Membrane protein C99-nanodisk compound as well as preparation method and application thereof

Abstract

The invention discloses a membrane protein C99-nanodisk compound as well as a preparation method and application thereof, and belongs to the technical field of biology. The membrane protein C99-nanodisk compound provided by the invention is prepared on the basis of label-resectable C99 recombinant membrane protein and 22A-nanodisk c, does not contain a detergent, and can be used for accurately detecting protein or small molecule drugs interacting with the C99 protein in vitro, so that a new tool can be provided for mechanism exploration and drug development of Alzheimer's disease.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a membrane protein C99-nanodisc complex (also referred to as C99-22A-Nanodiscs in this paper) and its preparation method and application, particularly relating to a tagless C99 recombinant membrane protein, the membrane protein C99-nanodisc complex prepared from the C99 recombinant membrane protein, and its preparation method and application. Background Technology

[0002] C99 protein is a core key protein in the development of Alzheimer's disease (AD), playing a crucial role in the cleavage of amyloid protein (APP-C99-Aβ). 42 It plays an important role. The intracellular domain (AICD) of the C99 protein has important physiological functions, and a large number of intracellular water-soluble proteins (such as intracellular signaling proteins) have been found to specifically recognize this region, including Fe65, the Mint2 / X11 family, JIP1b, etc. These interactions are not only crucial for the normal physiological function of APP (β-amyloid precursor protein), but also play a key role in the pathological process of AD development. Therefore, studying these interactions is of great significance.

[0003] Current research on the interaction between C99 protein and water-soluble proteins takes place in complex cellular environments. In in vitro biochemical experiments, studies on C99 protein have used its intracellular segment AICD to replace the C99 protein itself. However, the results obtained by this method differ significantly from those under physiological conditions. This is because the peptide segment cannot completely replace the C99 protein, and the interaction between C99 protein and intracellular signaling proteins occurs on the cell membrane, while existing studies lack an understanding of the phospholipid membrane environment.

[0004] In existing structural biology research, in order to simulate the phospholipid membrane environment, some studies have assembled the C99 protein into Micelles and Bicelles in vitro (Barrett PJ, et al. The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol). Science , 2012, 336(6085):1168-1171. Song Y, et al. Impact of bilayer lipid composition on the structure and topology of the transmembrane amyloid precursor C99 protein. Journal of the American Chemical Society , 2014, 136(11): 4093-4096), but both systems contain detergents, which can easily inactivate proteins and their stability is greatly affected by the components. In some cases, they may affect the structure and function of the membrane proteins themselves and cannot fully simulate the phospholipid membrane environment under physiological conditions. It has been reported that C99 protein with a His tag at the C-terminus is assembled into Polymer Nanodiscs, but the assembly efficiency is low. In Polymer Nanodiscs, only solid-state NMR can be used to detect C99 protein. It cannot simulate the body fluid environment and cannot detect protein interactions in an in vitro aqueous environment (Krishnarjuna B, et al. Nanodisc reconstitution and characterization of amyloid-β precursor protein C99[J]. Analytical Chemistry, 2024, 96(23):9362-9369.). In addition, the C-terminus of the C99 protein prepared and used in existing studies (see, for example, CN102731659A) contains a His tag, whose potential steric hindrance or conformational changes can interfere with the protein structure and function, thereby affecting its interaction with downstream signaling proteins.

[0005] Therefore, there is an urgent need in this field to develop new strategies for studying the C99 protein and its interactions with other proteins in an in vitro aqueous environment. Summary of the Invention

[0006] To address one or more problems existing in the prior art, one aspect of the present invention provides a method for preparing a membrane protein C99-nanofas complex, comprising the following steps: S1: Mix the 22A peptide solution with the phospholipid solution, wherein the 22A peptide is a short peptide of 22 amino acids; S2: Add C99 recombinant membrane protein to the mixture obtained in step S1 and incubate overnight; and S3: The solution after overnight incubation in step S2 is purified by molecular sieve to prepare the membrane protein C99-nanoplasm complex.

[0007] In some embodiments, in step S1, the amino acid sequence of the 22A peptide is as shown in SEQ ID NO:2.

[0008] In some embodiments, the phospholipid in step S1 is DMPC.

[0009] In some embodiments, in step S1, the 22A peptide solution and the phospholipid solution are mixed at a mass ratio of 1:(0.95-1.05); optionally, in step S1, the concentration of the phospholipid solution is twice the concentration of the 22A peptide solution, and the 22A peptide solution and the phospholipid solution are mixed at a volume ratio of 2:(0.95-1.05); further optionally, the concentration of the phospholipid solution is 20±2 mg / mL, and the concentration of the 22A peptide solution is 10±2 mg / mL.

