Eidr polypeptides and uses thereof

Abstract

The present application belongs to the field of enzyme engineering and biotechnology, and relates to eIDR polypeptide and application thereof. The present application provides eIDR polypeptide and variants thereof, eIDR fusion enzyme comprising a target protein and eIDR polypeptide, a method for purifying protein by using eIDR polypeptide, and a method for improving enzyme catalytic activity by using eIDR polypeptide; and also provides the use of eIDR polypeptide as a protein purification tag and for providing soluble expression of a target protein. The eIDR polypeptide and variants thereof of the present application can improve the soluble expression of various enzymes, and after being fused with enzymes, can improve the catalytic activity of the enzymes.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and biotechnology, and more specifically, to eIDR peptides and their applications. Background Technology

[0002] Escherichia coli (E. coli) expression systems have become the preferred platform for the industrial production of exogenous proteins due to their advantages of rapid growth, controllable cost, and high yield. However, overexpression of E. coli induces protein aggregation and inclusion bodies, affecting the subsequent development and application of the proteins. To overcome these limitations, current research mainly focuses on developing and optimizing various fusion tags to promote soluble protein expression.

[0003] Enzymes, as a class of highly efficient biocatalysts, possess significant advantages over traditional chemical catalysts, including high substrate specificity, remarkable catalytic efficiency, and environmental friendliness, playing a crucial role in numerous industrial sectors. With the continuous expansion of enzyme applications, the market is placing higher demands on their production volume. However, the insufficient production of some enzymes due to their low natural expression levels has become a key bottleneck restricting their large-scale industrial production and application.

[0004] Protein liquid-liquid phase separation creates a stable and high-concentration environment, which highly enriches enzyme molecules, their substrates, and cofactors in a small space, greatly increasing the frequency of effective collisions and thus achieving extremely high local reaction rates at low overall concentrations. Summary of the Invention

[0005] The purpose of this invention is to provide the intrinsically disordered region (eIDR) of the effector factor NLP869 of Phytophthora capsici and its applications.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides an eIDR polypeptide for promoting the soluble expression of a target protein, enhancing enzyme catalytic activity, or for protein purification, wherein the eIDR polypeptide is selected from (a) or (b): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1; (b) A derivative peptide having equivalent function formed by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO:1.

[0007] In embodiments of the present invention, the derived peptide may be selected from (i) or (ii): (i) The peptide shown in SEQ ID NO:1, obtained by replacing all aspartic acid and glutamic acid with lysine, is denoted as eIDR. H ; (ii) The peptide obtained by adding at least one TPAP tetrapeptide repeat sequence to the polypeptide shown in SEQ ID NO:1 or the derivative peptide of (i).

[0008] In a preferred embodiment, eIDR is obtained by adding five TPAP tetrapeptide repeat sequences to the polypeptide shown in SEQ ID NO:1 or the derivative peptide of (i). H-15R .

[0009] In a further preferred embodiment, the derived peptide eIDR H and eIDR H-15R The amino acid sequences are shown in SEQ ID NO:2 and SEQ ID NO:3, respectively.

[0010] Secondly, the present invention provides applications of the above-mentioned eIDR polypeptide in the following: 1) As a protein purification tag; 2) To improve the soluble expression of the target protein; 3) To enhance the catalytic activity of enzymes.

[0011] In embodiments of the present invention, the target protein may be, for example, an enzyme. In specific embodiments, the enzyme may be NADH oxidase (NOX), chitinase (CHI), TEV protease (TEVp), tanninase (TAH), enterokinase (EK), or human rhinovirus 3C protease (HRV 3Cp).

[0012] Thirdly, the present invention provides a fusion protein comprising a target protein and an eIDR polypeptide fused to the target protein.

[0013] In an embodiment of the present invention, the target protein and the eIDR polypeptide are directly linked by peptide bonds.

[0014] In embodiments of the present invention, the target protein is an enzyme. In specific embodiments, the enzyme may be selected from one of NADH oxidase (NOX), chitinase (CHI), TEV protease, tanninase (TAH), enterokinase (EK), or human rhinovirus 3C protease (HRV3Cp).

