A thermostable phosphorylated Tau protein and its applications

Abstract

The present invention provides a phosphorylated Tau protein mutant and application thereof. The provided phosphorylated Tau protein mutant comprises at least one mutation substitution or deletion at E117, A145, G204, S305, A384, and L408 relative to the amino acid sequence SEQ ID NO.1 of the phosphorylated wild-type Tau protein, has improved thermal stability, and the thermal stability is significantly higher than that of the wild-type Tau protein during long-term storage. The phosphorylated Tau protein mutant can be used as a standard product and has greater utilization value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mutant phosphorylated Tau protein with improved thermal stability, and a preparation method and application thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, and there is currently no mechanism-based treatment. AD is defined by the presence of abundant neurofibrillary lesions and neuritic plaques in the cerebral cortex. Neurofibrillary lesions are composed of paired helical and straight tau protein filaments (PHFs and SFs). Tau protein is an axonal microtubule-binding protein. Tau protein aggregation into insoluble filaments and increased protein concentration are typical pathological hallmarks of tauopathies. Tau protein filaments with different morphologies are characteristic of other neurodegenerative diseases. (PMID: 37337995, PMID: 34890553).

[0003] Most of the tau protein in the cerebrospinal fluid exists in the form of fragments, among which the N-terminal and middle Tau regions are the most abundant. This also shows that TAU ​​is unstable in the human body and is easily hydrolyzed into multiple domains. Methods for designing microtubule-associated protein TAU441 mutants with increased thermal stability have not yet been reported. Faced with the hot demand for global AD diagnosis, as an important marker, the highly thermally stable phosphorylated microtubule-associated protein TAU441 is of extremely high value. Summary of the invention

[0004] The present invention aims to provide a phosphorylated Tau protein mutant with high thermal stability obtained through rational design and screening, and a preparation method and application thereof.

[0005] On the one hand, the present invention provides a phosphorylated Tau protein mutant, whose amino acid sequence comprises at least one mutation substitution or deletion at E117, A145, G204, S305, A384, L408 relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitution or deletion at E117, A145, G204, S305, A384 and L408 relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant comprises substitutions or deletions at E117H, A145P, G204R, S305K, A384P and L408W relative to SEQ ID NO.1. In some embodiments, the amino acid sequence of the mutant is as shown in SEQ ID NO.2.

[0006] In another aspect, the present invention provides one or more biological materials selected from the group consisting of:

[0007] 1) A polynucleotide molecule encoding a Tau protein mutant;

[0008] 2) an expression vector comprising the polynucleotide molecule as described in 1);

[0009] 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

[0010] The present invention provides a polynucleotide molecule encoding the Tau protein mutant of the present invention; wherein the polynucleotide molecule may have various modifications in the coding region, as long as the amino acid sequence of the variant of the present invention does not change with the degeneracy of the codon or does not change with the preferred codon in the organism expressing the variant. In some embodiments, the polynucleotide molecule is as shown in SEQ ID NO: 4.

[0011] The expression vector provided by the present invention comprises a polynucleotide molecule encoding a Tau protein mutant of the present invention. In some embodiments, the expression vector further comprises one or more regulatory sequences, such regulatory sequences including but not limited to enhancers, promoters, leader peptide sequences, signal peptide sequences, and terminator sequences; wherein the regulatory sequence is operably connected to the polynucleotide molecule encoding the variant. In some embodiments, the expression vector can be a linear or circular DNA molecule, typically comprising elements such as a multiple cloning site, a resistance gene, and a replication initiation site.

[0012] In some embodiments, the expression vector of the present invention is preferably pET28a.

[0013] The host cell provided by the present invention refers to any cell that is beneficial to the expression of the variant of the present invention, that is, any cell that is susceptible to transformation, transfection or transduction with the expression vector described in the present invention, including any progeny cells that are different from the parent cell due to mutations that occur during replication.

[0014] In some embodiments, the host cell is a prokaryotic cell, which can be selected from gram-positive bacteria or gram-negative bacteria. In some embodiments, the host cell is a gram-positive bacteria, including but not limited to: Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanic Bacillus, Staphylococcus, Streptococcus and Streptomyces. In some embodiments, the host cell is a gram-negative bacteria, including but not limited to: Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silvae, Neisseria, Pseudomonas, Salmonella and Ureaplasma. In some embodiments, the host cell is Escherichia coli BL21 (DE3).