[0010] In some embodiments, in step S2, the concentration of the added C99 recombinant membrane protein is 0.30-0.40 mM; optionally, an excess of the C99 recombinant membrane protein is added to the mixture obtained in step S1.

[0011] In some embodiments, in step S2, the incubation is carried out overnight at 37°C and 220 rpm.

[0012] In some implementations, in step S3, molecular sieve purification is performed using a Superdex™ 200 Increase 10 / 300 separation column.

[0013] In some embodiments, the C99 recombinant membrane protein is an unlabeled C99 recombinant membrane protein.

[0014] In some embodiments, the tagless C99 recombinant membrane protein is obtained by the following methods: The purified tagged C99 recombinant membrane protein was subjected to enzymatic digestion using thrombin enzyme; wherein the conditions for enzymatic digestion were: the mass ratio of thrombin enzyme to the purified tagged C99 recombinant membrane protein was 1:1000 to 1:100, the treatment temperature was 4±1℃, and the treatment time was more than 10 days; optionally, the treatment time was more than 13 days.

[0015] In some embodiments, the purified tagged C99 recombinant membrane protein is obtained by immobilized metal affinity chromatography after induction of expression in Escherichia coli, or optionally by purification using a nickel-NTA column.

[0016] In some embodiments, the amino acid sequence of the C99 recombinant membrane protein is shown in SEQ ID NO:1.

[0017] In some embodiments, the tag is a His tag, which optionally includes a short peptide sequence consisting of 6-10 consecutive histidine residues, and may further be a 4×His tag, a 6×His tag, or an 8×His tag.

[0018] In another aspect, the present invention provides a membrane protein C99-nanoplasm complex prepared by the above method, and its application in in vitro detection of the interaction between C99 protein and small molecule drugs or proteins, or in screening drugs for the treatment of Alzheimer's disease.

[0019] In another aspect, the present invention provides a method for preparing a tagless C99 recombinant membrane protein, which includes enzymatic digestion of purified tagged (e.g., N-terminal tagged) C99 recombinant membrane protein using thrombin enzyme. The conditions for the enzymatic digestion treatment are as follows: the mass ratio of the thrombin enzyme to the purified tagged C99 recombinant membrane protein is 1:1000 to 1:100, the treatment temperature is 4±1℃, and the treatment time is more than 10 days.

[0020] In some implementations, the processing time is 13 days or more.

[0021] In some embodiments, the purified tagged C99 recombinant membrane protein is obtained by immobilized metal affinity chromatography after induction of expression in Escherichia coli, or optionally by purification using a nickel-NTA column.

[0022] In some embodiments, the amino acid sequence of the C99 recombinant membrane protein is shown in SEQ ID NO:1.

[0023] In some embodiments, the tag is a His tag, which optionally includes a short peptide sequence consisting of 6-10 consecutive histidine residues, and may further be a 4×His tag, a 6×His tag, or an 8×His tag.

[0024] The method for preparing the C99 recombinant membrane protein based on the above technical solution is the first to remove the N-terminal tag (e.g., His-tag) of the C99 protein in a detergent system, thus obtaining a tag-free C99 recombinant membrane protein. Therefore, when using this C99 recombinant membrane protein for in vitro interaction studies, there is no problem of tag interference with protein structure and function. This invention also assembles the tag-free C99 recombinant membrane protein into a detergent-free 22A-nanodisc system (obtaining a membrane protein C99-nanodisc complex), and uses this membrane protein C99-nanodisc complex to detect the interaction between the C99 protein and soluble proteins in vitro under detergent-free conditions. This best reflects the interaction process under physiological conditions, without the problem of detergent affecting the structure and function of the membrane protein itself and failing to fully simulate the phospholipid membrane environment under physiological conditions. Therefore, the membrane protein C99-nanoplasm complex provided by this invention can provide a membrane environment for C99 protein that is closer to its natural state, thereby enabling more realistic study of the interaction between C99 protein and intracellular proteins or small molecules, and thus providing a new tool for exploring AD mechanisms and developing drugs. Attached Figure Description

[0025] Figure 1 SDS-PAGE gel images were obtained to identify the N-terminal His tag of the C99 recombinant membrane protein after digestion with thrombin enzyme at different time points.

[0026] Figure 2 SDS-PAGE gel images of purified C99 recombinant membrane protein and C99 recombinant membrane protein after His tag excision.