[0015] In an embodiment of the present invention, the eIDR polypeptide is fused to the N-terminus or C-terminus of the target protein.

[0016] Fourthly, the present invention provides a method for purifying a target protein using eIDR peptides, comprising the following steps: (1) Provide a cell lysis buffer containing the fusion protein; (2) The cell lysis buffer was placed in a low-salt buffer to induce the fusion protein to undergo liquid-liquid phase separation and form aggregates; (3) The aggregates formed in step (2) are separated and collected by centrifugation; (4) The collected aggregates are resuspended in a high-salt buffer solution to re-dissolve the aggregates, thereby obtaining a solution containing the purified target protein.

[0017] In an embodiment of the present invention, the target protein is eIDR-GFP.

[0018] In an embodiment of the present invention, step (2) further includes adding a molecular crowding agent to the cell lysate to promote the liquid-liquid phase separation.

[0019] In a preferred embodiment of the present invention, the molecular crowding agent is PEG 8000.

[0020] Fifthly, the present invention provides the use of eIDR polypeptides or fusion proteins, i.e., eIDR fusion enzymes, in the preparation of compositions for improving enzyme catalytic activity.

[0021] In a sixth aspect, the present invention provides a method for improving enzyme catalytic activity, the method comprising fusing an eIDR polypeptide with a target protein to form a fusion protein, and placing the fusion protein in a reaction system capable of inducing liquid-liquid phase separation for enzymatic reaction.

[0022] In embodiments of the present invention, the target protein may be an enzyme. In specific embodiments, the enzyme may be NADH oxidase (NOX), chitinase (CHI), TEV protease, tanninase (TAH), enterokinase (EK), or human rhinovirus 3C protease (HRV 3Cp), preferably NOX.

[0023] In an embodiment of the present invention, the reaction system can be a low-salt buffer solution.

[0024] In embodiments of the present invention, a low-salt buffer refers to a buffer containing 30 mM Hepes, 30 mM NaCl, and a pH of 7.5. In embodiments of the present invention, a high-salt buffer refers to a buffer containing 30 mM Hepes, 600 mM NaCl, and a pH of 7.5.

[0025] By employing the above technical solutions, the present invention has at least the following advantages and beneficial effects. The eIDR polypeptide of the present invention can promote the soluble expression of various enzymes and can enhance the activities of chitinase and NADH oxidase (NOX). Furthermore, eIDR can also be used as a protein purification tag for purifying target proteins, laying the foundation for large-scale industrial applications. Attached Figure Description

[0026] Figure 1This is a preferred embodiment of the EK expression vector and expression method, where a: Schematic diagram of the EK expression vector. eIDR-K has an eIDR inserted at the N-terminus of EK, and EK-eIDR has an eIDR inserted at the C-terminus of EK; b: Expression. M represents the protein marker, "-" indicates that IPTG induction was not used, and "+" indicates that IPTG induction was used.

[0027] Figure 2 This is a preferred embodiment of the present invention for the quantitative analysis of soluble EK expression, wherein a: EK quantitative analysis protein electrophoresis diagram; M represents protein marker, P represents precipitate, and S represents supernatant; b: relative quantitative analysis of EK recombinant protein content. The contents of soluble and insoluble proteins are represented by black and white bar charts, respectively.

[0028] Figure 3 This is a preferred embodiment of the expression vector and expression of CHI, wherein a: schematic diagram of the CHI expression vector, eIDR-CHI has an eIDR inserted at the N-terminus of CHI, and CHI-eIDR has an eIDR inserted at the C-terminus of CHI; b: expression; M represents protein marker, "-" indicates that IPTG induction was not used, and "+" indicates that IPTG induction was used.

[0029] Figure 4 This is a preferred embodiment of the present invention for the quantitative analysis of CHI soluble expression, wherein a: CHI quantitative analysis protein electrophoresis diagram; M represents protein marker, P represents precipitate, and S represents supernatant; b: relative quantitative analysis of CHI recombinant protein content. The contents of soluble and insoluble proteins are represented by black and white bar charts, respectively.

