Structural domain for promoting protein liquid-liquid phase separation, recombinant protein and application of structural domain

Abstract

The invention relates to a structural domain for promoting protein liquid-liquid phase separation, recombinant protein and application of the structural domain. Specifically, the invention relates to a structural domain for promoting protein liquid-liquid phase separation, a nucleic acid molecule, a recombinant protein, a polynucleotide for coding the recombinant protein, a recombinant expression vector, a transformant and applications thereof. The structural domain can enable disordered protein to be subjected to dynamic reversible phase separation under an adversity condition. The method has a good application prospect in promotion of intracellular liquid-liquid phase separation and enrichment.

Description

Technical Field

[0001] The present disclosure relates to the field of gene 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 one of the important reasons for the formation of membrane-less cell structures and is an important way for organisms to respond to changes in the external environment and perform signal regulation. Intrinsically disordered proteins (IDPs) are the main type of proteins with liquid-liquid phase separation capabilities and play an important role in maintaining intracellular protein homeostasis and resisting abiotic stresses (high and low temperatures, drought, etc.).

[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 damage caused by adversity. 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 ability.

[0004] Currently, many proteins have been found to spontaneously form LLPS through their intrinsically disordered regions (IDRs) or low complexity regions (LCRs). However, the formation mechanism of LLPS is complex and depends not only on the sequence and structure of the protein, but also on the regulation of external conditions and other molecules. Therefore, further exploration of the structural domains and other factors that can induce LLPS is still needed to fully understand the mechanism 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 domain that promotes liquid-liquid phase separation of proteins, which can promote dynamic and reversible liquid-liquid phase separation of proteins under adverse conditions.

[0006] In order to achieve the above-mentioned object, the present disclosure provides, in a first aspect, a domain for promoting liquid-liquid phase separation of proteins, the amino acid sequence of the domain 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 domain described in the first aspect and a target protein.

[0009] Optionally, the target protein is a disordered protein; the target protein is 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 transformant is introduced with the recombinant expression vector described in the fifth aspect.

[0014] A seventh aspect of the present disclosure provides a method for promoting liquid-liquid phase separation of proteins, comprising connecting the domain described in the first aspect to a target protein; Wherein, the target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radioresistant.

[0015] An eighth aspect of 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.

[0016] 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 intracellular liquid-liquid phase separation and enrichment.

[0017] Other features and advantages of the present disclosure will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] 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 specific embodiments, they are used to explain the present disclosure but do not constitute a limitation of the present disclosure. In the accompanying drawings: Figure 1 This is a laser confocal microscopy observation of the recombinant engineering strains DosH-GFP and DohD-GFP under oxidative stress.

[0019] Figure 2 Is the recombinant engineering strain DosH ΔNS -GFP and NS-DohD-GFP under oxidative stress observed by laser confocal microscopy. DETAILED DESCRIPTION

[0020] The specific implementation of the present disclosure is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described herein is only used to illustrate and explain the present disclosure, and is not used to limit the present disclosure.

[0021] In a first aspect, the present disclosure provides a domain that promotes liquid-liquid phase separation of proteins, and the amino acid sequence of the domain is shown in SEQ ID NO.1.

[0022] The inventors of the present disclosure surprisingly found that the Deinococcus radiodurans The N-terminal domain of DosH protein (also called NS domain, with the amino acid sequence shown in SEQ ID NO.1) can promote the formation of liquid-liquid phase separation of DosH protein in host cells; wherein, the NS domain is the key region for DosH protein to form an α-helical structure, and this domain can enable DosH protein to undergo dynamic and reversible phase separation under adverse conditions. The inventors of the present disclosure have demonstrated that the lack of the NS domain makes it impossible for DosH protein to form liquid-liquid phase separation under adverse conditions.

[0023] 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.

[0024] A third aspect of the present disclosure provides a recombinant protein, comprising: the domain described in the first aspect and a target protein.

[0025] 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.

[0026] 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.

[0027] A fourth aspect of the present disclosure provides a polynucleotide encoding the recombinant protein described in the third aspect.

[0028] In the present disclosure, the inventors of the present disclosure connected the NS domain with the DohD protein from radioresistant Deinococcus to construct the recombinant protein NsdohD. The recombinant protein NsdohD in the host cells 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 cells.

[0029] 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.

[0030] The fifth aspect of the present disclosure provides a recombinant expression vector, into which the polynucleotide described in the fourth aspect is inserted.

[0031] The sixth aspect of the present disclosure provides a transformant, wherein the host of the transformant is a genetically engineered bacterium; the transformant is introduced with the recombinant expression vector described in the fifth aspect.

[0032] A seventh aspect of the present disclosure provides a method for promoting liquid-liquid phase separation of proteins, comprising connecting the domain described in the first aspect to a target protein; Wherein, the target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radioresistant.

[0033] In the present disclosure, the domain and the target protein can be connected using a kit conventionally used by those skilled in the art, such as the Clone Express®UItra One Step Cloning Kit (C115) of Vazyme.