[0027] Figure 3 The molecular sieve purification results of C99-22A-Nanodiscs prepared under different conditions (AI).

[0028] Figure 4 The results of TEM (A), DLS (B), and single molecular weight spectrophotometry analysis (C) of C99-22A-Nanodiscs are shown.

[0029] Figure 5 To verify the results of the interaction between C99-22A-Nanodiscs and Fe65 protein using liquid nuclear magnetic resonance (NMR).

[0030] Figure 6 The results of the interaction between C99-22A-Nanodiscs and Fe65 protein were verified using isothermal calorimetric titration (ITC), where AC represents the results of three replicate experiments. Detailed Implementation

[0031] To address the limitations of existing technologies that can only express C99 proteins with a His tag at the C-terminus (where the steric hindrance of the His tag interferes with protein structure and function), and the shortcomings of current C99 protein conformation studies using detergent-containing systems such as Micelles and Bicelles, which may affect the structure and function of membrane proteins in some cases and are therefore unsuitable for studying the interaction between membrane proteins and water-soluble proteins, this invention aims to provide a tag-free recombinant C99 membrane protein. Based on this tag-free recombinant C99 membrane protein, a detergent-free, soluble, stable, and size-tunable near-natural phospholipid membrane environment, 22A-Nanodiscs-like membrane system, is provided to more realistically study the interaction between C99 protein and intracellular proteins or other small molecules (such as small molecule drugs) in an in vitro aqueous environment, providing a new tool for exploring AD mechanisms and drug development.

[0032] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments are only used to further illustrate the present invention and are not intended to limit the scope of the present invention.

[0033] Unless otherwise specified, "%" in the following text indicates the percentage content by mass (g / 100 ml).

[0034] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0035] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., 3rd edition, 2001, NY, Cold SpringHarbor).

[0036] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0037] Example 1: Preparation of tag-free (His) C99 recombinant membrane protein 1.1. Preparation of pET28a-C99 carrier A DNA fragment encoding human APP-C99 (amino acids 672 to 770 of APP, amino acid sequence as shown in SEQ ID NO:1) was synthesized by Shanghai Sangon Biotech Co., Ltd. and inserted into the pET28a vector (Sangon Biotech (Shanghai) Co., Ltd., Cat: B540183) containing an N-terminal thrombin protease cleavage site and a 6×His tag, thus preparing a recombinant vector capable of expressing the C99 protein, named the pET28a-C99 vector. The C99 protein is a membrane protein with a hydrophobic transmembrane region. In this embodiment, the pET28a-C99 vector (plasmid) was transformed into *E. coli* BL21 (Rosetta) (Novagen) for protein expression.

[0038] The specific conversion steps are as follows: (1) Add 5 µL of 50 ng / µL pET28a-C99 plasmid to competent cells BL21 (Rosetta) and place on ice for 30 min; (2) Heat shock in a 42℃ water bath for 90 s; (3) Place it on ice to cool for another 5 minutes; (4) Add 100 µL of LB medium to recover competent cells, and shake well on a shaker at 37℃ and 220 rpm for 1 h; (5) Spread on plates and incubate overnight in a 37°C oven; (6) The next day, single colonies on the plate were picked and sequenced. If the sequencing was correct, it was BL21 (Rosetta) successfully transformed into pET28a-C99 plasmid. It was named BL21-C99 and stored at -80℃ for later use.

[0039] 1.2. Expression and purification of C99 protein (1) 10 mL of BL21-C99 was introduced into 600 mL of LB medium. When the cell density reached OD600 of 0.8, 1 mM IPTG was added and the cells were cultured at 37°C for 4 hours to induce overexpression of recombinant protein C99 (which has a 6×His tag at its N end).

[0040] (2) Centrifuge at 7000 rpm for 10 min and collect the precipitate (bacterial cells).

[0041] (3) The collected bacterial cells were crushed under pre-cooled pressure (500 MPa) for 20 min.

[0042] (4) Centrifuge at 14,000 rpm for 45 min and collect the precipitate.

[0043] (5) Remove the precipitate and dissolve it in urea A buffer (20 mM Tris, 300 mM NaCl, 8 M Urea, 0.2% SDS, pH=8.0). Rotate the solution in a beaker at 4°C overnight (16-20 h).

[0044] (6) Collect the mixture in the beaker, centrifuge at 14,000 rpm for 50 min, and collect the supernatant at 16℃.

[0045] (7) Mix the supernatant with the nickel-NTA column that has been equilibrated with urea A buffer and mix with a rotor in a beaker for 8 h.

