Transmembrane protein 106b amyloid fibril and preparation method and application thereof

Abstract

The application provides a transmembrane protein 106B (TMEM106B) amyloid fiber and a preparation method and application thereof. A raw material for reconfiguring the TMEM106B amyloid fiber in vitro is a fusion protein, and the fusion protein has a structure of formula I from an N terminal to a C terminal: L-T-E-P (I); in the formula, each "-" is independently a bond or a connecting peptide; L is a secretion signal peptide; T is a truncated fragment of the TMEM106B, and the truncated fragment comprises amino acids at positions 120-x of the TMEM106B, wherein x is a positive integer, and 254<=x<=274; E is a protease cleavage site; and P is an optional purification tag. The fusion protein is cut off the secretion signal peptide during a translation expression process of a eukaryotic expression system, and a T-E-P framework is reserved. The fusion protein with the T-E-P framework can be reconfigured to form the TMEM106B amyloid fiber in vitro. The application further provides a preparation method and application of the TMEM106B amyloid fiber.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a transmembrane protein 106B (TMEM106B) amyloid fiber, its preparation method, and its applications. Background Technology

[0002] In its native state, TMEM106B is a type II transmembrane protein mainly located in lysosomes, consisting of an N-terminal domain (NTD, 1-96aa), a transmembrane domain (TM, 97-117aa), and a C-terminal domain (CTD, 118-274aa). It is closely related to functions such as lysosomal morphology maintenance, localization, acidification, and transport. Recent studies have found that in the brains of patients with various neurodegenerative diseases (NDs), such as Parkinson's disease (PD), Alzheimer's disease (AD), and frontotemporal dementia (FTD), the TMEM106B protein can undergo cleavage, with its C-terminal fragments (CTFs) folding abnormally to form TMEM106B amyloid filaments with structures different from the native TMEM106B protein, resulting in pathological deposition. Cryo-electron microscopy has clearly identified the core primary structure of human brain-derived TMEM106B amyloid filaments as 120-254 amino acids. Based on the structural characteristics of TMEM106B amyloid filaments under cryo-electron microscopy, the amino acid initiation point of this filament can be determined to be S120, but the terminal amino acid site cannot be completely determined and could be any of 254-274 amino acids.

[0003] Currently, the only existing technology most relevant to the purpose of this invention is the method for obtaining the TMEM106B CTF protein; there is no in vitro amyloid fibril reconstruction method for this protein. Specifically: A protocol for obtaining TMEM106B CTF protein based on a prokaryotic expression system: This protocol uses an E. coli expression system to produce TMEM106B CTF protein. For example, in Schweighauser et al.'s study on age-dependent protein aggregation (Nature, 2022, 605(7909): 310-314), the target protein mainly exists in the form of insoluble inclusion bodies in this system, requiring complex denaturation, purification, and refolding processes to obtain soluble protein. The main drawbacks of this protocol are: firstly, the denaturation and refolding process may disrupt the native conformation of the protein, affecting its subsequent spontaneous fibrillation ability; secondly, the core purpose of this protocol is not amyloid fibrillation reconstruction, but rather to use the purified protein as an immunogen for the preparation of specific antibodies.

[0004] TMEM106B CTF protein acquisition protocol based on eukaryotic expression system: This protocol uses eukaryotic expression systems such as mammalian cells to obtain TMEM106B CTF protein that is closer to its natural state. For example, Baggen et al. used this protocol when studying the interaction between TMEM106B and viral proteins (Source: Cell, 2023, 186(16):3427-3442). Its advantage is that it can obtain proteins with post-translational modifications such as glycosylation. However, this protocol also has limitations: its research objective focuses on protein-protein interactions and does not involve, nor can it achieve, the remodeling of amyloid fibrils of this protein under in vitro conditions.

[0005] In summary, the core objectives of existing technical solutions are all focused on protein acquisition, antibody preparation, or interaction analysis. Due to limitations in technical approaches and objectives, no publicly available and effective technical solution has yet been able to successfully reconstruct TMEM106B amyloid fibers in vitro, which severely hinders in-depth research into their pathological functions.

[0006] Therefore, developing a method for efficiently reconstructing TMEM106B amyloid fibers in vitro has become a pressing technical problem to be solved in this field.

[0007] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of the present invention, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to provide a TMEM106B amyloid fiber, its in vitro reconstruction method and application, in order to solve the problem that the existing technology cannot effectively prepare the amyloid fiber.

[0009] In a first aspect of the invention, a fusion protein is provided, the fusion protein having a structure of Formula I from its N-terminus to its C-terminus: LTEP (I) In the formula, each "-" independently represents a bond or a linking peptide; L is the N-terminal secretory signal peptide; T is a truncated fragment of TMEM106B, which contains amino acids from position 120 to position x of TMEM106B, where x is a positive integer and 254 ≤ x ≤ 274; E represents the protein cleavage site; P is an optional purification tag.

[0010] In another preferred embodiment, x is 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273 or 274.

[0011] In another preferred embodiment, the N-terminal secretory signal peptide is selected from one or more of the following: Igκ chain signal peptide, tissue plasminogen activator signal peptide, albumin signal peptide, interleukin-2 signal peptide, or a secretory signal peptide with equivalent function.

[0012] In another preferred embodiment, the N-terminal secretory signal peptide is an Igκ chain signal peptide.

[0013] In another preferred embodiment, the sequence of the N-terminal secretory signal peptide is shown as positions 1-24 of SEQ ID No. 5.

[0014] In another preferred embodiment, T is a core segment of TMEM106B (120-274).

[0015] In another preferred embodiment, the sequence of T is as shown in positions 25-179 of SEQ ID No. 5.

[0016] In another preferred embodiment, the protease cleavage site is selected from one or more of the following: HRV 3C protease cleavage site, tobacco etch virus protease cleavage site, thrombin cleavage site, enterokinase cleavage site, or other functionally equivalent cleavage sites that can be recognized and cleaved by specific proteases.

[0017] In another preferred embodiment, the protease cleavage site is the HRV 3C protease cleavage site.

[0018] In another preferred embodiment, the sequence of the protease cleavage site is shown as positions 180-187 of SEQ ID No. 5.

[0019] In another preferred embodiment, the purification tag is selected from one or more of the following: multihistidine tags (such as 6xHis tags), glutathione S-transferase tags, maltose-binding protein tags, FLAG tags, Strep tags (such as Strep-tag II or Twin-Strep-tag), or other functionally equivalent tags that can be used for protein purification.

[0020] In another preferred embodiment, the purification tag is a 6xHis purification tag.

[0021] In another preferred embodiment, the amino acid sequence of the fusion protein is shown in SEQ ID No. 5.

[0022] In another preferred embodiment, the fusion protein is a glycosylated modified protein.

[0023] In another preferred embodiment, the fusion protein is a glycosylated modified protein expressed in HEK293F mammalian suspension cells.

