Photoinduced phase transition protein and use thereof

Abstract

The application discloses a light-induced phase transition protein and application thereof. A protein with a phase transition function is provided for the first time, and the amino acid sequence of the protein is shown as SEQ ID NO. 7. The application also provides an IDR polypeptide with a phase transition function, and the amino acid sequence of the polypeptide is shown as SEQ ID NO. 5. The application also provides application of the PE1 protein in light-induced phase transition. The tool has the advantages of high spatiotemporal accuracy, good biocompatibility and the like, can be integrated with physiological signals, and can be used in controllable phase separation research, local enrichment and material construction and the like, and has a wide prospect in the fields of life science research, drug development and synthetic biology.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to a photoinduced phase transition protein and its applications. Background Technology

[0002] Liquid-liquid phase separation is the core physical mechanism by which biomolecules spontaneously form membrane-free functional condensates within cells through multivalent weak interactions. These dynamic condensates, such as nucleoli and stress granules, precisely regulate key life activities such as gene expression, RNA metabolism, and cellular stress in space and time. Their molecular basis mainly relies on proteins or nucleic acids containing intrinsically disordered regions. Precise analysis and regulation of the liquid-liquid phase separation mechanism is not only at the forefront of understanding cellular organization principles and revealing the pathological mechanisms of neurodegenerative diseases and cancer, but also provides a completely new perspective for developing novel intelligent biomaterials and therapeutic strategies.

[0003] Currently, conventional methods for regulating protein phase separation (such as altering temperature, pH, ionic strength, or adding small molecule perturbators) have significant limitations: low spatiotemporal resolution, making dynamic manipulation at the subcellular scale difficult; strong nonspecificity, easily interfering with global cellular physiology; and poor in vivo compatibility, making reversible, localized regulation difficult in complex in vivo environments. Therefore, developing a regulatory tool with high spatiotemporal precision, non-invasiveness, and reversibility is a key technological bottleneck in this field.

[0004] To overcome this bottleneck, optogenetics has been introduced into this field. Current strategies primarily involve fusing exogenous light-sensitive protein modules (such as CRY2 / CIB and PhyB / PIF) with known phase transition proteins, promoting phase separation through dimerization induced by blue or red light. This "light-controlled dimerization-induced phase transition" strategy has been successfully applied to: on-demand assembly of transcriptional condensates and stress granules in living cells to study their dynamics and functions; constructing light-responsive artificial organelles for controllable metabolic pathway compartmentalization; and attempting to regulate the phase behavior of disease-related proteins through light-controlled aggregation.

[0005] However, this type of "grafting" strategy has inherent drawbacks: First, the molecular design is complex, requiring extensive optimization of the fusion protein to ensure that the photosensitizing module does not affect the natural folding and function of the target protein; second, the regulatory logic is simple and indirect, relying on exogenous dimerization, making it difficult to precisely simulate the multivalent weak interaction network dependent on endogenous phase separation; finally, there may be off-target effects and immunogenicity risks, as the introduction of exogenous large protein modules may interfere with endogenous cellular signaling or limit its therapeutic potential. Summary of the Invention

[0006] In view of the above-mentioned technical problems, the present invention proposes a photoinduced phase transition protein and its application.

[0007] The specific technical solution is as follows: In one aspect, the present invention provides a phase transition protein having the amino acid sequence shown in SEQ ID NO.7.

[0008] In one aspect, the present invention provides a polynucleotide encoding the aforementioned phase transition protein, the nucleotide sequence of which is shown in SEQ ID NO. 6.

[0009] In one aspect, the present invention provides a biomaterial comprising the above-mentioned polynucleotides, said biomaterial comprising a recombinant expression vector, recombinant engineered bacteria, or recombinant engineered cells; The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.6.

[0010] In one aspect, the present invention provides a fusion protein comprising the above-described phase transition protein and a fluorescent protein; the amino acid sequence of the phase transition protein is shown in SEQ ID NO.7.