[0030] Figure 5 In the preferred embodiment of the present invention, protein purification and enzyme activity analysis are shown, wherein a: protein purification electrophoresis diagram; M represents protein marker, F represents column flow-through, W represents washing buffer flow-through, E represents elution buffer flow-through; b: CHI, eIDR-CHI and CHI-eIDR enzyme activity verification.

[0031] Figure 6 This is a preferred embodiment of the TAH expression vector and expression, wherein a: a schematic diagram of the TAH expression vector, eIDR-TAH inserts eIDR at the N-terminus of TAH, and TAH-eIDR inserts eIDR at the C-terminus of TAH; b: expression; M represents protein marker, "-" indicates that IPTG induction was not used, and "+" indicates that IPTG induction was used.

[0032] Figure 7This is a preferred embodiment of the present invention for the quantitative analysis of soluble TAH expression, where a: TAH quantitative analysis protein electrophoresis diagram; M represents protein marker, P represents precipitate, and S represents supernatant; b: relative quantitative analysis of TAH recombinant protein content. The contents of soluble and insoluble proteins are represented by black and white bar charts, respectively.

[0033] Figure 8 This is a preferred embodiment of the TEVp expression vector and expression, wherein a: a schematic diagram of the TEVp expression vector, eIDR-TEVp inserts eIDR at the N-terminus of TEVp, and TEVp-eIDR inserts eIDR at the C-terminus of TEVp; b: expression; M represents protein marker, "-" indicates that IPTG induction was not used, and "+" indicates that IPTG induction was used.

[0034] Figure 9 This is a preferred embodiment of the present invention for the quantitative analysis of soluble TEVp expression, where a: electrophoresis diagram of TEVp quantitative analysis protein; M represents protein marker, P represents precipitate, and S represents supernatant; b: relative quantitative analysis of TEVp recombinant protein content. The contents of soluble and insoluble proteins are represented by black and white bar charts, respectively.

[0035] Figure 10 This is a preferred embodiment of the HRV 3Cp expression vector and expression, wherein a: HRV 3Cp expression vector schematic diagram, eIDR-3Cp inserts eIDR at the N-terminus of 3Cp, and 3Cp-eIDR inserts eIDR at the C-terminus of 3Cp; b: expression; M represents protein marker, "-" indicates that IPTG induction was not used, and "+" indicates that IPTG induction was used.

[0036] Figure 11 This is a preferred embodiment of the present invention for the quantitative analysis of soluble HRV 3Cp expression, where a: electrophoresis diagram of 3Cp quantitative analysis protein; M represents protein marker, P represents precipitate, and S represents supernatant; b: relative quantitative analysis of HRV 3Cp recombinant protein content. The contents of soluble and insoluble proteins are represented by black and white bar charts, respectively.

[0037] Figure 12 This is a preferred embodiment of the NOX expression vector and expression, wherein a: a schematic diagram of the NOX expression vector, eIDR-NOX has an eIDR inserted at the N-terminus of NOX, and NOX-eIDR has an eIDR inserted at the C-terminus of NOX; b: expression; M represents protein marker, "-" indicates that IPTG induction was not used, and "+" indicates that IPTG induction was used.

[0038] Figure 13This is a preferred embodiment of the present invention for the quantitative analysis of soluble NOX expression, where a: NOX quantitative analysis protein electrophoresis diagram; M represents protein marker, P represents precipitate, and S represents supernatant; b: relative quantitative analysis of NOX recombinant protein content. The contents of soluble and insoluble proteins are represented by black and white bar charts, respectively.

[0039] Figure 14 For the preferred embodiment of the present invention, protein purification and enzyme activity analysis are shown, where a: protein purification electrophoresis diagram; M represents protein marker, F represents column flow-through, W represents washing buffer flow-through, and E represents elution buffer flow-through; b: representative spectrum of the reaction progress of 1 µM protein solution in low-salt buffer; c: initial rate of 1 µM protein solution in low-salt buffer.