[0024] In a second aspect of the invention, a polynucleotide is provided that encodes a fusion protein as described in the first aspect of the invention.

[0025] In another preferred embodiment, the sequence of the polynucleotide is shown in SEQ ID No. 4.

[0026] In a third aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the second aspect of the invention.

[0027] In another preferred embodiment, the vector is the mammalian cell expression vector pcDNA3.1.

[0028] In a fourth aspect of the invention, a host cell is provided, the host cell containing a vector as described in the third aspect of the invention, or having an exogenous polynucleotide sequence as described in the second aspect of the invention integrated into its chromosome.

[0029] In another preferred embodiment, the host cell is a eukaryotic cell.

[0030] In another preferred embodiment, the host cell is a mammalian cell.

[0031] In another preferred embodiment, the host cell is a HEK293F mammalian suspension cell.

[0032] In a fifth aspect of the invention, a method for preparing TMEM106B amyloid fibers is provided, comprising the steps of: (1) Culture host cells as described in the fourth aspect of the present invention to express the TMEM106B CTF (120-274)3C 6xHis fusion protein; (2) Purification of the TMEM106B CTF (120-274) 3C 6xHis fusion protein obtained in step (1); (3) Optionally, validation of the TMEM106B CTF (120-274) 3C 6xHis fusion protein; (4) Optionally, purity characterization of the TMEM106B CTF (120-274) 3C 6xHis fusion protein; (5) In vitro reconstruction of TMEM106B amyloid fibers.

[0033] In another preferred embodiment, the host cell in step (1) is transfected with a recombinant expression plasmid encoding the fusion protein shown in SEQ ID No. 5.

[0034] In another preferred embodiment, the host cell in step (1) is a HEK293F mammalian suspension cell transfected with a recombinant expression plasmid encoding the fusion protein shown in SEQ ID No. 5.

[0035] In another preferred embodiment, in step (1), the culture conditions are 37°C and 8% CO2.

[0036] In another preferred embodiment, in step (1), transfection is performed by culturing cells at 37°C and 8% CO2 until the cell density reaches approximately 2.5-3.0 × 10⁻⁶ cells / year. 6 Cells were transfected at a rate of 1 cell per ml.

[0037] In another preferred embodiment, in step (1), the culture time is 4 to 6 days (e.g., 4 to 5 days).

[0038] In another preferred embodiment, in step (1), the cell culture medium is collected by centrifugation (4000 rpm, 4°C, 30 min).

[0039] In another preferred embodiment, in step (1), the cell culture medium is collected and filtered through a 0.45 μm filter to remove cell residue.

[0040] In another preferred embodiment, the purification step of the TMEM106B CTF (120–274) 3C 6xHis fusion protein includes the following steps: (2a) Affinity capture purification (e.g., nickel ion affinity chromatography column purification); (2b) Secondary purification using molecular sieves (e.g., purification using gel size exclusion columns).

[0041] In another preferred embodiment, the purification step of the TMEM106B CTF (120-274) 3C 6xHis fusion protein includes the following steps: (2a1) Purification by nickel ion affinity chromatography; (2b1) Gel size exclusion column purification.

[0042] In another preferred embodiment, in step (2a1), the first step involves equilibrating a nickel ion affinity chromatography column (HisTrap excel, Cytiva) with Buffer A (25 mM Tris-HCl, pH 8.0, 150 mM NaCl) and loading the column using the ÄKTA protein purification system.

[0043] In another preferred embodiment, in step (2a1), the second step is to perform stepwise elution: first, weakly bound contaminating proteins are washed away with an elution buffer containing 25 mM imidazole (with the same composition as Buffer A), and then the target protein is eluted with an elution buffer containing 250 mM imidazole (with the same composition as Buffer A).

[0044] In another preferred embodiment, in step (2b1), Superdex is equilibrated using a molecular sieve buffer (25 mM Tris-HCl, 500 mM NaCl, pH 8.0). TM 75 Increase 10 / 300 GL (Cytiva) chromatography column.

[0045] In another preferred embodiment, the in vitro reconstruction step of the TMEM106B amyloid fibrils includes the following steps: (5a) The pH of the purified TMEM106B CTF (120–274) 3C 6xHis protein solution was adjusted to 1.5–3.0 (approximately 2.0) using tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl). (5b) Incubate the reaction system with the pH lowered in (5a) at 95-100°C (e.g., 100°C) for 50-65 min (e.g., 1 h) to induce protein conformational changes; (5c) The protein solution after heat incubation to induce conformational changes in (5b) is placed at 36.5~37.5 (e.g. 37°C) and incubated at a constant temperature of 350~500 rpm (e.g. 400 rpm) for 7~14 days to complete the in vitro reconstruction of TMEM106B amyloid fibrous tissue.

[0046] In another preferred embodiment, in step (5a), the final concentration of TCEP-HCl in the protein solution is 30~60 mM (e.g., 50 mM).

[0047] In another preferred embodiment, in step (5a), the buffer for the TMEM106B CTF (120-274) 3C 6xHis protein solution is a Tris buffer containing 500 mM NaCl.

[0048] In a sixth aspect of the invention, there is provided the use of a fusion protein as described in the first aspect of the invention, a polynucleotide as described in the second aspect of the invention, a vector as described in the third aspect of the invention, and / or a host cell as described in the fourth aspect of the invention for preparing TMEM106B amyloid filaments.

[0049] In a seventh aspect of the present invention, a TMEM106B amyloid fiber is provided, which is prepared by the preparation method described in the fifth aspect of the present invention.

[0050] In an eighth aspect of the invention, a use of TMEM106B amyloid fibers is provided, comprising: (a) Used to prepare cell and animal models for studying the pathogenesis of NDs; (b) Screening of small molecule compounds for in vivo molecular tracer imaging development targeting TMEM106B amyloid fibers; (c) Pre-clinical screening of interventional drugs for drug development targeting TMEM106B amyloid fibrils.

[0051] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and are not intended to limit the invention. Attached Figure Description

[0052] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention. It is obvious that the drawings described below are merely some embodiments of the invention, and those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0053] Figure 1 Gel filtration chromatography chromatogram of TMEM106B CTF (120-274) 3C 6xHis recombinant protein. This size exclusion chromatogram corresponds to the Superdex assay. TM75 Increase 10 / 300 GL gel filtration chromatography purified TMEM106BCTF (120-274) 3C 6xHis recombinant protein. The size exclusion chromatogram shows a smooth single peak, indicating that the purified target protein TMEM106BCTF (120-274) 3C 6xHis monomer has good homogeneity.