[0046] (8) After the nickel-NTA has fully bound to the protein, use a 5 mL pipette tip to aspirate it and pump it back into the original column. Then rinse the inner wall of the beaker with 10 mL of urea Bbuffer (20 mM Tris, 300 mM NaCl, 30 mM imidazole, 8 M Urea, 0.2% SDS, pH=8.0) and pump it back into the original column.

[0047] (9) Wash the nickel column with 150 mL of urea B buffer (to rinse off any contaminating proteins).

[0048] (10) Wash the nickel-NTA column with 150 mL of washing buffer (20 mM Tris, 300 mM NaCl, 30 mM imidazole, pH=7.2) containing 0.05% LMPG (Lauryl Maltose Neopentyl Glycol). (rinse with urea and SDS).

[0049] (11) Rinse the nickel column with 20 mL of Elution buffer containing 0.05% LMPG (20 mM Tris, 300 mM NaCl, 500 mM imidazole, pH=7.2) and collect the flow-through.

[0050] (12) Add thrombin enzyme (Solarbio; Cat: T8021) to the collected solution and place it in a 4°C freezer for enzymatic digestion. Regarding enzymatic digestion, since the membrane protein is in a detergent environment after expression and purification, the conventional thrombin digestion method (e.g., digestion at 4°C for 16 hours) cannot remove the 6×His tag at the N-terminus of the C99 protein. Through inventive work, the inventors discovered that using thrombin enzyme to digest at 4°C for more than 10 days can remove the 6×His tag at the N-terminus of the C99 protein. This is described in detail below.

[0051] In existing technologies, the conditions typically used when using thrombin enzyme to cleave the His tag are as follows: 1) 4℃: Suitable for temperature-sensitive proteins, reducing non-specific cleavage, but requires a longer reaction time (overnight, 16-20 hours). 2) 16-25℃: A commonly used equilibrium point, balancing efficiency and specificity (e.g., incubation at 22℃ for 2 hours); and 3) The enzyme:substrate ratio is 1:1000 to 1:100 (w / w) (e.g., 1 μg thrombin is used to cut 1 mg of protein).

[0052] Therefore, at the beginning of the experiment, the inventors first removed the His tag at the N-terminus of the C99 protein under the following conditions: adding thrombin enzyme at an enzyme:substrate ratio of 1:1000 and incubating overnight (16-20 hours) at 4°C or for 2 hours at 22°C. However, neither of these conditions could remove the His tag at the N-terminus of the C99 protein.

[0053] Subsequently, the inventors conducted a series of experiments under the following conditions: thrombin enzyme was added at a 1:1000 enzyme:substrate ratio, and the mixture was treated at 4°C for extended periods (1, 3, 5, 7, 9, 10, 11, 12, and 13 days). The results are as follows. Figure 1 As shown, the His tag at the N-terminus of the C99 protein can be partially removed starting from day 10 of treatment, and completely removed by day 13 of treatment (SDS-PAGE gel images are commonly used in biology to reflect the results of His tag removal). Therefore, the product with the His tag removed from the N-terminus of the C99 protein after treatment at 4°C for 13 days was used for subsequent operations.

[0054] (13) Size exclusion chromatography (SEC) was performed on a Superdex™ 200 Increase 10 / 300 column. The components of the C99 protein obtained from the elution peak at 280 nm were analyzed by SDS-PAGE, and the components containing APP-C99 protein were combined to obtain the His-tag-free C99 recombinant membrane protein. Figure 2 As shown, 15% SDS-PAGE electrophoresis images of purified C99 recombinant membrane protein with His tag (His tag-C99) and C99 recombinant membrane protein with His tag removed (C99) are presented, confirming that the tag-removed C99 recombinant membrane protein, i.e., the tagless C99 recombinant membrane protein, was obtained.

[0055] (14) Adjust the C99 protein: The final concentration ratio of LMPG is 1:175 (at this time, the concentration of C99 protein is about 0.33 mM), and store it in a freezer at -80℃ for later use.

[0056] Example 2: Preparation of C99-22A-Nanodiscs This embodiment aims to prepare detergent-free C99-22A-Nanodiscs using the unlabeled C99 recombinant membrane protein prepared in Example 1. Specifically, in this embodiment, the inventors used nanodiscs developed based on conventional MSP-nanodiscs (membrane scaffold protein nanodiscs) that use a small peptide (a 22-amino acid peptide (22A), with the amino acid sequence PVLDLFRELLNELLEALKQKLK (SEQ ID NO:2)) as a scaffold to fix the phospholipid component, i.e., small peptide nanodiscs. The advantage of this nanodisc is that no detergent is needed during assembly, and the particle size of the nanodiscs can be achieved by changing the mass ratio of 22A to phospholipid. This embodiment only involves the assembly of C99-22A-Nanodiscs with a single DMPC (1,2-Dimyristoyl-sn-glycero-3-phosphocholine) phospholipid component, and the specific steps are described below.

