A microbinding protein targeting sodium-glucose cotransporter 2 and construction method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a micro binding protein targeting sodium-glucose cotransporter 2 and a construction method and application thereof. The amino acid sequence of the micro binding protein is shown in SEQ ID NO. 5, and the micro binding protein is a triple helix structure. The micro binding protein targeting sodium-glucose cotransporter 2 (sglt2) provided in the application can be combined with the sglt2 protein as a tool protein, is used for regulating cell glucose absorption, and can be used for fluorescence detection of the sglt2 protein through the micro binding protein marked with a specific label. The micro binding protein in the application can be applied to sglt2 function regulation and fluorescence label detection as a novel sglt2 tool.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and specifically relates to a minibinder (mb) targeting sodium-glucose cotransporter 2 (sglt2 protein) based on computer-aided design and application thereof. BACKGROUND

[0002] Sodium-glucose cotransporter-2 (sglt2) is the main cotransporter involved in renal glucose reabsorption, so its inhibitor SGLT2 inhibitor (sglt2i) can be used as a hypoglycemic drug for clinical diabetic patients. In recent years, its cardioprotective effect has attracted more and more attention from basic and clinical workers. In 2021, the ESC guideline included sglt2i in the new four-in-one scheme for heart failure. On August 17, 2023, the China National Medical Products Administration approved one of the sglt2is, dapagliflozin, for reducing the risk of cardiovascular death, hospitalization due to heart failure, or emergency treatment for heart failure in adult patients with symptomatic chronic heart failure. However, due to the low expression of sglt2 in myocardial cells, the mainstream research theory believes that the protective effect of sglt2i on the heart is mainly based on its protective effect on the kidney, and the direct protective mechanism of sglt2i on the cardiovascular system has not been clearly defined, and academic opinions are diverse. The current main academic opinion believes that in addition to promoting the renal glucose excretion of sglt2i, it indirectly protects the heart through kidney protection and inhibition of the RAS system. However, in recent years, many studies have suggested that sglt2i has independent protective effects on the heart, and the specific mechanism is not clear, especially the core issue of whether sglt2i has off-target effects. It is of great significance to clarify how sglt2i protects the heart and the mechanism of myocardial cells in cardiovascular therapeutic drugs, so constructing a clear sglt2 regulatory molecule and indicator plays an important role in clarifying the function of sglt2.

[0003] Computer-aided design is one of the key methods of contemporary design of binding proteins. Traditional methods of empirically preparing binding proteins, such as antibodies, have a mature method, but the steps are tedious and have significant limitations. Moreover, the high research and development costs and time requirements make this process even more complex. With the advancement of computer technology and algorithms in recent years, designing binding proteins with computers has become increasingly cost-effective and efficient. It also provides new solutions for problems that are limited by traditional methods. Among them, rifdock and motif graft are algorithms that can design binding proteins according to specified binding sites, which are of great significance for designing new molecular therapies and detecting related diseases. However, there is no report on using rifdock and motif graft methods to explore the influence of myocardial sglt2 protein on the myocardial protective effect of sglt2i. SUMMARY

[0004] In order to explore whether the myocardial protective effect of sglt2i directly acts on the sglt2 protein of the myocardium, so as to more effectively target the potential action pathway of protecting the myocardium, the present application synthesizes a novel sglt2 small molecule binding protein (minibinder, mb) as a tool protein based on the rifdock and motifgraft algorithms.

[0005] In the first aspect of the present application, a micro-binding protein targeting sglt2 protein is provided, and the amino acid sequence thereof is shown in SEQ ID NO. 5. The micro-binding protein is a triple helix structure.

[0006] In the second aspect of the present application, a construction method of the micro-binding protein is provided, comprising the following steps:

[0007] The amino acids at positions 247, 248, 254, 270, 271, 496, 516, 518 and 519 of the extracellular region sequence of sglt2 protein are selected as the binding target, and a plurality of micro-binding proteins (minibinder, mb) are designed based on the rifdock and motifgraft algorithms.

[0008] The binding of the plurality of minibinders to sglt2 is evaluated, and minibinders that bind to the extracellular region of sglt2 are obtained.

[0009] Preferably, the binding of the plurality of minibinders to sglt2 is evaluated using the alphafold2 algorithm.

[0010] Preferably, the minibinders that bind to the extracellular region of sglt2 need to be purified, and the purification is performed according to the following steps:

[0011] His-tag is inserted at the N-terminus of the DNA sequence of the minibinder, and flag-tag is inserted at the C-terminus to obtain a micro-binding protein-tag protein fusion. Then, the DNA sequence of the micro-binding protein-tag protein fusion is constructed into a plasmid through Xho I and Nco I two enzyme cutting sites to obtain an expression plasmid carrying the DNA sequence of the minibinder.