[0054] Figure 2 . Authentication and purity analysis of TMEM106B CTF (120-274) 3C 6xHis recombinant protein. a. Western blot (WB) patterns of the recombinant protein obtained using 6xHis tag antibody (left) and TMEM106B CTF-specific antibody (right), respectively. The molecular weight of the recombinant protein shown is approximately 30-40 kDa, consistent with the molecular weight of the target protein TMEM106BCTF (120-274) 3C 6xHis. b. Representative image of purified TMEM106B CTF (120-274) 3C 6xHis recombinant protein stained with Coomassie Brilliant Blue.

[0055] Figure 3 Gel filtration chromatography chromatogram of TMEM106B CTF (120-274) 6xHis recombinant protein. This size exclusion chromatogram corresponds to the Superdex assay. TM 75 Increase 10 / 300 GL gel filtration chromatography purified TMEM106B CTF(120-274) 6xHis recombinant protein. The size exclusion chromatogram shows a smooth single peak, indicating that the purified target protein TMEM106B CTF(120-274) 6xHis monomer has good homogeneity.

[0056] Figure 4 . Authentication and purity analysis of TMEM106B CTF (120-274) 6xHis recombinant protein. a. Western blot (WB) patterns obtained using 6xHis tag antibody (left) and TMEM106B CTF-specific antibody (right), respectively. The recombinant protein shown has a molecular weight of approximately 30-40 kDa, consistent with the target protein TMEM106B CTF (120-274) 6xHis. b. Representative image of purified TMEM106B CTF (120-274) 6xHis recombinant protein stained with Coomassie Brilliant Blue.

[0057] Figure 5. TMEM106B CTF (120-254) 3C 6xHis recombinant protein expression analysis. a. Western blotting analysis: The supernatant of HEK293F mammalian suspension cells expressing TMEM106B CTF (120-254) 3C 6xHis recombinant protein was detected using 6xHis tag antibody (left) and TMEM106B CTF-specific antibody (right). No target protein bands corresponding to the theoretical molecular weight (approximately 30-40 kDa) were observed. b. Coomassie brilliant blue staining analysis of the supernatant of HEK293F mammalian suspension cells and its eluent samples at different concentrations of imidazole showed no target protein bands corresponding to the theoretical molecular weight (approximately 30-40 kDa).

[0058] Figure 6 . Analysis of TMEM106B CTF (120-254) 6xHis recombinant protein expression. a. Western blotting analysis: The supernatant of HEK293F mammalian suspension cells expressing TMEM106B CTF (120-254) 6×His recombinant protein was detected using 6xHis tag antibody (left) and TMEM106B CTF-specific antibody (right). No target protein bands corresponding to the theoretical molecular weight (approximately 30-40 kDa) were observed. b. Coomassie brilliant blue staining analysis of the supernatant of HEK293F mammalian suspension cells and its eluent samples at different concentrations of imidazole showed no target protein bands corresponding to the theoretical molecular weight (approximately 30-40 kDa).

[0059] Figure 7 Representative image of TMEM106B amyloid filaments obtained by negative staining transmission electron microscopy (NS-TEM). NS-TEM characterization shows TMEM106B amyloid filaments formed by in vitro reconstruction of the expressed and purified TMEM106B CTF (120-274) 3C 6xHis recombinant protein (indicated by white arrows). Scale bar, 100 nm.

[0060] Figure 8 NS-TEM representative image of the in vitro reconstructed TMEM106B CTF (120-274) 6xHis recombinant protein sample obtained through expression and purification. NS-TEM shows that the sample mainly consists of non-classical amyloid fibrous structures or clump-like protein aggregates; classical amyloid fibers were not observed. Scale bar, 100 nm. Detailed Implementation

[0061] Exemplary embodiments will now be described more fully with reference to the accompanying drawings. However, these exemplary embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, they are provided so that the invention will be thorough and complete, and the concept of the exemplary embodiments will be fully conveyed to those skilled in the art.

[0062] Unless otherwise defined, all terms and phrases used herein include their meanings as they have in the art, unless explicitly stated otherwise or clearly indicated from the context of their use. While any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, specific methods and materials are now described.

[0063] Through extensive and in-depth research, the inventors have pioneered a method for the in vitro reconstruction of TMEM106B amyloid fibers. The method provided by this invention enables the efficient and controllable preparation of TMEM106B amyloid fibers in vitro, offering a crucial tool for research on the pathogenesis of disease-related disorders (NDs), disease model construction, and the development of therapeutic drugs.

[0064] This invention provides a TMEM106B amyloid fiber, its in vitro reconstruction method, and its applications, addressing the problem that existing technologies cannot effectively prepare this amyloid fiber. Specifically, this invention aims to: 1) provide a controllable and reproducible in vitro preparation method for TMEM106B amyloid fibers; 2) provide TMEM106B amyloid fibers prepared by this method and characterized by negative staining electron microscopy; 3) provide TMEM106B amyloid fibers as a research tool, which can, to some extent, serve as a substitute for human brain-derived TMEM106B amyloid fibers for research on the pathogenesis of non-dendronal diseases (NDs) and drug development. Based on these findings, this invention has been completed.

[0065] the term

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0067] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0068] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0069] The beneficial effects of this invention are: Currently, there is no in vitro reconstruction technology for TMEM106B amyloid fibers (i.e., existing technologies cannot achieve in vitro reconstruction of TMEM106B amyloid fibers). The TMEM106B amyloid fibers, their in vitro reconstruction method, and their applications provided by this invention have produced the following significant beneficial effects: (1) The successful reconstruction of TMEM106B amyloid fibers was achieved for the first time, filling a technological gap: This invention discloses and verifies for the first time a method for preparing TMEM106B amyloid fibers in vitro in a highly efficient and controllable manner, solving the core defect in the background technology that "TMEM106B amyloid fibers cannot be obtained in vitro", providing an indispensable key tool for studying the biological function and pathogenic mechanism of the fibers, and filling a technological gap in this field; (2) The fiber preparation process is stable, controllable, and reproducible: This invention establishes a standardized and well-defined preparation process. By precisely controlling the pH, temperature, and time of the pretreatment stage, as well as the subsequent shaking incubation time (7-14 days), the fiber preparation results of different batches have high consistency and reproducibility. Under optimized conditions, the protein to fiber conversion rate can stably reach about 50%. (3) It provides a new standardized tool for disease research, molecular tracer and drug development: The TMEM106B amyloid fibers prepared in this invention can be directly used as standardized pathological aggregates and applied to: ① NDs pathogenic mechanism research: used to construct cell or animal models to simulate the abnormal aggregation and neurotoxic effects of TMEM106B amyloid fibers in NDs, so as to open a new door to the research of NDs pathogenic mechanism. ② Development of in vivo molecular tracers: The in vitro reconstructed TMEM106B amyloid fibers obtained by this invention can be used for high-throughput screening of small molecule compounds in the development of in vivo imaging molecular tracers for human brain TMEM106B pathological fiber aggregates; on the other hand, it can be used to construct cell and animal models containing TMEM106B pathological fiber aggregates for preclinical research on the development of TMEM106B pathological fiber molecular tracers. ③ Therapeutic drug development: As an amyloid protein associated with NDs, TMEM106B fibrillary aggregates are one of the potential therapeutic targets for NDs. The in vitro reconstructed TMEM106B amyloid fibers obtained in this invention can be used as a tool for high-throughput screening of candidate drugs that can inhibit the formation of TMEM106B amyloid fibers or depolymerize existing fibers. Compared with using TMEM106B protein monomers, the accuracy and reliability of the screening results are greatly improved. (4) The method is simple and the cost is relatively low: Although a mammalian cell expression system is used, this invention achieves a preparation scheme that can be operated and completed in a conventional biological laboratory by optimizing the process flow. Compared with methods that require complex instruments or expensive reagents, this invention mainly utilizes standard protein purification and biochemical incubation equipment, effectively controlling the research and development cost and facilitating the promotion and application of this technology.