[0011] In one aspect, the present invention provides the use of the above-mentioned phase change proteins, polynucleotides, biomaterials, and fusion proteins in the preparation of liquid-liquid separation products for protein solutions.

[0012] Furthermore, the protein solution refers to a solution containing the PE1 P3A An aqueous system including proteins, their fusion proteins, or aggregates formed by phase separation.

[0013] In one aspect, the present invention provides an IDR polypeptide tag with phase transition function, the amino acid sequence of which is shown in SEQ ID NO.5.

[0014] In one aspect, the present invention provides a polynucleotide encoding the above-mentioned IDR polypeptide tag, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0015] In one aspect, the present invention provides the use of biomaterials comprising the above-described encoded IDR polypeptide tag or related biomaterials in the preparation of products for liquid-liquid separation and / or enrichment in protein solutions; The biomaterials include: A1) The polynucleotide encoding the IDR polypeptide tag as described above, the polynucleotide sequence of which is shown in SEQ ID NO.4; A2) A recombinant expression vector containing a polynucleotide encoding an IDR polypeptide tag, the polynucleotide sequence of which is shown in SEQ ID NO.4; A3) Recombinant engineered bacteria, wherein the recombinant engineered bacteria contains a polynucleotide encoding an IDR polypeptide tag, the polynucleotide sequence of which is shown in SEQ ID NO.4.

[0016] Furthermore, the expression vector is a pET series expression vector.

[0017] Furthermore, the expression vector is preferably pET-28a.

[0018] Furthermore, the host cell of the recombinant engineered bacteria is a prokaryotic cell or a eukaryotic cell.

[0019] Furthermore, the host cell of the recombinant engineered bacteria is preferably Escherichia coli BL21(DE3).

[0020] In one aspect, the present invention provides a fusion protein comprising an IDR peptide tag and a fluorescent protein; the amino acid sequence of the IDR peptide tag is shown in SEQ ID NO. 5.

[0021] Furthermore, the fluorescent proteins include YFP protein and mCherry fluorescent protein.

[0022] In one aspect, this application also provides the use of PE1 protein or related biomaterials in one of the following: a1) Preparation of products for liquid-liquid separation and / or enrichment of protein solutions under light-induced conditions; b1) Induces liposome deformation to generate liposome microvesicles; c1) Preparation of drug delivery carriers.

[0023] Furthermore, the amino acid sequence of the PE1 protein is shown in SEQ ID NO.3.

[0024] Furthermore, the related biological materials include: a polynucleotide encoding the PE1 protein, a recombinant expression vector containing the polynucleotide, a recombinant expression strain containing the vector or the polynucleotide, or a fusion protein composed of the IDR polypeptide tag; Furthermore, the polynucleotide encoding the PE1 protein is shown in SEQ ID NO.2.

[0025] Furthermore, the photo-induced condition refers to irradiating a system containing the PE1 protein or its related biological materials with light of a specific wavelength.

[0026] In some implementations, specifically: excitation light with a wavelength range of 514~594 nm and an intensity of 0.1~20 mW / mm is used. 2 The irradiation time is 10s to 30min.

[0027] The preferred photoinduction conditions are: using excitation light with a wavelength of 514 nm and a light intensity of 0.1~5 mW / mm. 2 Irradiate with light for 10 seconds to 10 minutes.

[0028] Furthermore, the protein solution refers to an aqueous phase system containing the PE1 protein, its fusion protein, or aggregates formed by phase separation thereof.

[0029] Furthermore, the liquid-liquid separation and / or enrichment products include: cell immunofluorescence kits; And / or, protein-lipid complexes used as cell membrane modification materials; And / or, immunophotocontrol materials.

[0030] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to clearly identify the liquid-liquid phase separation capability of the PE1 protein and a specific IDR sequence, opening up a completely new research direction for understanding the biological function of the PE1 protein. This invention is based on the PE1 protein's own natural IDR sequence, rather than grafting an exogenous photosensitizing module, which gives it better biocompatibility, less molecular burden, and allows for a more realistic simulation and study of the regulatory behavior of endogenous phase transition proteins. Attached Figure Description

[0031] Figure 1 This is a time-series diagram of the phase transition induced by PE1-YFP protein under light irradiation. Scale bar: 20µm.

