A structural domain promoting protein liquid-liquid phase separation, recombinant protein and its application
By connecting the N-terminal domain of the radiation-resistant aberrant cocci DosH protein with the DohD protein, the recombinant protein NsdohD is constructed, and dynamic reversible liquid-liquid phase separation under adversity conditions is achieved, solving the problem of unclear protein liquid-liquid phase separation mechanism in the prior art, and promoting protein enrichment and steady state maintenance.
Patent Information
- Application Number
- CN202510295448.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-13
- Publication Date
- 2025-08-08
- Estimated Expiration
- 2045-03-13
AI Technical Summary
The prior art has not yet fully understood the formation mechanism and regulation of liquid-liquid phase separation (LLPS), and it is difficult to achieve dynamic reversible phase separation of proteins under adverse conditions.
The N-terminal domain (NS domain) of the DosH protein derived from radiation-resistant cocci was used to connect it to the disordered protein DohD protein to construct the recombinant protein NsdohD, and the liquid-liquid phase separation of the protein was achieved in the host cell using a recombinant expression vector.
Under adversarial conditions, the recombinant protein NsdohD can undergo dynamic reversible liquid-liquid phase separation, promoting protein enrichment and steady-state maintenance.
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Figure CN119798388B_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the field of genetic engineering technology, and in particular to a structural domain for promoting liquid-liquid phase separation of proteins, a nucleic acid molecule, a recombinant protein, a polynucleotide encoding the recombinant protein, a recombinant expression vector, a transformant, and applications thereof. Background Art
[0002] Liquid-liquid phase separation (LLPS) is a key factor in the formation of membrane-less cells and a crucial mechanism for organisms to respond to environmental changes and regulate signals. Intrinsically disordered proteins (IDPs) are the primary type of protein capable of liquid-liquid phase separation and play a crucial role in maintaining intracellular protein homeostasis and resisting abiotic stresses (such as high and low temperatures and drought).
[0003] Late embryonic development abundant protein family III ( L ate E mbryogenesis A Bundant group 3 proteins (LEA3) are a large class of hydrophilic proteins that play an important role in the response of organisms to abiotic stress and the reduction of adverse damage. Deinococcus radiodurans ) source DosH ( Deinococcus o rdered s tress-inducible H The DosH protein is an intrinsically disordered protein that is abundantly expressed in Deinococcus radiodurans under abiotic stress. It contains an intrinsically disordered region, which is closely related to its super strong abiotic stress resistance.
[0004] Many proteins have been found to spontaneously form LLPS through their intrinsically disordered regions (IDRs) or low-complexity regions (LCRs). However, the mechanisms of LLPS formation are complex and depend not only on the protein's sequence and structure but also on external factors and other molecules. Therefore, further exploration of the structural domains and other factors that induce LLPS is needed to fully understand the mechanisms and regulation of LLPS. Summary of the Invention
[0005] In order to solve the above technical problems, the purpose of the present disclosure is to provide a structural domain that promotes protein liquid-liquid phase separation, which can promote dynamic and reversible liquid-liquid phase separation of proteins under adverse conditions.
[0006] In order to achieve the above objectives, the present disclosure provides, in a first aspect, a domain for promoting liquid-liquid phase separation of proteins, the amino acid sequence of which is shown in SEQ ID NO.1.
[0007] The second aspect of the present disclosure provides a nucleic acid molecule, which encodes the domain described in the first aspect; the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2.
[0008] A third aspect of the present disclosure provides a recombinant protein, comprising: the structural domain described in the first aspect and a target protein.
[0009] Optionally, the target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radiodurans.
[0010] A fourth aspect of the present disclosure provides a polynucleotide encoding the recombinant protein described in the third aspect.
[0011] Optionally, the nucleotide sequence of the polynucleotide is shown as SEQ ID NO.3.
[0012] The fifth aspect of the present disclosure provides a recombinant expression vector, into which the polynucleotide described in the fourth aspect is inserted.
[0013] The sixth aspect of the present disclosure provides a transformant, wherein the host of the transformant is a genetically engineered bacterium; the recombinant expression vector described in the fifth aspect is introduced into the transformant.
[0014] A seventh aspect of the present disclosure provides a method for promoting liquid-liquid phase separation of proteins, comprising linking the domain described in the first aspect to a target protein;
[0015] Wherein, the target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radiodurans.