[0070] The present invention will be further explained below with reference to specific embodiments.

[0071] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Guide* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the manufacturer's recommendations (e.g., product instructions). Unless otherwise stated, percentages and parts are by weight. Unless otherwise specified, the experimental materials and reagents used in the following embodiments are commercially available or can be prepared according to literature methods.

[0072] Example 1: Construction of TMEM106B CTF (120-274) 3C 6xHis expression plasmid

[0073] Using a plasmid containing the amino acid sequence encoding TMEM106B (118-274) (a courtesy of Professor Yang Haitao's research group at ShanghaiTech University; original plasmid construction source: Cell, 2023, 186(16): 3427-3442) as a template, specific primers (the nucleotide sequences of upstream primer 1, upstream primer 2, and downstream primer are shown in SEQ ID No. 1, SEQ ID No. 2, and SEQ ID No. 3, respectively) were added. The DNA fragment encoding the amino acid sequence TMEM106B (120-274) was specifically amplified by polymerase chain reaction (PCR). During the amplification process, the coding sequence corresponding to amino acids 118 and 119 at the N-terminus of the original sequence was removed, and homologous arms were introduced at both ends of the amplification product. Subsequently, the target fragment amplified by the above PCR was coupled with the linearized mammalian expression vector pcDNA. TM 3.1 (+) (Invitrogen) TM The recombinant expression plasmid (catalog number: V79020) is obtained by ligation via homologous recombination.

[0074] The recombinant expression plasmid encodes a fusion protein—TMEM106B CTF (120-274) 3C6xHis recombinant protein—whose structure, from N-terminus to C-terminus, consists of: a secretion signal peptide, a TMEM106B (120-274) core fragment (155 aa), an HRV 3C protease cleavage site, and a 6xHis purification tag. The complete DNA coding sequence and amino acid sequence of the TMEM106B CTF (120-274) 3C 6xHis recombinant protein are shown in SEQ ID No. 4 and SEQ ID No. 5, respectively.

[0075] SEQ ID No. 1: Nucleotide sequence of upstream primer 1 in PCR in Example 1

[0076] CTGGCAGCACCGGCGACGCCGCTCAGAGCATCGACGTGAAGTACATCGGCGTGAA

[0077] SEQ ID No. 2: Nucleotide sequence of upstream primer 2 in PCR in Example 1

[0078] AGCATCGACGTGAAGTACATCGGCGTGAA

[0079] SEQ ID No. 3: Nucleotide sequence of the downstream primer for PCR in Example 1

[0080] CTGAGCGGCGTCGCCGGTGCTGCCA

[0081] SEQ ID No. 4: DNA sequence encoding the TMEM106B CTF (120-274) 3C 6xHis recombinant protein

[0082] ATGGAGACAGACACCCTCCTGCTGTGGGTGCTGCTCCTGTGGGTCCCTGGCAGCACCGGCGACGCCGCTCAGAGCATCGACGTGAAGTACATCGGCGTGAAGAGCGCCTACGTGAGCTACGACGTGCAGAAGAGAACCATCTACCTGAACATTACCAACACACTGAATATCACCAACAACAATTACTACAGCGTGGAGGTGGAGAACATCACCGCCCAAGTGCAGTTCAGCAAGACCGTGATCGGCAAGGCTAGACTGAACAACATCACCATCATCGGCCCCCTGGACATGAAGCAGATCGACTACACCGTGCCCACCGTGATCGCCGAGGAGATGAGCTACATGTACGACTTCTGCACCCTGATCAGCATCAAGGTGCACAACATCGTGCTGATGATGCAAGTGACCGTGACCACAACCTACTTCGGCCACAGCGAGCAGATCAGCCAAGAGAGATATCAGTACGTGGACTGCGGCAGAAACACCACCTATCAGCTGGGGCAGAGCGAGTACCTGAACGTGCTGCAGCCTCAGCAGCTGGAGGTGCTGTTCCAAGGCCCCCACCATCACCATCACCACTGA

[0083] SEQ ID No.5: Amino acid sequence of TMEM106B CTF (120 - 274) 3C 6xHis recombinant protein

[0084] METDTLLLWVLLLWVPGSTGDAAQSIDVKYIGVKSAYVSYDVQKRTIYLNITNTLNITNNNYYSVEVENITAQVQFSKTVIGKARLNNITIIGPLDMKQIDYTVPTVIAEEMSYMYDFCTLISIKVHNIVLMMQVTVTTTYFGHSEQISQERYQYVDCGRNTTYQLGQSEYLNVLQPQQLEVLFQGPHHHHHH

[0085] Example 2: Construction of TMEM106B CTF (120 - 274) 6xHis expression plasmid

[0086] Based on Example 1, an expression plasmid without restriction enzyme sites was obtained by PCR amplification (the nucleotide sequences of upstream primer 1, upstream primer 2 and downstream primer are shown in SEQ ID No. 6, SEQ ID No. 7 and SEQ ID No. 8, respectively) and homologous recombination to remove the HRV 3C protease cleavage site.

[0087] The recombinant expression plasmid encodes a fusion protein—TMEM106B CTF (120-274) 6xHis recombinant protein, whose structure from the N-terminus to the C-terminus consists of: a secretion signal peptide, a TMEM106B (120-274) core fragment (155 aa), and a 6xHis purification tag. The complete DNA coding sequence and amino acid sequence of the TMEM106B CTF (120-274) 6xHis recombinant protein are shown in SEQ ID No. 9 and SEQ ID No. 10, respectively.