[0032] Figure 2 Phase transition characterization of PE1-YFP protein in laser-exposed / unexposed regions, scale bar: 20µm.

[0033] Figure 3 Time series diagram of single phase transition droplet fusion of PE1-YFP protein, scale bar: 2µm.

[0034] Figure 4 This is a time-series image showing the local darkening and recovery of PE1-YFP protein after laser stimulation. Scale bar: 2µm.

[0035] Figure 5 Statistical results of fluorescence intensity recovery in the laser-stimulated region of PE1-YFP protein.

[0036] Figure 6 For Ca 2+ Ion-induced photoinduced phase transition of PE1-YFP and Ca 2+ Verification of ion interaction; Scale bar: 5µm.

[0037] Figure 7 The photoinduced phase separation of PE1-YFP protein has high spatial positioning accuracy, scale bar: 1µm.

[0038] Figure 8The intrinsic disorder region (IDR) of the PE1 protein is the core functional domain for phase separation. Scale bar: 2µm.

[0039] Figure 9 For PE1 P3A Protein and PE1 WT Characterization of the ability of proteins to form phase change droplets in vitro, scale bar: 20µm.

[0040] Figure 10 For PE1 P3A Phase transition characterization of liposome membrane remodeling induced by phase transition protein droplets, scale bar: 20 µm.

[0041] Figure 11 A in PE1 △N -YFP proteins do not undergo phase transitions under basic conditions; Figure 11 B in the text is PE1 △N -YFP protein is unresponsive to light induction. Scale bar: 5µm. Detailed Implementation

[0042] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0043] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0044] Unless otherwise specified, 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 application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions were performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0045] Example 1: Photoinduced phase transition function of PE1 protein 1.1 The nucleic acid sequence corresponding to the human PE1 protein was cloned into the pET-28a protein expression vector. (1) Primer design and synthesis: Based on the coding sequence (CDS) of the target gene PE1 and the polyclonal restriction site information of the pET-28a(+) vector, a pair of specific primers were designed and synthesized. The 5' end of the upstream primer contains the vector homologous arm sequence and restriction site, and the 5' end of the downstream primer contains the YFP coding sequence, stop codon, vector homologous arm sequence and restriction site (F: 5'-GCGGCAGCCATATGGCTAGCATGGCCAGCTATCCTTACCG-3' (SEQ ID NO.14), R: 5'-GGTGGTGCTCGAGTGCGGCCGCTTTACTTGTACAGCTCGTCCATGCC-3' (SEQ ID NO.15), to ensure that the PCR amplification products have sequences that are completely homologous to the ends of the linearized vector.

[0046] (2) Amplification of the target fragment and linearization of the vector: Using the plasmid containing the PE1-YFP fusion gene as a template, the target fragment “PE1-CDS-YFP” was amplified by polymerase chain reaction (PCR) using the aforementioned primers. At the same time, the pET-28a(+) vector was digested with restriction endonucleases to obtain the linearized vector backbone.

[0047] (3) Homologous recombination cloning: The purified PCR target fragment is mixed with the linearized vector backbone at a molar ratio of 2:1 and incubated at 50°C for 15 minutes under the action of an enzyme with homologous recombination function to carry out the recombination reaction. This reaction can efficiently and seamlessly insert the target fragment into the predetermined position of the vector.

[0048] (4) Transformation and identification of positive clones: The recombinant product was transformed into competent E. coli cells and plated on a solid medium containing kanamycin for culture. Single colonies were picked and preliminarily screened by colony PCR. Plasmids were extracted and DNA was sequenced from positive clones. The correct recombinant expression plasmid was finally verified and named pET-28a-PE1-YFP.