[0040] Figure 15 The following are the enzyme activities of NOX and eIDR-NOX in preferred embodiments of the present invention under low NaCl concentration and high NaCl concentration, wherein a: a representative spectrum of the reaction progress of 1 µM protein solution in low salt buffer; b: initial rate of 1 µM protein solution in low salt buffer; c: representative spectrum of the reaction progress of 1 µM protein solution in high salt buffer; d: initial rate of 1 µM protein solution in high salt buffer.

[0041] Figure 16 This invention relates to the recruitment of NADH and FAD by eIDR-NOX in a preferred embodiment.

[0042] Figure 17 eIDR and eIDR are preferred embodiments of the present invention. H Sequence comparison.

[0043] Figure 18 eIDR is a preferred embodiment of the present invention. H Phase separation of NOX protein.

[0044] Figure 19 eIDR-NOX and eIDR are preferred embodiments of the present invention. H -NOX recruits NADH and FAD, where a: Fluorescence confocal microscopy images show eIDR-NOX (left) and eIDR H - Recruitment of NADH (top, blue fluorescence) and FAD (bottom, green fluorescence) in NOX (right); b: Allocation coefficients (Kp) of substrate NADH (top) and cofactor FAD (bottom) versus net charge function of the fusion protein.

[0045] Figure 20 eIDR-NOX and eIDR are preferred embodiments of the present invention. H-NOX enzyme activity at low NaCl concentrations, where a: a representative spectrum of the reaction progress of a 1 µM protein solution in low-salt buffer; b: the initial rate of a 1 µM protein solution in low-salt buffer.

[0046] Figure 21 This is a preferred embodiment of the eIDR-NOX fluorescence bleaching recovery experiment, wherein a: a representative image of fluorescence bleaching recovery; b: a relative quantitative curve of fluorescence intensity during eIDR-NOX fluorescence bleaching recovery; c: eIDR H - Relative quantitative curve of fluorescence intensity during NOX fluorescent bleaching recovery.

[0047] Figure 22 eIDR is a preferred embodiment of the present invention. H eIDR H-0R and eIDR H-15R Sequence comparison.

[0048] Figure 23 eIDR is a preferred embodiment of the present invention. H-0R -NOX, eIDR H-15R Phase separation of NOX protein.

[0049] Figure 24 eIDR is a preferred embodiment of the present invention. H-0R -NOX, eIDR H-15R -NOX fluorescence bleaching recovery experiment, where a: representative image of fluorescence bleaching recovery; b: eIDR H-0R - Relative quantitative curve of fluorescence intensity during NOX fluorescence bleaching recovery; c: eIDR H-15R - Relative quantitative curve of fluorescence intensity during NOX fluorescence bleaching recovery.

[0050] Figure 25 eIDR is a preferred embodiment of the present invention. H -NOX, eIDR H-0R -NOX, eIDR H-15R -NOX enzyme activity at low NaCl concentrations, where a: a representative spectrum of the reaction progress of a 1 µM protein solution in low-salt buffer; b: the initial rate of a 1 µM protein solution in low-salt buffer.

[0051] Figure 26 This is a preferred embodiment of the present invention for the detection of NOX fusion protein enzyme activity at low NaCl concentration, wherein a: a representative spectrum of the reaction progress of 1 µM protein solution in low salt buffer; b: the initial rate of 1 µM protein solution in low salt buffer.

[0052] Figure 27This is a schematic diagram of phase separation-based protein purification of the eIDR-GFP fusion protein in a preferred embodiment of the present invention.

[0053] Figure 28 The images shown are of the protein purification process based on phase separation in a preferred embodiment of the present invention, captured under natural light and ultraviolet light. From left to right, the images show the bacterial lysate, the first centrifugation, the high-salt buffer resuspension, and the second centrifugation. The top image is taken under natural light, and the bottom image is taken under the corresponding ultraviolet light.