[0088] SEQ ID No. 6: Nucleotide sequence of upstream primer 1 in PCR in Example 2

[0089] AACGTGCTGCAGCCTCAGCAGCACCATCACCATCACCACTGAGAAT

[0090] SEQ ID No. 7: Nucleotide sequence of upstream primer 2 in PCR in Example 2

[0091] CACCATCACCATCACCACTGAGAAT

[0092] SEQ ID No. 8: Nucleotide sequence of the downstream primer for PCR in Example 2

[0093] CTGCTGAGGCTGCAGCACGTTC

[0094] SEQ ID No. 9: DNA sequence encoding TMEM106B CTF (120-274) 6xHis recombinant protein

[0095] ATGGAGACAGACACCCTCCTGCTGTGGGTGCTGCTCCTGTGGGTCCCTGGCAGCACCGGCGACGCCGCTCAGAGCATCGACGTGAAGTACATCGGCGTGAAGAGCGCCTACGTGAGCTACGACGTGCAGAAGAGAACCATCTACCTGAACATTACCAACACACTGAATATCACCAACAACAATTACTACAGCGTGGAGGTGGAGAACATCACCGCCCAAGTGCAGTTCAGCAAGACCGTGATCGGCAAGGCTAGACTGAACAACATCACCATCATCGGCCCCCTGGACATGAAGCAGATCGACTACACCGTGCCCACCGTGATCGCCGAGGAGATGAGCTACATGTACGACTTCTGCACCCTGATCAGCATCAAGGTGCACAACATCGTGCTGATGATGCAAGTGACCGTGACCACAACCTACTTCGGCCACAGCGAGCAGATCAGCCAAGAGAGATATCAGTACGTGGACTGCGGCAGAAACACCACCTATCAGCTGGGGCAGAGCGAGTACCTGAACGTGCTGCAGCCTCAGCAGCACCATCACCATCACCACTGA

[0096] SEQ ID No.10: Amino acid sequence of TMEM106B CTF (120 - 274) 6xHis recombinant protein

[0097] METDTLLLWVLLLWVPGSTGDAAQSIDVKYIGVKSAYVSYDVQKRTIYLNITNTLNITNNNYYSVEVENITAQVQFSKTVIGKARLNNITIIGPLDMKQIDYTVPTVIAEEMSYMYDFCTLISIKVHNIVLMMQVTVTTTYFGHSEQISQERYQYVDCGRNTTYQLGQSEYLNVLQPQQHHHHHH

[0098] Example 3: Construction of TMEM106B CTF (120 - 254) 3C 6xHis expression plasmid

[0099] Using a plasmid containing the coding sequence of TMEM106B (120-274) as a template, a C-terminal truncated mutation was introduced by reverse PCR (the nucleotide sequences of the upstream and downstream primers are shown in SEQ ID No. 11 and SEQ ID No. 12, respectively) to obtain the coding sequence of TMEM106B (120-254). The PCR product was then circularized to obtain the recombinant expression plasmid.

[0100] The recombinant expression plasmid encodes a fusion protein—TMEM106B CTF (120-254) 3C6xHis recombinant protein—whose structure, from N-terminus to C-terminus, consists of: a secretion signal peptide, a TMEM106B (120-254) core fragment (135 amino acids), an HRV 3C protease cleavage site, and a 6xHis purification tag. The complete DNA coding sequence and amino acid sequence of the TMEM106B CTF (120-254) 3C 6xHis recombinant protein are shown in SEQ ID No. 13 and SEQ ID No. 14, respectively.

[0101] SEQ ID No. 11: Nucleotide sequence of the upstream primer for PCR in Example 3

[0102] CTGGAGGTGCTGTTCCAAGGCCCCCACCATCAC

[0103] SEQ ID No. 12: Nucleotide sequence of the downstream primer for PCR in Example 3

[0104] TTGGAACAGCACCTCCAGGCCGCAGTCCACGTACTGAT

[0105] SEQ ID No. 13: DNA sequence encoding the TMEM106B CTF (120-254) 3C 6xHis recombinant protein

[0106] ATGGAGACAGACACCCTCCTGCTGTGGGTGCTGCTCCTGTGGGTCCCTGGCAGCACCGGCGACGCCGCTCAGAGCATCGACGTGAAGTACATCGGCGTGAAGAGCGCCTACGTGAGCTACGACGTGCAGAAGAGAACCATCTACCTGAACATTACCAACACACTGAATATCACCAACAACAATTACTACAGCGTGGAGGTGGAGAACATCACCGCCCAAGTGCAGTTCAGCAAGACCGTGATCGGCAAGGCTAGACTGAACAACATCACCATCATCGGCCCCCTGGACATGAAGCAGATCGACTACACCGTGCCCACCGTGATCGCCGAGGAGATGAGCTACATGTACGACTTCTGCACCCTGATCAGCATCAAGGTGCACAACATCGTGCTGATGATGCAAGTGACCGTGACCACAACCTACTTCGGCCACAGCGAGCAGATCAGCCAAGAGAGATATCAGTACGTGGACTGCGGCCTGGAGGTGCTGTTCCAAGGCCCCCACCATCACCATCACCACTGA

[0107] SEQ ID No.14: Amino acid sequence of the TMEM106B CTF (120 - 254) 3C 6xHis recombinant protein

[0108] METDTLLLWVLLLWVPGSTGDAAQSIDVKYIGVKSAYVSYDVQKRTIYLNITNTLNITNNNYYSVEVENITAQVQFSKTVIGKARLNNITIIGPLDMKQIDYTVPTVIAEEMSYMYDFCTLISIKVHNIVLMMQVTVTTTYFGHSEQISQERYQYVDCGLEVLFQGPHHHHHH

[0109] Example 4: Construction of the TMEM106B CTF (120 - 254) 6xHis expression plasmid

[0110] Based on Example 3, an expression plasmid without restriction enzyme sites was obtained by PCR amplification (the nucleotide sequences of upstream primer 1, upstream primer 2 and downstream primer are shown in SEQ ID No. 15, SEQ ID No. 7 and SEQ ID No. 16, respectively) and homologous recombination to remove the HRV 3C protease cleavage site.

[0111] The recombinant expression plasmid encodes a fusion protein—TMEM106B CTF (120-254) 6xHis recombinant protein, whose structure from the N-terminus to the C-terminus consists of: a secretion signal peptide, a TMEM106B (120-254) core fragment (135 aa), and a 6xHis purification tag. The complete DNA coding sequence and amino acid sequence of the TMEM106B CTF (120-254) 6xHis recombinant protein are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively.