[0015] On the other hand, the present invention provides a method for preparing the Tau protein mutant, comprising: (1) culturing the host cell of the present invention under conditions suitable for mutant expression; and (2) recovering the mutant.

[0016] In some embodiments, the method further comprises a phosphorylation step.

[0017] In some embodiments, the method for recovering the mutant can be a method well known in the art, such as centrifugation, filtration, treatment with a crystal protein precipitant (salting out method), extraction, ultrasonic fragmentation, ultrafiltration, dialysis, various chromatography methods such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, etc., HPLC, and combinations of the above methods.

[0018] In some embodiments, the preparation method also includes a step of purifying the variant, and the purification step can be a method well known in the art, such as chromatography (such as ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), isoelectric focusing electrophoresis, ammonium sulfate precipitation, SDS-PAGE, etc.

[0019] In another aspect, the present invention provides a composition comprising the phosphorylated Tau protein mutant of the present invention.

[0020] In another aspect, the present invention provides a kit comprising the phosphorylated Tau protein mutant of the present invention.

[0021] On the other hand, the present invention provides the use of the phosphorylated Tau protein mutant in the preparation of a standard. In some embodiments, the standard is used in conjunction with a phosphorylated Tau protein detection kit or is included in a Tau protein detection kit. In some embodiments, the phosphorylated Tau protein detection kit is a p-Tau 217 detection kit or a p-Tau 181 detection kit.

[0022] On the other hand, the present invention provides the use of the phosphorylated Tau protein mutant in preparing phosphorylated Tau protein antigen.

[0023] On the other hand, the present invention provides the use of the phosphorylated Tau protein mutant in the preparation of phosphorylated Tau protein antibodies.

[0024] The phosphorylated Tau protein mutant provided by the present invention has significantly improved stability compared to the wild-type phosphorylated Tau protein, and its heat resistance reaches 37° C. Under the condition of 7 days, the reactivity CV on the chemiluminescence platform is less than 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1Schematic diagram of the construction of pET28a-Tau protein recombinant plasmid;

[0026] Figure 2 It is the electrophoresis diagram of wild-type Tau protein and the Tau protein mutant of the present invention. DETAILED DESCRIPTION

[0027] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0028] The technical scheme of the present invention is further described below in conjunction with specific examples. However, the following examples are merely examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims. In the following examples, unless otherwise specified, the reagents and consumables used are purchased from ordinary suppliers in the field, and the experimental methods and technical means used are conventional methods and means in the field.

[0029] Example 1: Preparation of mutant thermostable phosphorylated Tau protein

[0030] According to existing reports, the thermal stability of the microtubule-associated protein TAU441 is closely related to its recognition antibody binding. By analyzing the protein structure of the microtubule-associated protein TAU441, searching for segments related to proteolysis, and improving stability parameters, the present invention selects sites away from the diagnostic antibody recognition site to avoid affecting its recognition antibody binding. These sites include E117, G204, S305, and L408. In addition, the present invention selects hydrophobic core sites in some non-core regions, hoping to improve the stability of the protein value by improving internal hydrophobic interactions. These sites include A145 and A384.

[0031] (1) Strain construction: The wild-type gene sequence of microtubule-associated protein TAU441 is shown in SEQ ID NO.3, and the mutant gene sequence is shown in SEQ ID NO.4. After DNA sequence synthesis, PCR amplification was performed and then introduced into the BamHI and XhoI restriction sites of the expression vector pET28a to obtain a recombinant expression vector, generating a pET28a-microtubule-associated protein TAU441 plasmid ( Figure 1), the specific synthetic sequence includes the purification tag 6×His sequence added at the 5' end and the TAA stop codon at the 3' end. This plasmid was transformed into E. coli BL21 competent cells for expression host construction, and cloned strains were obtained after antibiotic resistance plate coating screening; after successful recombination, the obtained strains were activated overnight in LB medium at 37°C and added to the fermentation liquid (LB medium) and cultured until the OD600 value was 0.6-0.8, and IPTG with a final concentration of 0.5 mol was added to continue culturing for 4-6 hours, and then centrifuged at 12000rmp and 4-8°C to collect the strains.