[0054] Figure 29 The above are electrophoresis images of eIDR-GFP protein purification in a preferred embodiment of the present invention, where a: electrophoresis image of the protein without PEG, and b: electrophoresis images of the protein with 10%, 7%, and 3% PEG, respectively. M represents the protein marker, CL represents the bacterial lysate, P1 represents the precipitate after the first centrifugation (the insoluble portion of the bacterial lysate), S1 represents the supernatant after the first centrifugation (the soluble portion of the bacterial lysate), P2 represents the precipitate after the second centrifugation (insoluble cell debris), and S2 represents the supernatant after the second centrifugation (purified protein). Detailed Implementation

[0055] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s *Molecular Cloning: A Laboratory Manual* (Sambrook J & Russell DW, 2012), or according to the manufacturer's instructions.

[0056] The clonal competent cells used in the following examples were E. coli DH5α, purchased from Beijing Qingke Biotechnology Co., Ltd.; the expression competent cells were E. coli Rosetta (DE3), purchased from Beijing TransGen Biotech Co., Ltd.; and E. coli Rosetta-gamiB, purchased from Beijing Qingke Biotechnology Co., Ltd. The primers used are listed in the sequence listing (SEQ ID NO:5 to SEQ ID NO:59). The preparation methods for commonly used reagents and culture media are shown in Table 1.

[0057] Table 1. Commonly Used Reagents and Formulas LB medium Weigh 10 g NaCl, 10 g tryptone, and 5 g yeast extract, and bring the volume to 1 L with deionized water. For solids, add 15 g agar powder per 1 L, dispense into 250 mL Erlenmeyer flasks as needed, autoclave at 121 °C for 30 min, and store at room temperature. 4×Tris-HClBuffer Weigh 3.025 g Tris-HCl, add 30 mL of deionized water, adjust the pH to 6.8 with NaOH, and then bring the volume to 50 mL with deionized water. Store at room temperature. 2×Loadingbuffer Weigh 3.1 g DTT, 4 g SDS, and 0.02 g G-250, then add 24 mL glycerol and 20 mL 4×Tris-HCl Buffer (pH 6.8), and bring the volume to 100 mL with deionized water. Store at room temperature. 2×Bindingbuffer Weigh 1.75 g sodium chloride, 0.36 g Tris-Base and 12.1 g urea, and dilute to 100 mL with deionized water. Store at room temperature. Protein electrophoresis solution Weigh 14.4 g glycine, 3.1 g Tris-Base, and 1 g SDS, and bring the volume to 1 L with deionized water. Mix well with a magnetic stirrer and store at room temperature. NADH stock solution (10 mM) Weigh 70.94 mg of NADH powder, dilute to 10 mL with ultrapure water, dissolve thoroughly, and dispense into 2 mL centrifuge tubes as needed. Store at -20°C protected from light. FAD stock solution (10 mM) Weigh 82.95 mg of FAD powder, dilute to 10 mL with ultrapure water, dissolve thoroughly, and store at 4°C protected from light. The suppliers of the reagents used in the experiment are listed in Table 2.

[0058] Table 2. Reagents and Purchasing Companies Shanghai Aladdin Biochemical Technology Co., Ltd. NADH, N-acetylglucosamine (GlcNAc) Nanjing Novozymes Biotechnology Co., Ltd. 2×Phanta Max Master Mix Wuhan Aiboteke Biotechnology Co., Ltd. Protein Marker, 2x MultiF Seamless Assembly Mix Sigma-Aldrich NaCl, imidazole, PEG 8000 Shandong Cisco Biotechnology Co., Ltd. Gel recovery / PCR product purification kit Fuzhou Feijing Biotechnology Co., Ltd. DNS developer Beijing Solarbio Technology Co., Ltd. Tryptone, yeast extract, IPTG, agarose, Tris-base, glycerol, Hepes, Tris-HCl, glycine, SDS, Coomassie Brilliant Blue G-250, Tween-20 Thermo Fisher Scientific Endonuclease The methods used in the embodiments are described below.

[0059] The gene cloning and purification methods are as follows. The PCR reaction system used is shown in Table 3.

