designing micro-binding proteins based on Rifdock and motifgraft described by Cao Longxing et al. in their paper "Design of protein-binding proteins from the target structure alone", the procedure is as follows: utilizing the target protein, preparing the target structure, selecting hydrophobic amino acids in the 331-362 region of the extracellular region of the VEGFR2 protein, performing molecular docking using RifGen and RifDock, and optimizing the sequence using FastDesign. Ultimately, 10,000 motifs were selected for the design. By adjusting the thresholds, the final values ​​were: ddg: -45.00, contact_molecular_surface: 440.00; score_per_res: -2.25; mismatch_probability: 0.10; sap_score: 35.00; binder_delta_sap: 12.00, resulting in 5 minibinders, denoted as mb1, mb2, mb3, mb4, and mb5, with ΔG Gibbs binding energies of -62.902 kcal / mol, -57.384 kcal / mol, -67.366 kcal / mol, -55.140 kcal / mol, and -62.671 kcal / mol, respectively.

[0033] The amino acid sequence of VEGFR2-mb10913 is as follows:

[0034] VEGFR2-mb10913: SLEEVEELLKKLDTPVARRLLKSLELMRRLGVPPEYILILLAQLADELGDPELAFLILQILMEVQ.

[0035] Expression and purification of VEGFR2-mb

[0036] Based on the minibinder structure of VEGFR2, an N-terminal his-tag and a C-terminal flag-tag were designed for subsequent affinity purification experiments. The DNA sequence of mb5 was then constructed into the pet28α(+) plasmid using two restriction enzyme sites, XhoⅠ and NcoⅠ.

[0037] The purification method followed the conditions described in "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 4th Edition)," specifically:

[0038] After transforming the plasmid carrying the aforementioned mb5 DNA sequence into DH5α competent *E. coli*, the cells were inoculated onto kanamycin-containing solid medium and cultured overnight at 37°C. The next day, healthy single colonies were selected and placed in 50ml centrifuge tubes, with 10mL of LB medium added. The cells were then incubated overnight at 37°C with shaking. The following day, 200mL of LB medium with kanamycin to a final concentration of 200μg / mL was added to a 1L Erlenmeyer flask, followed by 1 / 100 of activated *E. coli* culture, and the cells were incubated at 37°C with shaking for 2 hours.

[0039] Because the expressing bacteria contain the lactose operon regulatory element, IPTG can initiate the expression of related proteins. Further IPTG-induced expression was achieved by adding IPTG1 (final concentration 0.1–2 mg) to the culture medium and incubating overnight at 37°C with shaking to induce protein expression. After centrifugation, the supernatant was discarded, and 40 mL of sonication buffer, 1 mmol / L LPMSF, and 2 mmol / L β-mercaptoethanol were added. The mixture was incubated in an ice-water bath at 90 Hz with a duty cycle of 5:5 s for 15 min. After centrifugation, the supernatant was collected and filtered through a 0.22 μm filter membrane. Nickel column affinity chromatography was performed using an AKTAgo chromatography system. 10 mM imidazole buffer was used as the loading buffer, 25 mM imidazole buffer was used to wash away contaminating proteins, and 250 mM imidazole buffer was used to elute the target protein. After chromatography, the mixture was dialyzed three times at 4°C for 8 h each time with 100 volumes of PBS buffer to remove endotoxins and high salt concentrations. After dialysis, the protein was concentrated using a 10 kDa ultrafiltration tube, centrifuged (3000g, 4℃) to 1 mL, and then replenished with PBS buffer to 15 mL. This process was repeated four times. The concentration of the target protein was determined by BCA. The protein was separated using SDS-PAGE, and its purity was further identified by Coomassie brilliant blue staining.

[0040] SDS-PAGE image of the target protein ( Figure 2 This indicates that high-purity VEGFR2-mb10913 recombinant protein was obtained.

[0041] Example 2: Protein sequencing using VEGF2-mb10913

[0042] The results are as follows Figure 3 As shown

[0043] Example 3: Effects of VEGFR2-mb10913 on HUVEC cell migration

[0044] Experimental steps:

[0045] Adherent HUVEC cells were digested with 0.25% trypsin and resuspended in serum-free ECM medium to a concentration of 1×10⁻⁶. 5 Single-cell suspensions of 103 cells / mL were seeded into Transwell chambers and divided into control, VEGF, and VEGFR2-mb10913 groups. 1×103 cells were added to each chamber. 5Cells (200 µL cell suspension) were added to PBS, VEGF (0.01 mg / ml), and VEGFR2-mb10913 (0.01 mg / ml), respectively. 500 µL of ECM medium containing 10% FBS was added to the lower chamber of a 24-well plate. After culturing at 37 °C for 24 h, the cells in the upper chamber were gently wiped with a cotton swab, fixed with 4% paraformaldehyde for 20–30 min, stained with crystal violet solution for 10–15 min, washed three times with PBS, air-dried, and then photographed and counted under a microscope. Cell migration was counted using ImageJ software. Experimental data were statistically analyzed using GraphPad Prism 8, with P < 0.05 considered statistically significant.