[0034] An eighth aspect of 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.

[0035] The present disclosure is further described in detail below through examples, but the present disclosure is not limited thereby.

[0036] In the following examples, where no specific experimental conditions are specified, they are based on conventional conditions familiar to those skilled in the art, such as the conditions 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.

[0037] The plasmids and strains cited in the examples are from the following sources: Shuttle plasmid pRADZ3 (carrying Amp resistance gene): preserved in this laboratory; DosH-GFP recombinant strain: constructed in our laboratory; DohD-GFP recombinant strain: constructed in our laboratory; DosH ΔNS -GFP recombinant strain: constructed in our laboratory; NS-DohD-GFP recombinant strain: constructed in our laboratory; Escherichia coli DH5α: a commercially available product of Beijing Novozymes Corporation.

[0038] Example 1 This example is used to illustrate the construction of recombinant engineering strains containing green fluorescent tags DosH-GFP and DohD-GFP.

[0039] 1. Experimental materials: Experimental strains: Wild R1 strain of Deinococcus radiodurans: purchased from China General Microbiological Culture Collection Center, the collection number of the strain is CGMCC 1.633; Deinococcus radiodurans d0s Gene deletion mutant strain: constructed for this experiment; Deinococcus radiodurans dD Gene deletion mutant strain: constructed for this experiment.

[0040] Experimental plasmid: Shuttle plasmid pRADZ3 (carrying Amp resistance gene): preserved in this laboratory; Escherichia coli DH5α: a commercially available product from Beijing Novozymes Corporation (Cat. No. C502).

[0041] 2. Experimental methods: 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.

[0042] 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 the UV gel irradiator, cut the gel and perform product purification operations according to the gel recovery instructions of PolyMei Biotechnology Co., Ltd.

[0043] 3. Purification and recovery of target fragments: The recovered fragments were fused by PCR using homology arms, and PCR amplified using the upstream and downstream primers of the two fragments. After the PCR amplification reaction, 1% agarose gel electrophoresis was performed, the gel was cut, and the product purification operation was performed according to the gel recovery instructions of Polymei Biotechnology.

[0044] 4. Plasmid double enzyme digestion: use Bm I and Spe The vector pRADZ3 was double-digested with endonuclease I. The system was placed in a PCR instrument at 37°C for 2 hours of enzyme digestion. The enzyme digestion product was purified and recovered, and the purified product was stored at -20°C for future use.

[0045] 5. Recombination and ligation of target fragments: Perform homologous recombination between the linearized vector after restriction digestion and purification and the target gene with homology arms, refer to the instructions of Vazyme's Clone Express®UItra One Step Cloning Kit (C115), place in a 50°C metal bath, and carry out ligation reaction for 30 minutes.

[0046] 6. Transformation of ligation products: In a clean bench, use heat shock method to directly transform cloned strains E. coli DH5α competent cells were incubated upside down in a 37°C incubator overnight.

[0047] 7. Identification of positive clones 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 it into the radiation-resistant strain.

[0048] 3. Experimental results: Recombinant engineering strains carrying green fluorescent tags DosH-GFP and DohD-GFP were successfully constructed.

[0049] Example 2 This example is a laser confocal microscopy observation experiment of the recombinant engineering strains DosH-GFP and DohD-GFP under oxidative stress.

[0050] 1. Experimental materials: Recombinant engineering strains: the recombinant engineering strains DosH-GFP and DohD-GFP obtained in Example 1.

[0051] 2. Experimental methods: 1. Activate the recombinant engineered strain by streaking on a TGY solid medium plate; 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) at 30°C overnight to mid-late exponential stage; 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 rAntibiotics), culture until the bacterial solution OD 600 =2.0~4.0; 4. Oxidative stress: Take 1 mL of bacterial solution, add 8.0 μL of 30% H2O2 solution to a final concentration of 80 mM H2O2 (1 μL of 30% H2O2≈10 mM H2O2), shake at 30°C in dark for 30 min, and observe the distribution of DosH and DohD proteins in different strains using a laser confocal microscope after treatment. Repeat the experiment more than three times.

[0052] 3. Experimental results: like Figure 1 As shown in the figure, under normal culture conditions, DosH protein is evenly distributed on the cell membrane with low fluorescence intensity. After being treated with 80mM H2O2 for 30min, DosH-GFP protein aggregates to form aggregates. Under normal culture conditions, DohD protein is evenly distributed in the cytoplasm, and under oxidative stress, DohD-GFP protein does not aggregate.

[0053] 4. Experimental conclusion: DosH protein can undergo phase separation under abiotic stress, while DohD protein cannot undergo phase separation under abiotic stress.

[0054] Example 3 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.