[0060] Table 3. PCR reaction system forward primer 2 reverse primer 2 template 2 2×Phanta Max Master Mix 25 sterile water Supplement to 50 Prepare the reaction system according to Table 3, place it in a PCR tube, mix thoroughly and centrifuge briefly, set the reaction program according to Table 4, and perform the PCR reaction; perform PCR reaction on 1% agarose gel electrophoresis, cut out the target band, put it into a 1.5 mL centrifuge tube, and collect the DNA solution using the Shandong Cisco Gel Recovery / PCR Product Purification Kit.

[0061] Table 4. PCR reaction procedure Pre-variation 95℃ 5 min 1× transsexual 95℃ 30 s 30× annealing Primer Tm-1℃ 30 s 30× extend 72℃ 1000 bp = 1 min 30× Supplement and extension 72℃ 10 min 1× The OverLap PCR method is as follows: (1) The relevant primers are listed in the sequence listing. It should be noted that the downstream primer of gene A needs to be added with 20 bp of the upstream sequence of gene B, and the upstream primer of gene B needs to be added with the last 20 bp of gene A, so that the two have an overlapping part. (2) Amplify A and B genes separately using primers for A and B genes, and recover the separately amplified A and B genes using gel extraction. (3) Genes A and B are primers for each other and templates for each other. Prepare the reaction system according to Table 5 and set the reaction procedure according to Table 6. Table 5. Overlap PCR reaction system Gene A 2 Gene B 2 2×Phanta Max Master Mix 25 sterile water 19 Table 6. Overlap PCR reaction procedure Pre-variation 95℃ 5 min 1× transsexual 95℃ 30 s 10× annealing Primer Tm-1℃ 30 s 10× extend 72℃ 1000 bp = 1 min 10× Supplement and extension 72℃ 10 min 1× (4) After the product from the previous step is cooled, add 2 μL of upstream primer of gene A and 2 μL of downstream primer of gene B to amplify gene A+B. Collect the DNA solution using Shandong Cisco Jie gel recovery / PCR product purification kit.

[0062] The vector digestion method is as follows, and the digestion system used is shown in Table 7.

[0063] Table 7. Enzyme digestion system carrier 8 Endonuclease 1 2 Endonuclease 2 2 10×buffer 4 sterile water Supplement to 40 After preparing the enzyme digestion system according to Table 7 and mixing it well, incubate it in a water bath at 37°C for 3 hours. Use the Shandong Cisco gel recovery / PCR product purification kit to recover the enzyme digestion vector. After the enzyme digestion and recovery product is aliquoted, it is stored at -20°C for subsequent experiments.

[0064] The ligation transformation and plasmid extraction methods are as follows, and the ligation system used is shown in Table 8.

[0065] Table 8. Connection System gene fragments 2 Enzyme-digested and recovered vector 3 Homologous recombination ligase 5 After preparing the ligation system according to Table 8 and mixing thoroughly, the mixture was incubated in a 50℃ metal bath for 15 min to obtain the ligation product. This product was immediately transformed into cloning competent E. coli DH5α cells. After incubation at 37℃ upside down for 16-20 h, single colonies from the plates were picked and cultured in 1 mL of LB medium. The cultured bacterial solution was used as a template for PCR verification. Bacterial solutions with correctly amplified bands were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0066] Selected bacterial cultures with correct sequencing were expanded in 20 mL LB medium, and recombinant plasmids were extracted using a plasmid mini-prep kit (Tiangen Biotech Co., Ltd.).

[0067] The steps for experimental expression of the target protein are as follows: (1) Transform competent cells E. coli Rosetta (DE3) with the extracted recombinant plasmid, invert the culture, pick a single colony on the plate and add it to 1 mL of the corresponding resistant LB medium, and culture at 37°C with shaking until turbidity; (2) Aspirate 800 μL of bacterial culture from each tube and transfer it to a new sterilized 1.5 mL centrifuge tube. Select one tube as a blank control and add 0.8 μL of 1 M IPTG to the remaining centrifuge tubes. Incubate at 37℃ with shaking for 5-6 h. (3) Centrifuge the above bacterial culture at 7000 rpm for 1 min, carefully discard the supernatant, add 50 μL of 2×Binding Buffer to each tube to resuspend the bacterial cells, then add 50 μL of 2×Loading Buffer, mix thoroughly, boil in a water bath for 15 min, centrifuge at 12000 rpm for 2 min, and aspirate the supernatant for SDS-PAGE detection. (4) After electrophoresis, use rapid protein staining solution (Qingdao Shengquan Chemical Reagent Co., Ltd.) to stain the strain with the darkest target band, i.e. the highest expression level, for preservation or expansion culture.