[0112] SEQ ID No. 15: Nucleotide sequence of upstream primer 1 in PCR in Example 4

[0113] TCAGTACGTGGACTGCGGCCACCATCACCATCACCACTGAGAAT

[0114] SEQ ID No. 7: Nucleotide sequence of upstream primer 2 in PCR in Example 4 (same as nucleotide sequence of upstream primer 2 in PCR in Example 2)

[0115] CACCATCACCATCACCACTGAGAAT

[0116] SEQ ID No. 16: Nucleotide sequence of the downstream primer for PCR in Example 4

[0117] GCCGCAGTCCACGTACTGATATC

[0118] SEQ ID No. 17: DNA sequence encoding TMEM106B CTF (120-254) 6xHis recombinant protein

[0119] ATGGAGACAGACACCCTCCTGCTGTGGGTGCTGCTCCTGTGGGTCCCTGGCAGCACCGGCGACGCCGCTCAGAGCATCGACGTGAAGTACATCGGCGTGAAGAGCGCCTACGTGAGCTACGACGTGCAGAAGAGAACCATCTACCTGAACATTACCAACACACTGAATATCACCAACAACAATTACTACAGCGTGGAGGTGGAGAACATCACCGCCCAAGTGCAGTTCAGCAAGACCGTGATCGGCAAG GCTAGACTGAACAACATCACCATCATCGGCCCCCTGGACATGAAGCAGATCGACTACACCGTGCCCACCGTGATCGCCGAGGATGAGCTACATGTACGACTTCTGCACCCTGATCAGCATCAAGGTGCACAACATCGTGCTGATGATGCAAGTGACCGTGACCACAACCTACTTCGGCCACAGCGAGCAGATCAGCCAAGAGAGATATCAGTACGTGGACTGCGGCCACCATCACCATCACCACTGA

[0120] SEQ ID No. 18: Amino acid sequence of TMEM106B CTF (120-254) 6xHis recombinant protein

[0121] METDTLLLWVLLLWVPGSTGDAAQSIDVKYIGVKSAYVSYDVQKRTIYLNITNTLNITNNNYYSVEVENITAQVQFSKTVIGKARLNNITIIGPLDMKQIDYTVPTVIAEEMSYMYDFCTLISIKVHNIVLMMQVTVTTTYFGHSEQISQERYQYVDCGHHHHHHH

[0122] Example 5. Expression and purification of recombinant proteins with different truncated forms of TMEM106B

[0123] (1) Plasmid transfection and protein expression: The recombinant expression plasmids described in Examples 1-4 were transfected into HEK293F mammalian suspension cells (Gibco). TM ,Expi293F TMRecombinant expression was performed in cells (catalog number: A14527CN). Specifically, cells were cultured under appropriate conditions (37°C, 8% CO2) until a cell density of approximately 2.5-3.0 × 10⁶ cells / year was reached. 6 Cells were transfected at a density of 10 cells / ml. Cells were cultured for 4-5 days after transfection. After culture, the cell culture medium was collected by centrifugation (4000 rpm, 4°C, 30 min) and filtered through a 0.45 μm filter to remove cell debris, yielding a clear supernatant.

[0124] Note: Regarding the expression of different truncated recombinant TMEM106B proteins in HEK293F mammalian suspension cells: HEK293F mammalian suspension cells possess a complete endoplasmic reticulum-Golgi apparatus secretion pathway. Target protein expression strictly follows these pathways: 1) Co-translational transport: When mRNA is translated into a secretory signal peptide on ribosomes, the secretory signal peptide is immediately recognized by signal recognition granules, carrying the entire ribosome complex to the endoplasmic reticulum membrane. The protein is synthesized while simultaneously penetrating the endoplasmic reticulum lumen. 2) Secretory signal peptide cleavage: Within the endoplasmic reticulum lumen, signal peptidase precisely cleaves the secretory signal peptide during or immediately after translation. At this point, the N-terminus of the mature protein is the first amino acid of the different truncated recombinant TMEM106B proteins (i.e., amino acid 120 of the TMEM106B protein). 3) Folding and modification: After cleavage of the secretory signal peptide, the target protein undergoes correct disulfide bond formation, N-glycosylation, and other modifications within the endoplasmic reticulum lumen. Molecular chaperones ensure proper protein folding. 4) Vesicle transport and exocytosis: The target protein is transported from the endoplasmic reticulum to the Golgi apparatus via the capsid protein complex II vesicles. After further processing and sorting, it is released into the supernatant of HEK293F mammalian suspension cell culture by fusing with the cell membrane through secretory vesicles.

[0125] Therefore, the first amino acid of the TMEM106B CTF (120-274) 3C 6xHis recombinant protein and the TMEM106B CTF (120-274) 6xHis recombinant protein successfully expressed and purified in this invention is the 120th amino acid (S120) of the TMEM106B protein, and the promoter and secretory peptide preceding it are removed.

[0126] In some embodiments, the host cell is not limited to HEK293F mammalian suspension cells, but may also be other eukaryotic cells suitable for recombinant protein expression.

[0127] (2) Preliminary purification using nickel ion affinity chromatography: First, the nickel ion affinity chromatography column (HisTrap excel, Cytiva) was equilibrated with Buffer A (25 mM Tris-HCl, pH 8.0, 150 mM NaCl) and loaded using the ÄKTA protein purification system. Then, stepwise elution was performed: weakly bound contaminating proteins were first washed away with elution buffer containing 25 mM imidazole (same composition as Buffer A), followed by elution of the target protein with elution buffer containing 250 mM imidazole (same composition as Buffer A). The eluent was collected using the ÄKTA automated collection system and further concentrated and filtered.

[0128] In some embodiments, the affinity chromatography packing material may be a nickel affinity chromatography medium or other metal ion affinity materials.

[0129] (3) Gel size exclusion column purification: Subsequently, gel filtration chromatography was performed to further purify the protein. Superdex was equilibrated with molecular sieve buffer (25 mM Tris-HCl, 500 mM NaCl, pH 8.0). TM 75 Increase 10 / 300GL (Cytiva) chromatography column. Load the concentrated and filtered protein sample and elute with the same buffer to remove low molecular weight impurities. Use the ÄKTA system to automatically collect the elution peak of the target protein, further concentrate and purify the obtained target protein, then flash freeze in liquid nitrogen and store at -80°C.

[0130] In some embodiments, the gel filter column may be Superdex. TM 75 or other chromatography media suitable for the target protein molecular weight range.

[0131] Example 6. Analysis of expression and purification results of recombinant proteins with different truncated forms of TMEM106B

[0132] Under the same expression and purification conditions as in Example 5, expression analysis, identity verification, and purity characterization of different truncated recombinant proteins of TMEM106B were performed.

[0133] (1) TMEM106B CTF (120-274) 3C 6xHis recombinant protein

[0134] The eukaryotic protein expression system in Example 5 successfully expressed the TMEM106B CTF (120-274) 3C6xHis recombinant protein: First, after purification using the gel size exclusion column in Example 5, the purified protein exhibited a single elution peak (e.g., Figure 1As shown in the figure, the purified protein has good homogeneity. Next, the identity of the recombinant protein was verified by Western blotting: 6xHis-tagged antibody (Invitrogen, catalog number: MA1-21315) and TMEM106B CTF-specific antibody (NovusBiologicals, catalog number: NBP1-91311) were used for detection. The results showed that both antibodies could detect specific bands consistent with the theoretical molecular weight of the target protein. Figure 2 (a) This result indicates that the purified recombinant protein was indeed the target protein TMEM106B CTF (120-274) 3C 6xHis. Finally, the purity of the target protein was analyzed by Coomassie Brilliant Blue staining, and the results showed that only one band with the same molecular weight (approximately 30-40 kDa) as the target protein TMEM106B CTF (120-274) 3C 6xHis was visible. Figure 2 (b). This result indicates that the purified target protein TMEM106B CTF (120-274) 3C6xHis has high purity.