[0049] 1.2 PE1-YFP Protein Purification: The PE1-YFP structure was cloned into an N-terminal His-Sumo tag. The His-Sumo tag-encoded protein structure, His-Sumo-PE1-YFP, was then transformed into competent *E. coli* BL21(DE3) cells and cultured overnight at 37°C. The His-Sumo tag protein was expressed in BL21(DE3) cells in 2-YT medium via overnight shaking at 16°C. Cells were collected by centrifugation, resuspended in lysis buffer, and homogenized using an autoclave or sonication. After removing cell debris by high-speed centrifugation, the clarified lysis buffer was loaded onto a Ni-Sepharose column for affinity chromatography to collect the protein. After elution with protein elution buffer, TEV protease was added to remove the His-Sumo tag. The eluent, containing His-Sumo tag-free protein, was collected using a second Ni-Sepharose Excel column. The harvested protein eluent was concentrated using Millipore protein concentrate tubes, and then dialyzed against a storage buffer of 50 mM HEPES, 200 mM NaCl, 1 mM TCEP, and 10% glycerol at pH 7.4. After rapid freezing, it was stored at -80°C. PE1 IDR The purification steps for -mCherry protein are the same as those for PE1-YFP protein.

[0050] 1.3 Validation of photoinduced phase transition of PE1-YFP protein: Add 50 µM PE1-YFP to buffer (20 mM HEPES, 1 mM TCEP, pH 7.4), incubate at room temperature for 10-30 minutes, and then stimulate the PE1-YFP protein phase transition droplet with a 514 nm laser using a laser microscope for 1-10 seconds and record the result.

[0051] like Figure 1 As shown, when the protein is irradiated with a 514 nm laser in buffer solution, the PE1-YFP protein aggregates from a homogeneous, diffuse state in the solution to the irradiated region and undergoes a protein phase transition. The area outside the yellow curve represents the un-illuminated region, where the PE1-YFP protein is not induced to aggregate and does not undergo a phase transition. Figure 2 ).

[0052] Example 2: PE1 protein liquid-liquid phase separation capability Add 50µM PE1-YFP to the buffer (20mM HEPES, 1mM TCEP, pH 7.4), incubate at room temperature for 10-30 minutes, and then stimulate the PE1-YFP protein phase transition droplet with a 514nm laser microscope for 1-10 seconds before taking pictures.

[0053] like Figure 3As shown, the droplets generated by the phase transition of PE1-YFP protein undergo a phase transition and then merge, proving that PE1 has undergone liquid-liquid phase separation to form aggregates.

[0054] Example 3: Analysis of Phase Transition Characteristics of PE1 Protein FRAP experiments were performed on droplets generated by the phase transition of PE1-YFP protein, and the results are as follows: Figure 4 As shown, it was found that PE1 locally darkened and quickly recovered after laser bleaching, indicating that PE1 has the characteristics of a phase transition protein. The droplets formed have strong fluidity, and the statistical results of fluorescence intensity recovery in the protein-stimulated area also confirmed this result. Figure 5 ).

[0055] Example 4, Ca 2+ Photoinduced phase transition of PE1 protein Add 50µM PE1-YFP to the buffer (20mM HEPES, 1mM TCEP, pH 7.4), and add 500µM CaCl2. Incubate at room temperature for 10-30 minutes. Stimulate the PE1-YFP protein phase transition droplet with a 514nm laser using a laser microscope for 1-10 seconds, and then take pictures.

[0056] like Figure 6 As shown, the PE1-YFP protein aggregated from a homogeneous state dispersed in solution to the light-irradiated region and underwent a phase transition, and the aggregation was faster and brighter than when no calcium ions were added; Ca 2+ After the ions are chelated by EDTA, they cannot exert their promoting function, and the photoinduced phase transition of PE1-YFP protein disappears, indicating that Ca... 2+ Ions promoted the enrichment and phase transition of PE1-YFP protein in the light-irradiated central region, suggesting that the PE1 phase transition is involved in calcium ion-mediated intercellular signaling.

[0057] Example 5: Precision detection of photoinduced phase transition of PE1 protein Incubate 50 µM PE1-YFP in buffer (20 mM HEPES, 1 mM TCEP, pH 7.4) at room temperature for 10–30 minutes, and use a 514 nm laser microscope to precisely illuminate the area to 1 µm. 2 Irradiation observation was conducted within the specified range.