[0046] The results are as follows Figure 4 As shown, VEGFR2-mb10913 can promote the migration ability of endothelial cells.

[0047] Example 4: Effects of VEGFR2-mb10913 on angiogenesis in HUVEC cells

[0048] Experimental steps:

[0049] VEGF is a specific agonist of VEGFR2, and VEGFR2-mb10913 and VEGF play a role in regulating the angiogenesis of HUVEC cells by VEGFR2. The specific approach is as follows:

[0050] Place Matrigel, 24-well plates, pipette tip boxes, and EP tubes in a 4°C freezer overnight. The next day, remove the Matrigel and place it on an ice pack. Add 30 μL of Matrigel to each well of the pre-chilled 24-well plate until it is evenly spread, avoiding air bubbles. Incubate at 4°C for 10 minutes to allow the Matrigel to level naturally. Incubate at 37°C for 30 minutes to allow the Matrigel to solidify. Select P5 generation HUVEC cells in the logarithmic growth phase and divide them into control, VEGF, and VEGFR2-mb10913 groups. Digest the treated cells with 0.25% trypsin, resuspend the cells in ECM medium without endothelial growth factor, and count them using a cell counting chamber. Add 1 × 10⁻⁶ μL of Matrigel to each well. 5 Cells (500µL cell suspension) were added to PBS, VEGF (0.01mg / ml), and VEGFR2-mb10913 (0.01mg / ml), respectively. After cell adhesion, they were incubated at 37°C in a 5% CO2 incubator for 10 h. The cells were then observed and photographed under an inverted microscope. ImageJ software was used to measure the vascular intersections and total vessel length to evaluate their tube-forming ability. All experimental data were statistically analyzed using GraphPad Prism 8, with P < 0.05 considered statistically significant.

[0051] The results are as follows Figure 5 As shown, compared with the control group, the positive drug VEGF can significantly promote the angiogenesis of HUVEC cells, and the tool protein VEGFR2-mb10913 can bind to VEGFR2 protein and promote the angiogenesis of endothelial cells.

Claims

1. A micro-binding protein targeting VEGFR2 protein, the amino acid sequence of which is shown in SEQ ID NO. 5, wherein the micro-binding protein has a triple helix structure.

2. A method for constructing the micro-binding protein according to claim 1, characterized in that, Includes the following steps: The amino acid sequence of the extracellular region of VEGFR2 protein, from 331 to 362, was selected as the binding target. Five micro-binding proteins were designed based on the rifdock and motifgraft algorithms. The binding of the five micro-binding proteins to VEGFR2 was evaluated, and the micro-binding proteins that bind to the extracellular region of VEGFR2 were obtained.

3. The construction method according to claim 1, characterized in that, The binding status of several micro-binding proteins to VEGFR2 was evaluated using the alphafold2 algorithm.

4. The construction method according to claim 1, characterized in that, After obtaining the micro-binding protein that binds to the extracellular region of VEGFR2, it is purified. The purification process is carried out according to the following steps: inserting a his-tag at the N-terminus and a flag-tag at the C-terminus of the micro-binding protein to obtain micro-binding protein-tag protein fusion. The DNA sequence of the microbinding protein-tag protein fusion was then constructed into a plasmid using two restriction enzyme sites, XhoⅠ and NcoⅠ, to obtain an expression plasmid carrying the DNA sequence of the microbinding protein. The plasmid carrying the DNA sequence of the microbinding protein was transformed into competent cells, cultured, induced to express, and then purified to obtain the microbinding protein.

5. The construction method according to claim 4, characterized in that, The competent cells are DH5α Escherichia coli competent cells, and the plasmid is pet28α(+) plasmid.

6. A nucleotide sequence encoding the microbinding protein of claim 1.

7. An expression vector containing the nucleotide sequence of claim 6.

8. Use of the microbinding protein of claim 1 or the expression vector of claim 5 in binding to VEGFR2 protein.

9. The use according to claim 8, characterized in that, The microbinding protein or the expression vector is used to regulate endothelial cell growth.

10. The use according to claim 8, characterized in that, The microbinding protein or the expression vector is used for the expression detection of VEGFR2 protein.

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