[0135] (2) TMEM106B CTF (120-274) 6xHis recombinant protein

[0136] The eukaryotic cell protein expression system in Example 5 can also successfully express the TMEM106B CTF (120-274) 6xHis recombinant protein: the gel filtration chromatography pattern of this recombinant protein is shown in Example 5. Figure 3 The same Western blot analysis was performed on the TMEM106B CTF (120-274) 3C 6xHis recombinant protein as described above. The results showed that both the 6xHis tag antibody and the TMEM106B CTF specific antibody could detect specific bands consistent with the theoretical molecular weight of the target protein TMEM106B CTF (120-274) 6xHis. Figure 4 (a) Coomassie brilliant blue staining results showed only one band identical to the target protein TMEM106B CTF (120-274) 6xHis molecular weight (approximately 30-40 kDa). Figure 4 (b) indicates that the purified TMEM106B CTF (120-274) 6xHis recombinant protein has high purity.

[0137] (3) TMEM106B CTF (120-254) 3C 6xHis recombinant protein

[0138] Western blot (WB) and Coomassie brilliant blue staining analysis revealed that no target protein band matching the theoretical molecular weight of the target protein TMEM106B CTF (120-254) 3C 6xHis was observed in the cell culture medium of the eukaryotic protein expression system described in Example 5 (e.g., Figure 5 (As shown).

[0139] Note: Figure 5 The absence of a target protein band does not indicate that the TMEM106B CTF (120-254) 3C 6xHis plasmid was not expressed in the eukaryotic protein expression system of Example 5. It only reflects that, under the conditions described, the target protein TMEM106B CTF (120-254) 3C 6xHis was not observed to be expressed in secretory form in HEK293F mammalian suspension cell culture medium.

[0140] (4) TMEM106B CTF (120-254) 6xHis recombinant protein

[0141] Western blot (WB) and Coomassie brilliant blue staining analysis revealed that no target protein band matching the theoretical molecular weight of TMEM106B CTF (120-254) 6xHis was observed in the cell culture medium of the eukaryotic protein expression system in Example 5 (e.g., Figure 6 (As shown).

[0142] Note: Figure 6 The absence of a target protein band does not indicate that the TMEM106B CTF (120-254) 6xHis plasmid was not expressed in the eukaryotic protein expression system of Example 5. It only reflects that the target protein TMEM106B CTF (120-254) 6xHis was not expressed in secretory form in HEK293F mammalian suspension cell culture medium under the conditions described.

[0143] Example 7. In vitro reconstruction of amyloid fibrils using TMEM106B

[0144] Take the purified TMEM106B CTF (120-274) 3C 6xHis recombinant protein solution and add TCEP-HCl to bring the final concentration to 50 mM. The pH of the solution will naturally decrease to approximately 2.0 at this point, requiring no further adjustment. Then, place the reaction system in a PCR instrument and incubate at 100°C for 1 h to precisely induce protein conformational changes. After heating, transfer the protein solution to 37°C and incubate with constant temperature shaking at 400 rpm for 7–14 days to complete the in vitro reconstruction of TMEM106B amyloid fibrils.

[0145] The purified TMEM106B CTF (120-274) 6xHis recombinant protein was reconstructed in vitro using the same method and under the same conditions as described above.

[0146] Example 8. Characterization of amyloid fibril formation using TMEM106B

[0147] The samples incubated in Example 7 were observed using negative staining transmission electron microscopy (NS-TEM). The results showed that typical elongated, rod-shaped, unbranched TMEM106B amyloid fibers could be observed in the samples incubated with TMEM106B CTF(120-274) 3C 6xHis. Figure 7 This indicates that the TMEM106B CTF (120-274) 3C 6xHis recombinant protein can form amyloid filaments under the stated conditions; only non-classical amyloid fibrous structures or clump-like protein aggregates were observed in the TMEM106B CTF (120-274) 6xHis incubated samples, while typical amyloid filaments were not observed. Figure 8 ).

[0148] Note: Figure 7 , Figure 8 This is used to illustrate the amyloid fibrillation characteristics of different truncated recombinant proteins of TMEM106B under the same in vitro reconstruction conditions. The absence of observed amyloid fibrillation does not indicate that this recombinant protein cannot be reconstructed in vitro to form amyloid fibrillation; it merely reflects that amyloid fibrillation did not form under the stated conditions.

[0149] discuss

[0150] I. Inherent defects of existing technology

[0151] Based on the analysis of the aforementioned background technical solutions, the existing technologies have the following inherent defects in realizing the in vitro reconstruction and application of TMEM106B amyloid fibers: 1. The prokaryotic expression system results in impaired protein activity and ineffective fibrosis: The TMEM106B CTF protein obtained from the E. coli-based expression protocol exists in inclusion body form and must undergo renaturation treatment. This process easily disrupts the protein's normal native spatial conformation, leading to the loss of its correct folding state and self-assembly activity. Furthermore, the prokaryotically expressed protein lacks the glycosylation modification of TMEM106B CTF, and glycosylation is a crucial factor affecting its normal function and correct folding. Therefore, the protein obtained by this protocol inherently lacks the ability to form amyloid aggregates consistent with the in-situ fibrous structure, which is a fundamental defect preventing its use for fibrous remodeling. 2. The technical objectives of existing solutions are limited and do not address the core of fiber remodeling: The technical objectives of existing solutions (including prokaryotic and eukaryotic expression systems) are highly focused on obtaining protein samples that can be used for immune or interaction studies. Their overall technical path design and optimization direction do not cover the core objective of "inducing protein amyloid fibrosis", nor do they provide corresponding technical conditions or methods to solve this problem. 3. Lack of dedicated in vitro fiber remodeling methods hinders subsequent research: Due to the aforementioned deficiencies, there is currently a lack of stable and controllable in vitro sources of TMEM106B amyloid fibers in this field. The absence of this crucial tool directly limits in-depth research into the pathogenic mechanisms and transmission patterns of TMEM106B fibers in NDs, the development of in vivo molecular imaging tracers for human brains, and their potential as therapeutic targets.

[0152] II. Key Technical Points of the Invention

[0153] This invention aims to solve the technical challenge of obtaining structurally uniform TMEM106B amyloid fibers suitable for high-level biological research through in vitro reconstruction using existing technologies. To achieve this objective, this invention has developed a novel technical solution, the successful implementation of which relies on the following indispensable key technical points. These points are also the core protections of this invention; any third party implementing any of these points without permission may constitute infringement.