[0058] like Figure 7 As shown, PE1-YFP at 1µm 2 Within a certain range, it undergoes a phase transition and aggregates under light-induced conditions, with precision reaching the local location of a single cell. It can be used to enrich biomolecules into designated micro-regions / for local enrichment and membrane remodeling material construction.

[0059] Example 6, PE1IDR Phase separation function Use 50µM PE1 IDR -mCherry was incubated in buffer (20 mM HEPES, 1 mM TCEP, pH 7.4) at room temperature for 10-30 minutes and observed using a 594 nm laser microscope.

[0060] like Figure 8 As shown, PE1 IDR The formation of droplets during phase transitions is a typical characteristic of phase transition proteins, indicating that the intrinsic disorder region (IDR) of the PE1 protein is the core functional domain for liquid-liquid phase separation.

[0061] Example 7, PE1 P3A Protein expression and purification With linearized carrier PE1 WT -pET28A was used as the backbone, with upstream primer PE1 P3A -pET28A-F (SEQ ID NO.12): 5'-CTGCAGGACAAGCAGCAGGAGCCGCTCCGGGTAGCTACTACCCTG-3';, downstream primer PE1 P3A -pET28A-R (SEQ ID NO.13): 5'-GCTGCTTGTCCTGCAGCTCCTGCGCAGCCCTGCCGGTAAG-3', was amplified by PCR and the amplified product was directly transformed into competent *E. coli* cells. After transformation, the bacterial culture was evenly spread on LB solid agar plates containing 50 µg / mL kanamycin resistance. The plates were inverted and incubated at 37°C for 12-16 hours. Single, independently growing colonies were picked from the plates and inoculated into LB liquid medium containing 50 µg / mL kanamycin, and cultured with shaking at 37°C and 200 rpm for 12-16 hours. Plasmid DNA was extracted using a plasmid extraction kit. The extracted plasmid was sent to a professional sequencing company for Sanger dideoxy chain termination sequencing using specific primers. The sequencing results were compared with the expected sequence, and positive recombinant plasmids with completely correct sequences were selected and named PE1. P3A -pET28A. PE1 P3A The purification steps for YFP protein are the same as those for PE1-YFP protein in Example 1.

[0062] Example 8, PE1 P3A Enhanced phase transition ability of proteins PE1 of the same concentration WT -YFP and PE1 P3A-YFP protein was mixed with a buffer solution containing 5 µM CaCl2 (solvent: 20 mM HEPES, 1 mM TCEP, pH 7.4 buffer) at a volume ratio of 1:9 to construct the reaction system. After incubation at room temperature for 5–20 minutes, the droplet formation was observed and recorded under a 514 nm excitation microscope.

[0063] like Figure 9 As shown, under the same experimental conditions, PE1 P3A The mutant produced a significantly higher number of phase transition droplets than the wild-type PE1. WT This phenomenon indicates that PE1 P3A The mutant has a stronger ability to separate liquid phases (phase transitions).

[0064] Example 9: PE1 binding to liposomes promotes the production of tentacle-like lipids. 1) Weigh and prepare a lipid mixture containing phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol-3-phosphate. Dissolve the lipid powder in chloroform and mix thoroughly to form a homogeneous lipid solution to prepare liposomes.

[0065] 2) Add the liposomes to the PE1 protein phase transition system and incubate at room temperature for 5 minutes.

[0066] 3) Observe the interaction between liposomes and PE1 protein phase transition particles under a laser microscope with the same imaging parameters.

[0067] The results are as follows Figure 10 As shown, at the binding site of the PE1 phase transition protein droplet and the liposome, the liposome is induced to deform, promoting the formation of tiny liposome tentacle-like structures and generating strings of liposome microvesicles.