[0016] In an eighth aspect, the present disclosure provides the use of the domain described in the first aspect, the recombinant protein described in the third aspect, the recombinant expression vector described in the fifth aspect, or the transformant described in the sixth aspect in promoting intracellular liquid-liquid phase separation and enrichment.
[0017] Through the above technical solution, the present disclosure provides a domain that promotes protein liquid-liquid phase separation, which can enable disordered proteins to undergo dynamic and reversible phase separation under adverse conditions. It has good application prospects in promoting liquid-liquid phase separation and enrichment in cells.
[0018] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The accompanying drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following detailed description, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings:
[0020] Figure 1 These are laser confocal microscopy observations of the recombinant engineering strains DosH-GFP and DohD-GFP under oxidative stress.
[0021] Figure 2 Is the recombinant engineering strain DosH ΔNS -GFP and NS-DohD-GFP under oxidative stress were observed using a laser confocal microscope. DETAILED DESCRIPTION
[0022] The following describes the specific embodiments of the present disclosure in detail with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present disclosure and are not intended to limit the present disclosure.
[0023] In a first aspect, the present disclosure provides a domain that promotes liquid-liquid phase separation of proteins. The amino acid sequence of the domain is shown in SEQ ID NO.1.
[0024] The inventors of the present disclosure surprisingly found that the Deinococcus radiodurans The N-terminal domain of the DosH protein (also known as the NS domain, with the amino acid sequence shown in SEQ ID NO. 1) can promote liquid-liquid phase separation of the DosH protein in host cells. The NS domain is a key region in the formation of the α-helical structure of the DosH protein, which enables dynamic and reversible phase separation of the DosH protein under adverse conditions. The inventors of the present disclosure have demonstrated that the absence of the NS domain prevents the DosH protein from undergoing liquid-liquid phase separation under adverse conditions.
[0025] The second aspect of the present disclosure provides a nucleic acid molecule, which encodes the domain described in the first aspect; the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.2.
[0026] A third aspect of the present disclosure provides a recombinant protein, comprising: the structural domain described in the first aspect and a target protein.
[0027] In the present disclosure, the recombinant protein is formed by connecting the C-terminus of the domain to the N-terminus of the target protein.
[0028] In the present disclosure, the target protein is a disordered protein; in one embodiment of the present disclosure, the target protein is the DohD protein (AAF09701) derived from Deinococcus radiodurans.
[0029] A fourth aspect of the present disclosure provides a polynucleotide encoding the recombinant protein described in the third aspect.
[0030] In the present disclosure, the inventors of the present disclosure connected the NS domain with the DohD protein derived from radioresistant Deinococcus to construct the recombinant protein NsdohD. The recombinant protein NsdohD in the host cell was able to undergo liquid-liquid phase separation, further proving that the NS domain can promote the liquid-liquid phase separation of the target protein in the host cell.
[0031] In one embodiment of the present disclosure, the nucleotide sequence of the polynucleotide (ie, the recombinant protein NsdohD) is shown as SEQ ID NO. 3. The amino acid sequence of NsdohD is shown as SEQ ID NO. 4.
[0032] The fifth aspect of the present disclosure provides a recombinant expression vector, into which the polynucleotide described in the fourth aspect is inserted.
[0033] The sixth aspect of the present disclosure provides a transformant, wherein the host of the transformant is a genetically engineered bacterium; the recombinant expression vector described in the fifth aspect is introduced into the transformant.
[0034] A seventh aspect of the present disclosure provides a method for promoting liquid-liquid phase separation of proteins, comprising linking the domain described in the first aspect to a target protein;
[0035] Wherein, the target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radiodurans.
[0036] In the present disclosure, the domains can be linked to the target protein using a kit conventionally used by those skilled in the art, such as the Clone Express® Ultra One Step Cloning Kit (C115) produced by Vazyme.
[0037] In an eighth aspect, the present disclosure provides the use of the domain described in the first aspect, the recombinant protein described in the third aspect, the recombinant expression vector described in the fifth aspect, or the transformant described in the sixth aspect in promoting intracellular liquid-liquid phase separation and enrichment.
[0038] The present disclosure is further described in detail below through examples, but the present disclosure is not limited thereto.