[0068] The steps for high-level expression of the target protein are as follows: (1) The preserved high-expression E. coli strain was activated and revived, and cultured in 1 L LB medium at 37°C with shaking at 180 rpm until OD. 600 It is 0.6-0.8; (2) Change the parameters of the shaking incubator to 16℃ and 120 rpm. After the temperature of the culture medium drops to 16℃, add IPTG to the culture medium to induce the strain to express the target protein. The final concentration of IPTG is 1 mM. Induce culture for 12-16 h. (3) Transfer the cultured bacterial solution to a centrifuge cup, centrifuge at 8000 rpm for 5 min, discard the supernatant, and obtain the precipitate. Resuspend the precipitate with a suitable buffer solution to obtain the bacterial solution, which is then dispensed into 50 mL BD tubes. Add 5 mL of glycerol to each tube and store at -20℃.

[0069] The method for analyzing soluble expression is as follows: Take 1 g of the precipitate from Method 2.6 and resuspend it in 10 mL of buffer. Place the resuspended solution on ice and break it up under the following conditions: 3 s operation, 3 s rest, 260 W power, and 10 min total time. Transfer 200 μL to a new 1.5 mL centrifuge tube and centrifuge at 14000 rpm for 10 min at 4 °C. Take 50 μL of the supernatant and add 50 μL of 2× Loading buffer, labeling this sample as "S". Discard the remaining supernatant and resuspend the precipitate in 200 μL of 2× Binding buffer. Transfer 50 μL of the resuspended solution to another clean 1.5 mL centrifuge tube and add 50 μL of 2× Loading buffer, labeling this sample as "P". Boil the sample in a water bath for 15 min, centrifuge at 12000 rpm for 2 min, and take 10 μL for gel electrophoresis to determine protein solubility. Analyze the electrophoresis bands using ImageJ software and use formula (2-1) to analyze the soluble expression level of the protein.

[0070] Protein solubility (%) = S / (S + P) × 100 (2-1) S: Amount of the target protein expressed in the supernatant; P: The amount of the target protein expressed in the precipitate.

[0071] Purification of the target protein includes His-Tag affinity chromatography and gel filtration chromatography, which are described below.

[0072] His-Tag affinity chromatography: (1) Sterilization: Pour 50 mL of bacterial solution from Method 2.6 into a beaker, add 300 μL of Tween-20, place on ice for ultrasonic lysis, break for 3 s, pause for 3 s, for a total of 30 min, with a power of 260 W. (2) High-speed centrifugation: After the bacteria are broken, centrifuge at 4℃ and 14000 rpm for 25 min, and take samples of the precipitate and supernatant respectively, and label them as “S” and “P”. (3) Column loading: After centrifugation in a 4℃ chromatography cabinet, gently pour the supernatant into the nickel column. Repeat the column loading process 3 times. Take 50 μL of the last flow-through liquid as the sample and label it "F". (4) Washing: Use washing solution (add imidazole to a final concentration of 30 mM in buffer) to wash away impurities. Repeat washing 3 times and keep 50 μL of the first washing solution as a sample, labeled "W". (5) Elution: Use a small amount of elution buffer (add imidazole to the final concentration of 300 mM in the buffer) to elute the target protein, and keep 50 μL of the elution buffer as a sample, labeled "E"; (6) Purity detection: The purification effect was verified by SDS-PAGE.