[0068] Comparative Example 1, PE1 △N -Photoinduced phase transition function of YFP protein With linearized carrier PE1 WT Using pET28A as the backbone, a recombinant plasmid was constructed through site-directed mutagenesis or fragment substitution. After transformation into competent *E. coli* cells, the plasmid was plated on kanamycin-resistant LB agar plates and incubated overnight at 37°C. Single colonies were picked for amplification, plasmid was extracted, and verified by Sanger sequencing to obtain a recombinant plasmid with the correct sequence, named PE1. △N -pET28A. PE1 △N The expression and purification steps for the -YFP protein are the same as those for the purification of the PE1-YFP protein in Example 1.

[0069] Purified PE1 △NYFP protein was mixed with a buffer solution containing 5 µM CaCl2 (solvent: 20 mM HEPES, 1 mM TCEP, pH 7.4) at a volume ratio of 1:9 to construct the reaction system. After incubation at room temperature for 5–20 minutes, the formation of phase transition droplets was observed and recorded under a microscope with 514 nm excitation light. All experimental conditions (including protein concentration, buffer composition, incubation time, and observation methods) were the same as those for wild-type PE1. WT -YFP remains consistent.

[0070] like Figure 11 As shown in A, under the same experimental conditions, PE1 △N -YFP protein did not undergo liquid-liquid phase separation, and no phase transition droplet formation was observed. For example... Figure 11 As shown in B, under photoinduced conditions, PE1 △N The -YFP protein also failed to produce phase transition droplets, indicating that this mutant not only lacks the ability to undergo liquid-liquid phase separation, but also lacks the ability to undergo photoinduced phase transition.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. 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 phase transition protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

7.

2. A polynucleotide encoding the phase transition protein as described in claim 1, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

6.

3. A biomaterial comprising the polynucleotide of claim 2, characterized in that, The biomaterials include recombinant expression vectors, recombinant engineered bacteria, or recombinant engineered cells; The sequence of the polynucleotide is shown in SEQ ID NO.

6.

4. A fusion protein, characterized in that, It includes the phase transition protein and fluorescent protein as described in claim 1; the amino acid sequence of the phase transition protein is shown in SEQ ID NO.

7.

5. The use of the phase change protein of claim 1, the polynucleotide of claim 2, the biomaterial of claim 3, and the fusion protein of claim 4 in the preparation of products for liquid-liquid separation in photoinduced protein solutions.

6. Application of IDR peptide tags or related biomaterials in the preparation of products for liquid-liquid separation and / or enrichment of protein solutions under photoinduced conditions; The amino acid sequence of the IDR polypeptide tag is shown in SEQ ID NO.5; The biomaterials include: A1) A polynucleotide encoding an IDR polypeptide tag, the sequence of which is shown in SEQ ID NO.4; A2) A recombinant expression vector containing a polynucleotide encoding an IDR polypeptide tag, the polynucleotide sequence of which is shown in SEQ ID NO.4; A3) Recombinant engineered bacteria, wherein the recombinant engineered bacteria contains a polynucleotide encoding an IDR polypeptide tag, the polynucleotide sequence of which is shown in SEQ ID NO.

4.

7. Application of PE1 protein or related biomaterials in one of the following: a1) Preparation of products for liquid-liquid separation and / or enrichment of protein solutions under light-induced conditions; b1) Induces liposome deformation to generate liposome microvesicles; c1) Preparation of drug delivery carriers based on liposome microvesicles; The amino acid sequence of the PE1 protein is shown in SEQ ID NO.3; The relevant biomaterials include: The polynucleotide encoding the PE1 protein, a recombinant expression vector containing the polynucleotide, a recombinant expression strain containing the recombinant vector or the polynucleotide, or a fusion protein consisting of an IDR polypeptide tag with an amino acid sequence as shown in SEQ ID NO.5; The nucleotide sequence encoding the PE1 protein is shown in SEQ ID NO.

2.

8. The application as described in claim 7, characterized in that, The liquid-liquid separation and / or enrichment products include: cell immunofluorescence kits and / or protein-lipid complexes and / or immunophotocontrol materials for cell membrane modification.

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