[0039] In the following examples, where specific experimental conditions are not specified, they were based on conventional conditions well known to those skilled in the art, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.
[0040] The plasmids and strains cited in the examples are derived from the following:
[0041] Shuttle plasmid pRADZ3 (carrying Amp resistance gene): preserved in this laboratory;
[0042] DosH-GFP recombinant strain: constructed in our laboratory;
[0043] DohD-GFP recombinant strain: constructed in our laboratory;
[0044] DosH ΔNS -GFP recombinant strain: constructed in our laboratory;
[0045] NS-DohD-GFP recombinant strain: constructed in our laboratory;
[0046] Escherichia coli DH5α: a commercial product of Beijing Novozymes Co., Ltd.
[0047] Example 1
[0048] This example is used to illustrate the construction of recombinant engineering strains containing green fluorescent tags DosH-GFP and DohD-GFP.
[0049] 1. Experimental materials:
[0050] Experimental strains:
[0051] Wild-type Deinococcus radiodurans R1 strain: purchased from China General Microbiological Culture Collection Center, with the collection number of CGMCC 1.633;
[0052] Deinococcus radiodurans dosH Gene deletion mutant strain: constructed for this experiment;
[0053] Deinococcus radiodurans dohD Gene deletion mutant strain: constructed for this experiment.
[0054] Experimental plasmid:
[0055] Shuttle plasmid pRADZ3 (carrying Amp resistance gene): preserved in this laboratory;
[0056] Escherichia coli DH5α: a commercial product from Beijing Novozymes Co., Ltd. (Cat. No. C502).
[0057] 2. Experimental methods:
[0058] 1. Target fragment amplification: D. radiodurans Genes were amplified using the genome as a template dosH、dohD The egfp gene segment was amplified using the plasmid carrying the egfp gene as a template.
[0059] 2. Purification and recovery of target fragments: After the PCR amplification reaction is completed, perform 1% agarose gel electrophoresis for about 30 minutes. When the bands are clearly separated under a UV illuminator, cut the gel and perform product purification operations according to the gel recovery instructions of Polymeric Biotechnology Co., Ltd.
[0060] 3. Purification and Recovery of Target Fragments: PCR-fuse the recovered fragments using the homology arms and amplify them using the upstream and downstream primers of the two fragments. After the PCR amplification reaction, perform electrophoresis on a 1% agarose gel, cut the gel, and purify the product according to the gel recovery instructions provided by Polymer Biotechnology.
[0061] 4. Plasmid double enzyme digestion: use BamH I and Spe Double-digest the vector pRADZ3 with endonuclease I. Place the system in a PCR instrument at 37°C and allow the digestion reaction to proceed for 2 hours. Purify and recover the digestion product and store it at -20°C until use.
[0062] 5. Recombination and ligation of target fragments: Perform homologous recombination between the linearized vector after enzyme digestion and purification and the target gene with homology arms. Refer to the instructions of the Clone Express® UItra One Step Cloning Kit (C115) from Vazyme and place it in a 50°C metal bath for 30 minutes of ligation reaction.
[0063] 6. Transformation of ligation products: In a clean bench, use heat shock method to directly transform cloned strains E. coli DH5α competent cells were cultured in an incubator at 37°C overnight.
[0064] 7. Positive clone identification and sequencing verification: Single colonies were picked from the transformed plates for colony PCR and enzyme digestion verification, and then sent to BGI for sequencing. After successful sequencing, the recombinant plasmid was extracted and re-transformed into the radiation-resistant strain.
[0065] 3. Experimental results:
[0066] Recombinant engineering strains carrying green fluorescent tags DosH-GFP and DohD-GFP were successfully constructed.
[0067] Example 2
[0068] This example is a laser confocal microscopy observation experiment of the recombinant engineering strains DosH-GFP and DohD-GFP under oxidative stress.
[0069] 1. Experimental materials:
[0070] Recombinant engineering strains: the recombinant engineering strains DosH-GFP and DohD-GFP obtained in Example 1.