[0012] The plasmid carrying the DNA sequence of the minibinder is transformed into competent cells, cultured, induced for expression, and purified to obtain the micro-binding protein.

[0013] Preferably, the competent cells are BL21 E. coli competent cells, and the plasmid is a pet28α(+) plasmid.

[0014] In a third aspect, the present application provides a nucleotide sequence encoding the minibinder.

[0015] In a fourth aspect, the present application provides an expression vector comprising the nucleotide sequence.

[0016] In a fifth aspect, the present application provides use of the minibinder or the expression vector in binding sglt2 protein.

[0017] Preferably, the minibinder or the expression vector is used for regulating cell glucose uptake.

[0018] Preferably, the minibinder or the expression vector is used for detecting expression of sglt2 protein.

[0019] Compared with the prior art, the present application has the following advantages:

[0020] 1. Based on rifdock and motifgraft algorithms, the present application first takes the extramembrane region of protein as a target to design minibinder, and synthesizes a novel sglt2 minibinder as a tool protein. The minibinder has a length of only 65 amino acids and is a stable triple helix structure. Compared with other types of binding proteins, such as antibodies synthesized by traditional methods, the minibinder has the advantages of short sequence and efficient synthesis. Compared with small molecule drugs, the minibinder has the advantages of high affinity and good targeting. Since sglt2 is a cylindrical structure assembled by multiple alpha helices, in the design process, in order to enable the minibinder to bind to the active site of sglt2 and thereby interfere with the function of sglt2, the present application selects the amino acids in the extramembrane region of sglt2 as a target, so that the minibinder interacts with the amino acid residues in the extramembrane region of sglt2 sodium-sugar channel, thereby interfering with the function of sglt2.

[0021] 2. The minibinder targeting sglt2 protein provided by the present application can interact with sglt2 and promote glucose uptake, and is used for regulating cell glucose absorption. As a tool, it can also be used to explore whether myocardium expresses sglt2.

[0022] 3. The minibinder provided by the present application can interact with sglt2 through fitc-glucose sugar uptake experiment detection in addition to fluorescence detection, and the diversity of verification methods provides more reliable basis for the minibinder as a tool protein for sglt2 detection and research. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1is the docking result of mb1, mb2, mb3, mb4, mb5 and sglt2 based on rifdock and motifgraft;

[0024] Figure 2 is the docking result of mb5 and sglt2 predicted in alphafold2 (the binding site of minibinder and sglt2 is shown in the red circle);

[0025] Figure 3 is the SDS-PAGE diagram of sglt2 minibinder nickel column affinity chromatography and its target peak;

[0026] Figure 4 is the sglt2 minibinder pull down experiment result of sglt2 protein;

[0027] Figure 5 is the sglt2 minibinder HK-2 fluorescence labeling experiment result;

[0028] Figure 6 is the HK-2 glucose uptake change under the intervention of sglt2 minibinder; DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be further described in detail below in combination with specific embodiments. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0030] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. The experimental methods in the following examples without specific conditions are usually carried out according to the conventional conditions, such as the conditions 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, Fourth Edition), or according to the conditions recommended by the manufacturer.

[0031] Example 1: Obtaining sglt2 minibinder

[0032] Design of sglt2 minibinder

[0033] According to the method of designing minibinders based on rifdock and motif graft described by Cao Longxing et al. in the paper "Design of protein-binding proteins from the target structure alone", the steps of using target protein, preparing target structure, selecting hydrophobic amino acids at positions 247, 248, 254, 270, 271, 496, 516, 518, 519 of the sequence of the extracellular region of the sglt2 protein, using RifGen and RifDock for molecular docking, using FastDesign for sequence optimization are followed. Finally, 10000 motifs are selected for design, by adjusting the threshold, finally adjusted: 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, 5 minibinders are obtained, respectively marked as mb1, mb2, mb3, mb4, mb5, and their amino acid sequences are as follows: ΔG The Gibbs binding energies are -62.902 kcals / mol, -57.384 kcals / mol, -67.366 kcals / mol, -55.140 kcals / mol and -62.671 kcals / mol, respectively. As shown in Figure 1

[0034] The amino acid sequences of the 5 minibinders are as follows:

[0035] mb1: SEEEREAEELLQKAIEALREGDREKLRKLAEEAAEAAERAGDPFAAA FIRLLLRRAL, as shown in SEQ ID NO. 1.

[0036] mb2: SFRARLSRIIEEIRAAALLGDEELVERLLEELERLAREAGDEEAKQFV ELQKRYL, as shown in SEQ ID NO. 2.

[0037] mb3: NAARLSRLYEEARRAALRGNPEEAREIIEEAIELAEELGDERTLKRLR QVLKLQEEY, as shown in SEQ ID NO. 3.