[0057] (1) Preparation of phospholipid and 22A peptide stock solution: Weigh an appropriate amount of DMPC phospholipid powder (Avanti; Cat: 850345P) and dissolve it in 10 mM phosphate buffer (pH 7.4) to prepare a 20 mg / ml suspension for later use. Prepare a 10 mg / ml stock solution of 22A peptide (obtained by conventional technology) using 40 mM phosphate buffer (pH 7.8).

[0058] (2) Mix the small peptide stock solution with the phospholipid stock solution, add the purified C99 protein (the His-tagged C99 recombinant membrane protein prepared in Example 1 (i.e. the protein solution of step (14) in Example 1)), and incubate overnight at 37°C and 200 rpm on a shaker.

[0059] In step (2), the small peptide stock solution and phospholipid stock solution are mixed according to the following AI amounts, and the purified C99 protein is added (the addition of excess C99 protein in each system will not affect the assembly of C99-22A-Nanodiscs, and excess C99 protein can be removed by subsequent molecular sieve purification): A: 250 μL 20 mg / mL DMPC, 500 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; B: 150 μL 20 mg / mL DMPC, 500 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; C: 200 μL 20 mg / mL DMPC, 500 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; D: 300 μL 20 mg / mL DMPC, 500 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; E: 250 μL 20 mg / mL DMPC, 200 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; F: 250 μL 20 mg / mL DMPC, 300 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; G: 250 μL 20 mg / mL DMPC, 400 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; H: 250 μL 20 mg / mL DMPC, 600 μL 10 mg / mL 22A small peptide, 50 μL 0.33 mM C99; I: 250 μL 20 mg / mL DMPC, 500 μL 10 mg / mL 22A small peptide.

[0060] (3) Molecular sieve purification: Molecular sieve purification was performed using Superdex™ 200 Increase 10 / 300 separation columns. The specific procedures were as follows: (3.1) Buffer treatment: The ultrapure water and buffer (50mM PBS, 100mM NaCl, pH=6.5) used for molecular sieves were prepared fresh and used immediately. Impurities were removed by passing the filter membrane with a pore size of 0.22 μm and the gas was removed by sonication for 30 minutes. (3.2) Equilibrium separation column: Rinse the equilibrium separation column with purification buffer, 1.5-2 column volumes, and preheat with UV lamp 30 minutes before loading the sample; (3.3) Sample preparation: Centrifuge at 13000 rpm for 30 min at 4℃ to remove precipitates and air bubbles from the sample; carefully aspirate the prepared sample with the sample needle, being careful not to generate air bubbles, and slowly inject it into the sample loop; set the flow rate, pressure limit, and time of the molecular sieve instrument according to the default parameters, and set the automatic sample collection program.

[0061] The molecular sieve purification results under various AI conditions are as follows: Figure 3As shown, Group I (empty nanodiscs, without added C99 protein) exhibits a 9.2 mAU UV absorption peak at 15.47 mL, indicating successful assembly of 22A-Nanodiscs. Under Group A conditions, when assembling C99-22A-Nanodiscs, a 49.91 mAU UV absorption peak is observed at 13.82 mL, consistent with the increase in relative molecular weight after C99 protein assembly into 22A-Nanodiscs, indicating successful formation of C99-22A-Nanodiscs under Group A conditions. Under Group E conditions, only a 3.4 mAU UV absorption peak is formed at 14.53 mL, indicating that effective assembly of 22A-Nanodiscs is not possible under these conditions. Under the conditions of groups B, C, D, F, G, and H, UV absorption peaks of 8.8-14.3 mAU appeared in the range of 14.7-15.3 mL, which were not much different from group I. This indicates that C99 protein could not efficiently assemble into 22A-Nanodiscs under these conditions, and the assembly efficiency was low.