[0154] 1. Construction, expression, and purification of recombinant protein particles with different truncated forms of TMEM106B

[0155] Technical Features: Based on the primary structural characteristics of TMEM106B amyloid filaments derived from human brain tissue, this invention does not use full-length or other truncated forms of TMEM106B protein. Instead, it specifically constructs four plasmids expressing TMEM106B CTF(120-274) 3C 6xHis recombinant protein, TMEM106B CTF(120-274) 6xHis recombinant protein, TMEM106B CTF(120-254) 3C 6xHis recombinant protein, and TMEM106B CTF(120-254) 6xHis recombinant protein. Through protein expression, purification, and filament reconstruction, this invention uniquely verifies that the specific truncated form of TMEM106B CTF(120-274) 3C 6xHis recombinant protein serves as the smallest functional unit capable of correctly assembling into amyloid filaments.

[0156] The technical problem solved: Full-length TMEM106B is difficult to express, purify, and form uniform filaments. This specific truncated form removes interfering domains, enabling efficient expression of soluble proteins in mammalian eukaryotic expression systems, which is the decisive structural basis for the final successful reconstructing into filaments.

[0157] 2. Co-optimized protein expression

[0158] Technical features: It adopts a specific fusion protein architecture of "TMEM106B CTF (120-274) 3C 6xHis" and is secreted and expressed in HEK293F mammalian suspension cells.

[0159] Technical problems solved: This design addresses the challenges of easy inclusion and lack of post-translational modification in prokaryotic systems, as well as low expression levels and purification difficulties in eukaryotic systems. The synergistic effect of this design ensures efficient protein secretion and correct folding.

[0160] 3. Two-step high purification process

[0161] Technical Features: For the expression products obtained from the above-mentioned secretory expression, this invention develops a highly efficient two-step purification process: 1) Affinity Capture: Initial capture using a fusion tag for rapid separation of the target protein. 2) Secondary Molecular Sieve Purification: Specifically designed for efficient removal of protein aggregates, polymers, and small molecule degradation fragments, thereby ensuring the acquisition of a homogeneous, monomeric target protein.

[0162] Technical problems solved: Overcoming the shortcomings of non-uniform products from traditional methods, providing high-purity standardized protein raw materials for subsequent fiber reconstruction and functional research.

[0163] 4. The decisive fiber reconstruction pretreatment process (acid reduction heat treatment)

[0164] Technical characteristics: 50 mM TCEP-HCl is directly added to a Tris buffer containing 500 mM NaCl, allowing the pH of the system to naturally decrease to approximately 2.0 using its acidity, while simultaneously heating at 100°C for 1 h. Strong reducing properties, extreme acidity, and high-temperature heat treatment are all essential.

[0165] The technical problem solved: Traditional neutral or mild denaturation conditions cannot cause the TMEM106B protein to unfold and initiate fibril assembly. This step is the only effective means to trigger fibril formation, and this non-obvious combination of conditions is the core breakthrough of this invention, directly determining the success or failure of fibril reconstruction.

[0166] 5. Controllable fiber assembly and maturation conditions

[0167] Technical characteristics: The pretreated protein solution must be incubated for 7-14 days under constant temperature shaking conditions of 37°C and 400 rpm.

[0168] Technical problems solved: The system addresses the issues of random polymerization, low efficiency, and non-uniform products during fiber formation. This condition provides an optimal kinetic environment for the ordered and controllable assembly of fibers, ensuring the uniformity and high repeatability of the final product in terms of structure and morphology.

[0169] Other embodiments of the invention will readily occur to those skilled in the art upon consideration of the specification and practice of the invention disclosed herein. This application is intended to cover any variations, uses, or adaptations of the invention that follow the general principles of the invention and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of the invention are indicated by the appended claims.

Claims

1. A fusion protein, characterized in that, The fusion protein has a structure of Formula I from the N-terminus to the C-terminus: LTEP (I) In the formula, each "-" independently represents a bond or a linking peptide; L is the N-terminal secretory signal peptide; T is a truncated fragment of TMEM106B, which contains amino acids from position 120 to position x of TMEM106B, where x is a positive integer and 254 ≤ x ≤ 274; E represents the protein cleavage site; P is an optional purification tag.

2. The fusion protein according to claim 1, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID No.

5.

3. A polynucleotide, characterized in that, The polynucleotide encodes the fusion protein as described in any one of claims 1-4.

4. The polynucleotide according to claim 3, characterized in that, The sequence of the polynucleotide is shown in SEQ ID No.

4.

5. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 3.

6. A host cell, characterized in that, The host cell contains the vector as described in claim 5, or its chromosome has an integrated exogenous polynucleotide sequence as described in claim 3.

7. The host cell according to claim 6, characterized in that, The host cell was HEK293F mammalian suspension cell.

8. A method for preparing TMEM106B amyloid fibers, characterized in that, Including the following steps: (1) Culture the host cells as described in claim 6 to express the TMEM106B CTF (120-274) 3C 6xHis fusion protein; (2) Purification of the TMEM106B CTF (120-274) 3C 6xHis fusion protein obtained in step (1); (3) Optionally, validation of the TMEM106B CTF (120-274) 3C 6xHis fusion protein; (4) Optionally, purity characterization of the TMEM106B CTF (120-274) 3C 6xHis fusion protein; (5) In vitro reconstruction of TMEM106B amyloid fibers.

9. The method according to claim 8, characterized in that, The purification steps for the TMEM106B CTF (120–274) 3C 6xHis fusion protein include the following steps: (2a) Affinity capture purification; (2b) Secondary purification of molecular sieves.

10. The method according to claim 8, characterized in that, The in vitro reconstruction process of TMEM106B amyloid fibers includes the following steps: (5a) The pH of the purified TMEM106B CTF (120-274) 3C 6xHis protein solution was adjusted to 1.5-3.0 using tris(2-carboxyethyl)phosphonic acid hydrochloride; (5b) Incubate the reaction system with the pH lowered in (5a) at 95-100°C for 50-65 min to induce protein conformational changes; (5c) The protein solution after heating and incubation to induce conformational changes in (5b) was placed at 36.5~37.5°C and incubated at a constant temperature of 350~500 rpm for 7~14 days to complete the in vitro reconstruction of TMEM106B amyloid fibers.

11. A TMEM106B amyloid fiber, characterized in that, It is prepared by the preparation method described in claim 8.

12. An application of TMEM106B amyloid fibers, characterized in that, include: (a) Used to prepare cell and animal models for studying the pathogenesis of NDs; (b) Screening of small molecule compounds for in vivo molecular tracer imaging development targeting TMEM106B amyloid fibers; (c) Pre-clinical screening of interventional drugs for drug development targeting TMEM106B amyloid fibrils.

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