[0071] 2. Experimental methods:
[0072] 1. Activate the recombinant engineered strain by streaking on a TGY solid medium plate;
[0073] 2. Pick a single colony and inoculate it into 5 mL of fresh TGY liquid medium (the recombinant strain is supplemented with 10 μg / mL Kan antibiotic and 8 μg / mL Cm r Antibiotics) were cultured at 30°C overnight to the mid-late exponential stage;
[0074] 3. Transfer the culture medium to 20 mL of fresh TGY liquid medium at 1% (add 10 μg / mL Kan antibiotic and 8 μg / mL Cm r antibiotics), cultured to the OD 600 =2.0~4.0;
[0075] 4. Oxidative stress: Add 8.0 μL of 30% H₂O₂ solution to 1 mL of bacterial culture to a final H₂O₂ concentration of 80 mM (1 μL of 30% H₂O₂ ≈ 10 mM H₂O₂). Incubate at 30°C in a shaker in the dark for 30 min. After treatment, observe the distribution of DosH and DohD proteins in different strains using a laser confocal microscope. Perform the experiment at least three times independently.
[0076] 3. Experimental results:
[0077] like Figure 1 As shown, under normal culture conditions, DosH protein is evenly distributed on the cell membrane with low fluorescence intensity. However, after treatment with 80 mM H₂O₂ for 30 minutes, DosH-GFP protein aggregated. Under normal culture conditions, DohD protein is uniformly distributed in the cytoplasm. Under oxidative stress, DohD-GFP protein does not aggregate.
[0078] 4. Experimental Conclusions
[0079] DosH protein can undergo phase separation under abiotic stress, while DohD protein cannot undergo phase separation under abiotic stress.
[0080] Example 3
[0081] This example is used to illustrate the acquisition of a recombinant engineering strain DosH lacking the NS domain. ΔNS -GFP and the recombinant engineering strain NS-DohD-GFP containing the NS domain.
[0082] 1. Experimental materials:
[0083] Experimental strains:
[0084] Wild-type Deinococcus radiodurans R1 strain: purchased from China General Microbiological Culture Collection Center, with the collection number of CGMCC 1.633;
[0085] Deinococcus radiodurans dosH Gene deletion mutant strain: constructed for this experiment;
[0086] Deinococcus radiodurans dohD Gene deletion mutant strain: constructed for this experiment.
[0087] Experimental plasmid:
[0088] Shuttle plasmid pRADZ3 (carrying Amp resistance gene): preserved in this laboratory;
[0089] Escherichia coli DH5α: a commercial product from WeiZan Company (Cat. No.: C502).
[0090] 2. Experimental methods:
[0091] 1. Target fragment amplification: D. radiodurans Genes were amplified using the genome as a template dosH、dohD The egfp gene segment was amplified using the plasmid carrying the egfp gene as a template.
[0092] 2. Purification and recovery of target fragments: After the PCR amplification reaction is completed, perform 1% agarose gel electrophoresis for about 30 minutes. When the bands are clearly separated under a UV illuminator, cut the gel and perform product purification operations according to the gel recovery instructions of Polymeric Biotechnology Co., Ltd.
[0093] 3. Purification and Recovery of Target Fragments: PCR-fuse the recovered fragments using the homology arms and amplify them using the upstream and downstream primers of the two fragments. After the PCR amplification reaction, perform electrophoresis on a 1% agarose gel, cut the gel, and purify the product according to the gel recovery instructions provided by Polymer Biotechnology.
[0094] 4. Plasmid double enzyme digestion: use BamH I and Spe Double-digest the vector pRADZ3 with endonuclease I. Place the system in a PCR instrument at 37°C and allow the digestion reaction to proceed for 2 hours. Purify and recover the digestion product and store it at -20°C until use.
[0095] 5. Recombination and ligation of target fragments: Perform homologous recombination between the linearized vector after enzyme digestion and purification and the target gene with homology arms. Refer to the instructions of the Clone Express® UItra One Step Cloning Kit (C115) from Vazyme and place it in a 50°C metal bath for 30 minutes of ligation reaction.
[0096] 6. Transformation of ligation products: In a clean bench, use heat shock method to directly transform cloned strains E. coli DH5α competent cells were cultured in an incubator at 37°C overnight.
[0097] 7. Positive clone identification and sequencing verification: Pick a single colony from the transformed plate, perform colony PCR and enzyme digestion verification, and send it to BGI for sequencing. After successful sequencing, extract the recombinant plasmid and re-transform the radiation-resistant strain. dosH 、 dohD gene deletion mutants.