[0032] (2) Protein purification: The collected strains were washed with 0.2M PBS buffer with a pI value of 7.0 to obtain bacterial cells: after high-pressure crushing, affinity chromatography and ion exchange chromatography were performed to purify microtubule-associated proteins. After protein purification, the storage buffer formula was 20mM Tris, pH 8.0, 150mM NaCl, 5% glycerol, stored at -20°C. The concentration was determined by absorbance at 280nm.

[0033] (3) Phosphorylation and identification: Add ATP with a final concentration of 10mM and MgCl2 with a final concentration of 10mM to the protein solution. Add an appropriate amount of GSK3β kinase and mix well. Then place the sample in a 30℃ constant temperature water bath and incubate for 30min. After protein phosphorylation, use a storage buffer with a formula of 50mM Tris, 150mM NaCl, 10% glycerol, 1mM DTT, 0.25mM PMSF, pH7.5, and store at -20℃. Determine the concentration by absorbance at 280nm. Identify by SDS-Page gel running and compare with wild-type protein ( Figure 2 ).

[0034] Example 2: Reactivity assay

[0035] The synthesized phosphorylated Tau protein mutant and the unmutated phosphorylated wild-type Tau protein were diluted to 10 ng / mL using Tris buffer and then diluted in multiple steps. The p-Tau217 in vitro diagnostic kit (Hunan Novi Medical Technology Co., Ltd., M4701CB) was used to evaluate the reactivity of the synthesized phosphorylated Tau protein mutant and the unmutated phosphorylated wild-type Tau protein. The above proteins were used as samples for detection using a fully automatic chemiluminescence immunoassay analyzer (Shenzhen Yingkai Biotechnology Co., Ltd., Shine i2910). The test results are shown in Table 1.

[0036] Table 1:

[0037]

[0038] The results showed that the phosphorylated Tau mutant was more reactive than the phosphorylated wild-type Tau protein.

[0039] Example 3: Thermal stability determination

[0040] The stability of the above phosphorylated Tau protein mutants and phosphorylated wild-type Tau protein was tested using the p-Tau217 in vitro diagnostic kit (Hunan Novi Medical Technology Co., Ltd., M4701CB). The phosphorylated Tau protein was adjusted to a concentration of 1ug / mL using Tris buffer, and then placed in a 37°C incubator for 14 days. The reverse order was used, and the samples were taken at the time and diluted to 10ng / mL using Tris buffer. The samples were tested using a fully automatic chemiluminescence immunoassay analyzer (Shenzhen Yingkai Biotechnology Co., Ltd., shine i2910). The test results are shown in Table 2.

[0041] Table 2:

[0042]

[0043] After 7 days of heat damage, the reaction value of phosphorylated Tau protein mutant decreased less than that of phosphorylated wild-type Tau protein, which was relatively stable. After 10 / 14 days of heat damage, the phosphorylated wild-type protein decreased by more than 15% (96.9%, 99.0%), while the phosphorylated Tau protein mutant decreased by less than 10%, which was more stable than the wild-type protein.

Claims

1. A phosphorylated Tau protein mutant, whose amino acid sequence comprises at least one mutation substitution or deletion at E117, A145, G204, S305, A384, L408 relative to SEQ ID NO.

1.

2. The mutant according to claim 1, wherein the amino acid sequence comprises E117H, A145P, G204R, S305K, A384P and L408W substitutions relative to SEQ ID NO.

1.

3. The mutant according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

4. One or more biological materials selected from the following: 1) A polynucleotide molecule encoding the Tau protein mutant according to claim 1; 2) an expression vector comprising the polynucleotide molecule as described in 1); 3) A host cell comprising the polynucleotide molecule described in 1), or a host cell comprising the expression vector described in 2).

5. A polynucleotide molecule encoding the mutant according to claim 3, wherein the nucleotide sequence thereof is as shown in SEQ ID NO.

4.

6. A method for preparing a phosphorylated Tau protein mutant, comprising: (1) culturing the host cell according to claim 4 under conditions suitable for mutant expression; and (2) recovery of mutants.

7. The preparation method according to claim 6, further comprising a phosphorylation step.

8. A composition comprising the phosphorylated Tau protein mutant according to claim 1.

9. A kit comprising the phosphorylated Tau protein mutant according to claim 1.

10. Use of the phosphorylated Tau protein mutant according to claim 1 in preparing a standard product.

11. Use of the phosphorylated Tau protein mutant according to claim 1 in preparing phosphorylated Tau protein antigen or phosphorylated Tau protein antibody.

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