[0055] 1. Experimental materials: Experimental strains: Wild R1 strain of Deinococcus radiodurans: purchased from China General Microbiological Culture Collection Center, the collection number of the strain is CGMCC 1.633; Deinococcus radiodurans d0s Gene deletion mutant strain: constructed for this experiment; Deinococcus radiodurans dD Gene deletion mutant strain: constructed for this experiment.

[0056] Experimental plasmid: Shuttle plasmid pRADZ3 (carrying Amp resistance gene): preserved in this laboratory; Escherichia coli DH5α: a commercially available product from Viagra (Cat. No. C502).

[0057] 2. Experimental methods: 1. Target fragment amplification: D. radiodurans Genes were amplified using the genome as a template dosH、dohDThe egfp gene segment was amplified using the plasmid carrying the egfp gene as a template.

[0058] 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 the UV gel irradiator, cut the gel and perform product purification operations according to the gel recovery instructions of PolyMei Biotechnology Co., Ltd.

[0059] 3. Purification and recovery of target fragments: The recovered fragments were fused by PCR using homology arms, and PCR amplified using the upstream and downstream primers of the two fragments. After the PCR amplification reaction, 1% agarose gel electrophoresis was performed, the gel was cut, and the product purification operation was performed according to the gel recovery instructions of Polymei Biotechnology.

[0060] 4. Plasmid double enzyme digestion: use Bm I and Spe The vector pRADZ3 was double-digested with endonuclease I. The system was placed in a PCR instrument at 37°C for 2 hours of enzyme digestion. The enzyme digestion product was purified and recovered, and the purified product was stored at -20°C for future use.

[0061] 5. Recombination and ligation of target fragments: Perform homologous recombination between the linearized vector after restriction digestion and purification and the target gene with homology arms, refer to the instructions of Vazyme's Clone Express®UItra One Step Cloning Kit (C115), place in a 50°C metal bath, and carry out ligation reaction for 30 minutes.

[0062] 6. Transformation of ligation products: In a clean bench, use heat shock method to directly transform cloned strains E. coli DH5α competent cells were incubated upside down in a 37°C incubator overnight.

[0063] 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. d0s , dD gene deletion mutants.

[0064] 3. Experimental results: The recombinant strain DosH lacking the NS domain was successfully constructed. ΔNS -GFP and the recombinant engineering strain NS-DohD-GFP containing the NS domain.

[0065] Example 4 This example is a recombinant engineering strain DosH ΔNS-GFP and NS-DohD-GFP under oxidative stress under laser confocal microscopy.

[0066] 1. Experimental materials: Recombinant engineering strains: The recombinant engineering strain DosH obtained in Example 3 ΔNS -GFP and NS-DohD-GFP.

[0067] 2. Experimental methods: 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 g / mL Cm r Antibiotics), cultured at 30°C; 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) at 30°C until mid-late exponential period; 3. Transfer the culture medium to 20 mL 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; 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 incubator in the dark for 30 min, 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. The experiments were repeated more than three times independently.

[0068] 3. Experimental results: like Figure 2 As shown, under normal culture conditions, DosH ΔNS -GFP protein was evenly distributed in the cells. After treatment with 80 mM H2O2 for 30 min, DosH ΔNS -GFP protein is still evenly distributed in the cytoplasm and does not aggregate. Under normal culture conditions, NS-DohD-GFP protein is evenly distributed on the cell membrane with low fluorescence intensity. After treatment with 80mM H2O2 for 30min, NS-DohD-GFP protein aggregates to form aggregates.

[0069] 4. Experimental conclusion: The N-terminal NS domain of DosH protein can promote the phase separation of disordered proteins DosH and DohD.

[0070] 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 in the above embodiments. Within the technical concept of the present disclosure, a variety of simple modifications can be made to the technical solution of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0071] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0072] In addition, 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 domain that promotes protein liquid-liquid phase separation, characterized in that The amino acid sequence of this domain is shown in SEQ ID NO.

1.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the domain of claim 1; The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO.

2.

3. A recombinant protein, characterized in that The recombinant protein comprises: the structural domain according to claim 1 and a target protein.

4. The recombinant protein according to claim 3, wherein The target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radioresistance. A polynucleotide encoding the recombinant protein according to claim 4 .

6. The polynucleotide according to claim 5, wherein The nucleotide sequence of the polynucleotide is shown in SEQ ID NO.

3.

7. A recombinant expression vector, characterized in that: The recombinant expression vector is inserted with the polynucleotide according to claim 5 or 6.

8. A transformant, characterized in that The host of the transformant is a genetically engineered bacterium; the transformant is introduced with the recombinant expression vector of claim 7.

9. A method for promoting protein liquid-liquid phase separation, characterized in that: Connecting the domain of claim 1 to a target protein; Wherein, the target protein is a disordered protein; the target protein is the DohD protein derived from Deinococcus radioresistant.

10. Use of the structural domain according to claim 1, the recombinant protein according to claim 3 or 4, the recombinant expression vector according to claim 7 or the transformant according to claim 8 in promoting intracellular liquid-liquid phase separation and enrichment.

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