[0062] Therefore, in this embodiment, when preparing detergent-free C99-22A-Nanodiscs, in step (2) above, 500 μL of small peptide stock solution (10 mg / mL) and 250 μL of phospholipid stock solution (20 mg / mL) are mixed (i.e., 22A small peptide and phospholipid are mixed in approximately equal mass, for example, the mass ratio of the two can be 1:(0.95-1.05)), and more than 50 μL of purified C99 protein (0.33 mM) is added (the addition of excess C99 protein will not affect the assembly of C99-22A-Nanodiscs, and excess C99 protein can be removed in the subsequent molecular sieve purification step), and incubated overnight at 37°C and 220 rpm on a shaker. Subsequently, detergent-free C99-22A-Nanodiscs are prepared by molecular sieve purification in step (3).

[0063] The prepared C99-22A-Nanodiscs were analyzed by transmission electron microscopy (TEM), dynamic light scattering (DLS) (using a dynamic light scattering nanoparticle size analyzer, Zetasizer Nano-ZSE), and single-molecule mass spectrometry (using a RefeynTwoMP). The results are as follows: Figure 4 As shown, the AC amplitudes represent the TEM, DLS, and single-molecule mass spectrophotometric analysis results of C99-22A-Nanodiscs, respectively. Specifically, Figure 4 Image A in the middle section was taken by electron microscopy using a 120kV transmission electron microscope (TEM). The results show that C99-22A-Nanodiscs have a disk-like morphology in solution and are uniformly dispersed, with a diameter of approximately 10 nm. Figure 4 The B-mode particle size was measured by dynamic light scattering (DLS) experiments on 22A-Nanodiscs (obtained from system I above) and C99-22A-Nanodiscs (obtained from system A above). It can be seen that the diameter of 22A-Nanodiscs is about 9.86 nm, and the diameter of C99-22A-Nanodiscs formed after assembly into C99 protein is about 10.93 nm. At the same time, the comparison of scattering intensity shows that C99 protein can be efficiently assembled into 22A-Nanodiscs under these assembly conditions. Figure 4 The relative molecular weights of 22A-Nanodiscs and C99-22A-Nanodiscs were determined using a single-molecule mass spectrophotometer. The relative molecular weights of 22A-Nanodiscs were measured to be approximately 120 kDa, and those of C99-22A-Nanodiscs were approximately 138 kDa, a difference of about 18 kDa. This is close to the relative molecular weight of the C99 protein itself, demonstrating that the C99 protein can assemble into 22A-Nanodiscs. Furthermore, based on the count percentages, 22A-Nanodiscs had a count of 94%, and C99-22A-Nanodiscs had a count of 97%, indicating that both 22A-Nanodiscs and C99-22A-Nanodiscs are homogeneous and stable in solution, which is beneficial for subsequent detection of protein interactions in an aqueous environment.

[0064] The above results indicate that the C99 recombinant membrane protein can be uniformly and stably assembled into 22A-Nanodiscs under suitable conditions, and C99-22A-Nanodiscs were successfully prepared.

[0065] Example 3: Liquid nuclear magnetic resonance (NMR) study of the interaction between C99-22A-Nanodiscs and Fe65 Nuclear magnetic resonance (NMR) data were collected at 298 K using a Bruker Avance 850 MHz NMR spectrometer equipped with a cryogenic probe, processed using the NMR data processing software NMRPipe, and analyzed using the Sparky program. The specific operations included the following.

[0066] (1) The C99-22A-Nanodiscs prepared in Example 2 was dialyzed into NMR buffer (50 mM PBS, 100 mM NaCl, pH=6.5) and concentrated to a total concentration of 0.15 mM as the C99-22A-Nanodiscs solution for later use.

[0067] (2) make uniform 15N-labeled Fe65-PTB2 protein samples (where Fe65-PTB2 represents the PTB2 domain of Fe65 protein, and its amino acid sequence is shown in SEQ ID NO:3, prepared using conventional techniques. It is well known in existing technology that Fe65 can bind to C99 protein through its PTB2 domain, indicating an interaction) were dialyzed into NMR buffer and mixed with 10% (v / v) D2O to maintain a final concentration of 0.03 mM, serving as the Fe65-PTB2 protein solution for later use.

[0068] (3) Before the experiment, all samples need to be centrifuged at high speed (4℃, 13000 rpm) for 30 minutes to remove precipitates and air.

[0069] (4) Take 300 μl of Fe65-PTB2 protein solution sample and transfer it to a Shigemi NMR tube to collect the protein. 1 H, 15 N-HSQC spectra (Fe65-PTB2:C99-22A-Nanodiscs molar ratio of 1:0). Then, C99-22A-Nanodiscs solutions with molar ratios of 1:0.2 and 1:0.5 (Fe65-PTB2:C99-22A-Nanodiscs) were added respectively, and one image was collected for each ratio. 1 H, 15 N-HSQC spectrum.