[0038] ​mb4: DESRELLEKVAKELAELLRNGNFEEAERLVERVEERLKELGDEEAVR LLRSFLFRAR, as shown in SEQ ID NO. 4.

[0039] mb5: TEELVEELLRELGVPPEQIEALKRELEQLKRRGDPAYERVREAAERIKKGEIPPEVLARYLALLS, as shown in SEQ ID NO. 5.

[0040] Further simulation of minibinder: SGLT2 complex by alphafold2 algorithm obtained five binding sites of minibinder and sglt2, only mb5 and five predicted binding sites of sglt2 are located in the extracellular region of sglt2 protein, which meets the expected expectation, as shown in Figure 2 Therefore, the minibinder5 was used for subsequent research.

[0041] Expression and purification of sglt2 minibinder

[0042] According to the structure of sglt2 minibinder, his-tag at N-terminal and flag-tag at C-terminal were designed for subsequent affinity purification and pull down experiment, and then the DNA sequence of mb5 was constructed into pet28a(+) plasmid through Xho I and Nco I enzyme cutting sites.

[0043] The purification method refers to the conditions described in "Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, Fourth Edition)", specifically:

[0044] After the plasmid carrying the above-mentioned DNA sequence of mb5 was transformed into the competent E. coli BL21, the seed plate was cultured in kanamycin solid medium at 37°C overnight, and the next day, the single colony with good growth was selected and placed in a 50 ml centrifuge tube, 10 ml LB medium was added, and the culture was incubated at 37°C overnight. The next day, 200 mL of LB medium with a final concentration of kanamycin of 200 μg / mL was added to a 1 L conical flask, and 1 / 100 of the activated E. coli liquid was added and cultured at 37°C for 2 h.

[0045] Because the expression bacteria contain the lac operon regulatory elements, IPTG can initiate protein expression. Further IPTG-induced expression was performed by adding IPTG1 (final concentration 0.1–2 mM) to the culture medium and incubating at 37°C with shaking for 5 h to induce protein expression. The supernatant was removed by centrifugation, and 40 mL of sonication buffer (1 mmol / L PMSF and 2 mmol / L β-mercaptoethanol) was added. The mixture was placed in an ice-water bath and sonicated at 90 Hz with a duty cycle of 5:5 s for 15 min. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter. Nickel affinity chromatography was performed using an AKTAgo chromatography system at a flow rate of 1 mL / min. 10 mM imidazole buffer was used as the loading buffer, 25 mM imidazole buffer was used to wash contaminants, and 250 mM imidazole buffer was used to elute the target protein. After chromatography, the protein was dialyzed against 100 volumes of PBS at 4°C for 8 h three times to remove endotoxins and high salt content. After dialysis, concentrate the protein using a 3 kDa pore size ultrafiltration tube. Centrifuge (3000 g, 4°C) to 1 mL and then add PBS buffer to 15 mL. Repeat four times. Target protein concentration is determined by BCA assay. Proteins are separated by SDS-PAGE and further assessed for purity by Coomassie Brilliant Blue staining.

[0046] SDS-PAGE image of target protein ( Figure 3 ) showed that high-purity sglt2 minibinder tool protein was obtained.

[0047] Example 2: Pull down experiment using sglt2 minibinder 5

[0048] Minibinder 5, protein A / G magnetic beads and His antibody or IgG were incubated together at 4℃ overnight. The antibody not combined with magnetic beads and minibinder 5 combined with the antibody were washed by magnetic separation for three times to obtain minibinder 5 combined with His magnetic beads and IgG group magnetic beads. After trypsin digestion, the HK-2 cells were centrifuged to obtain the precipitate, 1 mL NP40 lysis solution (1% NP40, 150 mM KCl, 25 mM Tris-HCl, 5 mM EDTA, 0.5 mM DTT, 1 mM PMSF) was added, and the cell suspension was added to a glass grinder, grinded on ice for 30 min, and the sample was cooled for 2-3 s after every 1-2 times of grinding. The grinded sample was centrifuged at 13000 rpm for 10 min at 4℃, and the supernatant was transferred to the centrifuge tube containing minibinder 5 combined with His magnetic beads and IgG group magnetic beads, respectively. The centrifuge tube was placed on the suspension instrument and incubated at 40 rpm and 25℃ for 3 h. After incubation, it was separated on the magnetic stand for 30 s, and the supernatant was removed. The supernatant of the IgG group was reserved for the subsequent input group. 500 uL NP40 lysis solution was added, the magnetic beads were resuspended by gently blowing with a pipette, placed on a magnetic stand for 30 s, the supernatant was removed, and the above steps were repeated three times. 100 ul SDS-PAGE loading buffer was added per 10 uL magnetic bead volume, heated at 75℃ for 30 min, placed on a magnetic stand for 10 s, and the supernatant was detected by Western blot method using sglt2 antibody to detect the binding of mb5 to sglt2.