[0098] 3. Experimental results:
[0099] Successfully constructed a recombinant engineering strain DosH lacking the NS domain ΔNS -GFP and the recombinant engineering strain NS-DohD-GFP containing the NS domain.
[0100] Example 4
[0101] This example is a recombinant engineering strain DosH ΔNS -GFP and NS-DohD-GFP were observed under laser confocal microscopy under oxidative stress.
[0102] 1. Experimental materials:
[0103] Recombinant engineered strains:
[0104] The recombinant engineering strain DosH obtained in Example 3 ΔNS -GFP and NS-DohD-GFP.
[0105] 2. Experimental methods:
[0106] 1. Recombinant engineering strain DosH ΔNS -GFP and NS-DohD-GFP were streaked and activated on TGY solid medium plates (the recombinant strains were supplemented with 10 μg / mL Kan antibiotics and 8 μg / mL Cm r Antibiotics), cultured at 30°C;
[0107] 2. Pick a single colony and inoculate it into 5 mL of fresh TGY liquid medium (the recombinant strain is supplemented with 10 μg / mL Kan antibiotic and 8 μg / mL Cmr Antibiotics) at 30°C until the mid-to-late exponential period;
[0108] 3. Transfer the culture medium to 20 mL of fresh TGY liquid medium at 1% (add 10 μg / mL Kan antibiotic and 8 μg / mL Cm r Antibiotics), culture at 30℃ until the bacterial solution OD 600 =2.0~4.0;
[0109] 4. Oxidative stress: Take 1 mL of bacterial solution, add 8.0 μL of 30% H2O2 solution to the bacterial solution until the final H2O2 concentration is 80 mM (1 μL of 30% H2O2 ≈ 10 mM H2O2), shake in a 30°C shaker in the dark for 30 minutes, and observe with a laser confocal microscope after the treatment to observe the DosH protein of different strains. ΔNS The distribution of -GFP and NS-DohD-GFP was analyzed. The experiments were repeated more than three times independently.
[0110] 3. Experimental results:
[0111] like Figure 2 As shown, under normal culture conditions, DosH ΔNS -GFP protein was evenly distributed in the cells. After treatment with 80mM H2O2 for 30min, DosH ΔNS The NS-DohD-GFP protein remained evenly distributed in the cytoplasm and did not aggregate. Under normal culture conditions, the NS-DohD-GFP protein was evenly distributed on the cell membrane with low fluorescence intensity. After treatment with 80 mM H2O2 for 30 minutes, the NS-DohD-GFP protein aggregated and formed aggregates.
[0112] 4. Experimental Conclusions
[0113] The N-terminal NS domain of DosH protein can promote the phase separation of disordered proteins DosH and DohD.
[0114] The preferred embodiments of the present disclosure are described in detail above in conjunction with the accompanying drawings. However, the present disclosure is not limited to the specific details of the above embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the scope of protection of the present disclosure.
[0115] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.
[0116] In addition, the various embodiments of the present disclosure may be arbitrarily combined, and as long as they do not violate the concept of the present disclosure, they should also be regarded as the contents disclosed by the present disclosure.
Claims
1. A recombinant protein, characterized in that The recombinant protein is obtained by connecting the structural domain and the target protein; The target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radiodurans; The amino acid sequence of the recombinant protein is shown in SEQ ID NO.
4.
2. A polynucleotide encoding the recombinant protein according to claim 1.
3. The polynucleotide according to claim 2, wherein The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.
3.
4. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with the polynucleotide according to claim 2 or 3.
5. A transformant, characterized in that: The host of the transformant is a genetically engineered bacterium; the recombinant expression vector according to claim 4 is introduced into the transformant.
6. A method for promoting liquid-liquid phase separation of a target protein in a cell, characterized in that: Attach the domain to the target protein; Wherein, the target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radiodurans; The amino acid sequence of the recombinant protein obtained by ligation is shown in SEQ ID NO.
4. The cell is a DohD gene deletion mutant of Deinococcus radiodurans.
7. Use of the recombinant protein according to claim 1, the recombinant expression vector according to claim 4, or the transformant according to claim 5 in promoting liquid-liquid phase separation and enrichment of a target protein in a cell, wherein the target protein is the DohD protein derived from Deinococcus radiodurans; and the cell is a DohD gene deletion mutant of Deinococcus radiodurans.