[0070] (5) Data processing: The collected NMR spectra can be opened with Topspin software for preliminary processing. Analysis and image export: The original data is transformed using the nmrPipe software in the Ubuntu system through a script, and phase correction and peak intensity analysis are performed using nmrDraw and Sparky software.

[0071] The results are as follows Figure 5 As shown, it can be seen that when only for 15 N-labeled Fe65-PTB2 was analyzed by NMR and collected. 1 H, 15 N-HSQC spectrum, then C99-22A-Nanodiscs ( Figure 5When C99 nanodiscs (represented as C99 nanodiscs) were added to Fe65-PTB2 solutions at molar ratios of 1:0.2 and 1:0.5, the overall signal intensity decreased. Analysis of key amino acids (enlarged image) showed that the peak intensities of isoleucine (position 562, I562), glutamic acid (position 648, E648), alanine (position 649, A649), alanine (position 653, A653), methionine (position 655, M655), and glutamine (position 659, Q659) in Fe65-PTB2 were significantly weakened. This is due to the slower flipping speed of the larger C99-22A-Nanodiscs. The spectral lines of these specific residues further broadened due to a moderate to slow exchange process between the free and complex states. These residues with further reduced intensities may be located at the interaction interface or participate in direct interactions with C99-22A-Nanodiscs.

[0072] The results of the above liquid NMR experiments clearly demonstrate that C99-22A-Nanodiscs can be applied to detect proteins or small molecule drugs that interact with the membrane protein C99 in an in vitro aqueous environment.

[0073] Example 4: Isothermal calorimetric titration (ITC) study on the interaction between C99-22A-Nanodiscs and Fe65 This embodiment uses a Malvern MicroCal PEAQ-ITC instrument to perform ITC titration experiments at 25°C, measuring the binding affinity between Fe65-PTB2 and C99-22A-Nanodiscs. The specific procedures included the following (repeated three times).

[0074] (1) Sample preparation: The purified Fe65-PTB2 (same as in Example 3) and C99-22A-Nanodiscs (prepared in Example 2) were dialyzed into a buffer solution of 20 mM Tris / HCl, 150 mM NaCl, pH 7.4. The final concentration of C99-22A-Nanodiscs was 0.1 mM and the final concentration of Fe65-PTB2 was 0.75 mM. Before the experiment, all samples were centrifuged at high speed (4℃, 13000 rpm) for 30 minutes to remove precipitates and bubbles.

[0075] (2) Instrument cleaning: Add ultrapure water, methanol, and decon90 cleaning solution according to the instrument operation requirements. After fixing the sample needle and cleaning needle, select automatic cleaning through the software interface. After the cleaning program is completed, remove the sample needle and allow the remaining small amount of methanol to evaporate in the air.

[0076] (3) Experimental parameter settings: According to the experimental requirements, the reaction temperature was set to 25℃, the DP value was 5, the stirring speed was 750 rpm, the total number of titrations was 20, the first titration volume was 0.4 μl, and the subsequent titrations were 2 μl each time, with a titration interval of 120 seconds.

[0077] (4) Preliminary experiment: Before the experiment, conduct a preliminary experiment of water droplet and buffer droplet to confirm that the instrument is in good condition. Under normal circumstances, the DP value change should be less than 0.02. If the DP value is abnormal, consider cleaning the reference cell. After the DP value returns to normal, the experiment can be carried out again.

[0078] (5) Sample loading: Use a syringe to draw 300 μl of 0.1 mM C99-22A-Nanodiscs protein and add it to the sample cell. Gently stir the syringe to remove air bubbles. Take 60 μl of 0.75 mM Fe65-PTB2 and place it in a 250 μl PCR tube, inserting it into the injection port of the automatic sample loading needle. Place the sample loading needle below the liquid surface and control the sample loading needle through the operating interface to automatically inject 40 μl.

[0079] (6) Titration: After the sample injection is completed, move the sample needle to the designated position above the sample cell and fix it, and run the experimental program.

[0080] (7) Control group: The same sample cell was titrated with buffer as a blank control group.

[0081] (8) Data processing: After the experiment, the control group and the experimental group were set up on the MicroCal PEAQ-ITC Analysis Software. By fitting, information such as the dissociation constant KD, enthalpy change (ΔH), and entropy change (ΔS) can be obtained.