[0049] The results are shown in Figure 4 The SGLT2 protein in the cells can be pulled down by using specific anti-histidine tag antibody. In the figure, input refers to the cell protein after incubation with specific anti-histidine tag antibody magnetic beads; igg refers to non-specific antibody group magnetic beads (control group) incubated with cell protein; His-antibody refers to anti-histidine tag antibody magnetic beads (experimental group) incubated with cell protein. The results show that minibinder 5 combined with anti-histidine tag antibody magnetic beads can interact with sglt2.

[0050] Example 3

[0051] Fluorescent labeling detection of sglt2 minibinder 5 and human kidney proximal tubular cell HK-2 binding

[0052] HK-2 cells are the main sglt2(+) cells, and 90% of the glucose reabsorption in the primary urine is involved in the sglt2 expressed by them. Therefore, HK-2 cells are used for fluorescent labeling experiments.

[0053] Experimental steps:

[0054] The experiment uses the Alexa Fluor 488 coupling kit provided by Abeam Company (a23653) to fluorescently label minibinder 5. The kit uses a 100 μg specification. After mixing 100 μg of minibinder 5 with 10 μL of Modifier reagent, open the bottle cap of the Alexa Fluor 488 coupling mixture, and use a pipette tip to suck the minibinder 5 (with Modifier reagent) and directly add it to the fluorescent freeze-dried powder material. Use the pipette tip to suck the liquid and re-titrate it again or twice, and gently resuspend. After placing the bottle cap, place it at 25°C in the dark for 2 h, then add 11 μL of Quencher reagent to quench the fluorescent molecules that are not bound to minibinder 5, gently mix, and stand for 5 min to obtain fluorescently labeled minibinder 5.

[0055] HK-2 cells were cultured using a twelve-well plate. When the cell density reached 50%, the cells were washed three times with pbs, fixed with 4% paraformaldehyde for 15 min, washed three times with pbs, added with 0.2% triton X-100 to break the membrane for 20 min, added with 2% BSA to block for 1 h, washed three times with pbs, mixed the fluorescently labeled minibinder 5 with pbs at a volume ratio of 1:25, and added dropwise to the climbing sheet, incubated in a wet box at 4°C overnight, and the next day washed three times with pbs, each for 5 min. Finally, the climbing sheet was placed on a glass slide, and the labeling of minibinder 5 and HK-2 cells was observed using a Nikon fluorescence confocal microscope. At the same time, HK-2 cells were labeled with the same quenched labeled product as a control.

[0056] The results are shown in Figure 5 Green fluorescence is fluorescently labeled minibinder 5, which can indirectly indicate the expression and distribution of SGLT2 in cells.

[0057] Example 4: sglt2 minibinder 5 intervention HK-2 sugar uptake experiment

[0058] Experimental steps:

[0059] Empagliflozin (EMPA) is a specific inhibitor of sglt2. The effect of minibinder 5 and EMPA on the regulation of sglt2 on HK-2 cell glucose reabsorption. The specific method is as follows:

[0060] ​HK-2 cells were divided into Con group, EMPA group, EMPA + minibinder 5 group and minibinder 5 group by adding 20 mM fitc-glucose into DMEM medium, and 2 mL DMEM medium containing vehicle (marked as CON group), 1 μΜ EMPA, 1 μΜ EMPA + 50 nM minibinder 5 and 50 nM minibinder 5 were added respectively, and cultured at 37 °C for 36 h. After washing with PBS for three times, the un-uptaken fitc-glucose was removed. The uptake of fitc-glucose by HK-2 cells was detected by using an Olympus fluorescence microscope.

[0061] As shown in the results, minibinder 5 can improve the glucose uptake of cells. The positive drug EMPA can inhibit the glucose uptake of HK-2 cells, and minibinder 5 can promote the glucose uptake and reverse the inhibition of EMPA on the glucose uptake of HK-2 cells. Figure 6

[0062] Obviously, those skilled in the art can make various modifications and variations to the present application without departing from the spirit and scope of the present application. Thus, if these modifications and variations of the present application belong to the scope of the claims of the present application and their equivalent technologies, the present application also intends to include these modifications and variations.​

Claims

1. A microbinding protein targeting sglt2 protein, characterized in that Its amino acid sequence is shown in SEQ ID NO. 5, and the microbinding protein has a triple helical structure.

2. A gene encoding the microbinding protein according to claim 1.

3. An expression vector containing the sequence of the gene according to claim 2.

4. Use of the microbinding protein according to claim 1 or the expression vector according to claim 3 in preparing a product for detecting expression of sglt2 protein.

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