[0082] The results are as follows Figure 6 As shown, AC represents the results of three repeated experiments. It can be seen that the KD value of group A is 2.38 ± 0.07 μM, the KD value of group B is 2.67 ± 0.15 μm, and the KD value of group C is 2.08 ± 0.07 μm. The average value of the three groups is approximately 2.37 μM. Therefore, it can be determined that after C99 is assembled into 22A-Nanodiscs, it can interact with Fe65, and the affinity between the two can be accurately measured.

[0083] The results of the above isothermal calorimetric titration experiments directly demonstrate that C99-22A-Nanodiscs can be applied to detect proteins or small molecule drugs that interact with membrane protein C99 in an in vitro aqueous environment.

[0084] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a membrane protein C99-nanofas complex, comprising the following steps: S1: Mix the 22A peptide solution with the phospholipid solution, wherein the 22A peptide is a short peptide of 22 amino acids; S2: Add C99 recombinant membrane protein to the mixture obtained in step S1 and incubate overnight; and S3: The solution after overnight incubation in step S2 is purified by molecular sieve to prepare the membrane protein C99-nanoplasm complex.

2. The preparation method according to claim 1, wherein in step S1, the amino acid sequence of the 22A peptide is as shown in SEQ ID NO: 2; and / or In step S1, the phospholipid is DMPC; and / or In step S1, the 22A peptide solution and the phospholipid solution are mixed at a mass ratio of 1:(0.95-1.05). Optionally, in step S1, the concentration of the phospholipid solution is twice the concentration of the 22A peptide solution, and the 22A peptide solution and the phospholipid solution are mixed at a volume ratio of 2:(0.95-1.05). Further optionally, the concentration of the phospholipid solution is 20±2 mg / mL, and the concentration of the 22A peptide solution is 10±2 mg / mL; and / or In step S2, the concentration of the added C99 recombinant membrane protein is 0.30-0.40 mM; optionally, an excess of the C99 recombinant membrane protein is added to the mixture obtained in step S1.

3. The preparation method according to claim 1 or 2, wherein in step S2, the mixture is incubated overnight at 37°C and 220 rpm; and / or In step S3, molecular sieve purification is performed using a Superdex™ 200 Increase 10 / 300 separation column.

4. The preparation method according to any one of claims 1-3, wherein the C99 recombinant membrane protein is a tag-free C99 recombinant membrane protein; Preferably, the tagless C99 recombinant membrane protein is obtained by the following method: The purified tagged C99 recombinant membrane protein was subjected to enzymatic digestion using thrombin enzyme; wherein the conditions for enzymatic digestion were: the mass ratio of thrombin enzyme to the purified tagged C99 recombinant membrane protein was 1:1000 to 1:100, the treatment temperature was 4±1℃, and the treatment time was more than 10 days; optionally, the treatment time was more than 13 days.

5. The preparation method according to claim 4, wherein the purified tagged C99 recombinant membrane protein is obtained by immobilized metal affinity chromatography after induction of expression in *E. coli*, optionally purified by nickel-NTA column purification; and / or The amino acid sequence of the C99 recombinant membrane protein is shown in SEQ ID NO:1; Further optionally, the tag is a His tag, which may include a short peptide sequence consisting of 6-10 consecutive histidine residues, and may be a 4×His tag, a 6×His tag, or an 8×His tag.

6. A membrane protein C99-nanoplasm complex, which is prepared by the method of any one of claims 1-5.

7. The use of the membrane protein C99-nanopan complex of claim 6 in in vitro detection of the interaction between C99 protein and small molecule drugs or proteins, or in screening drugs for the treatment of Alzheimer's disease.

8. A method for preparing a tagless C99 recombinant membrane protein, comprising enzymatic digestion of purified tagged C99 recombinant membrane protein using thrombin enzyme; The conditions for the enzymatic digestion treatment are as follows: the mass ratio of the thrombin enzyme to the purified tagged C99 recombinant membrane protein is 1:1000 to 1:100, the treatment temperature is 4±1℃, and the treatment time is more than 10 days. Optionally, the processing time is 13 days or more.

9. The preparation method according to claim 8, wherein the purified tagged C99 recombinant membrane protein is obtained by immobilized metal affinity chromatography after induction of expression in *E. coli*, optionally purified by nickel-NTA column purification; and / or The amino acid sequence of the C99 recombinant membrane protein is shown in SEQ ID NO:

1.

10. The preparation method according to claim 8 or 9, wherein the tag is a His tag, optionally comprising a short peptide sequence consisting of 6-10 consecutive histidine residues, further optionally a 4×His tag, a 6×His tag, or an 8×His tag.

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