[0073] Gel filtration chromatography: (1) Concentration: Pour the eluent after affinity chromatography into an ultrafiltration tube for ultrafiltration concentration, centrifuge at 4°C and 2800 rpm until the volume is less than 2 mL; (2) Equilibrate the gel filtration chromatography column: Open AKTA and equilibrate one column volume with sonicated degassed buffer solution. Set the liquid flow rate to 0.5 mL / min and the pressure to 2.5 MPa. (3) Sample loading: Transfer the concentrated protein solution to a new 2 mL centrifuge tube and centrifuge at 4°C and 12,000 rpm for 15 min. Rinse the loading loop with 20 mL of buffer and use a syringe to push the centrifuged protein solution into the loading loop; (4) Elution and collection: Run the program and start collecting protein when the UV value is greater than 100. Collect one tube for every 500 μL, take a sample, collect the protein according to the UV absorption curve, and store it at -80℃ after quick freezing in liquid nitrogen. (5) Cleaning system: Use ultrasonically degassed ultrapure water to balance one column volume, set the liquid flow rate to 0.5 mL / min and the pressure to 2.5 MPa, and turn off AKTA; (6) Purity detection: The purification effect was verified by SDS-PAGE.

[0074] The method for determining chitinase activity is as follows: (1) Determination of the standard curve of N-acetylglucosamine (GlcNAc) Accurately weigh 0.2212 g of GlcNAc, dissolve it in ultrapure water, and dilute to 100 mL. Mix well to obtain a 10 mM stock solution. Dilute 20 mL of the GlcNAc stock solution to 100 mL and mix well to prepare a 2 mM GlcNAc standard solution. Add 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mL of the 2 mM GlcNAc standard solution sequentially to centrifuge tubes numbered 0-10, and bring the volume to 1.0 mL with ultrapure water. Mix well. Take 500 μL of each gradient solution and mix with an equal volume of DNS colorimetric reagent. React in a boiling water bath for 10 min. After the reaction, centrifuge at 12000 rpm for 10 min and measure the absorbance of the supernatant at 540 nm. Construct a standard curve with GlcNAc concentration on the x-axis and A540 on the y-axis.

[0075] The linear regression equation obtained by fitting the standard curve is: y = 0.7958x – 0.12704, R0 2 = 0.99918. Substituting the measured absorbance value into this equation gives the GlcNAc concentration.

[0076] (2) Preparation of 2% (w / v) colloidal chitin Weigh 1 g of chitin (MedChemExpress (MCE)) powder into a beaker, slowly add 40 mL of concentrated hydrochloric acid, and stir continuously until the chitin is completely dissolved. Add 500 mL of 50% ethanol, mix thoroughly, and let stand overnight at 4°C. Then centrifuge at 8000 rpm for 10 min, retain the precipitate after centrifugation, wash the precipitate with ultrapure water until the pH of the supernatant after centrifugation is neutral, and add ultrapure water to make up to 50 mL to obtain 2% colloidal chitin.

[0077] (3) Enzyme activity detection The enzyme activity assay consisted of 400 μL of 2% colloidal chitin and 100 μL of enzyme solution. The blank control was prepared by replacing the enzyme solution with an equal volume of buffer solution. The reaction mixture was incubated in a 55°C water bath for 20 min, then immediately transferred to a boiling water bath for 10 min to terminate the reaction, and cooled to room temperature in ice water. 500 μL of DNS chromogenic reagent was added to each tube, followed by heating in a boiling water bath for 10 min for color development, and then cooling to room temperature. The reaction mixture was centrifuged at 12000 rpm for 10 min, and the absorbance of the supernatant was measured at 540 nm.

[0078] The amount of reducing sugar produced was calculated based on the difference in absorbance between the experimental and control groups, and the chitinase activity was calculated using the standard curve. Each experimental group was repeated three times. Chitinase activity (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol GlcNAc per minute under the experimental conditions.

[0079] The method for determining NADH oxidase activity is as follows: (1) Determination of the NADH standard curve A 10 mM NADH stock solution was used to prepare standard solutions of 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 μM using ultrapure water. A₂O₃ was then measured using a spectrophotometer. 340 Establish a concentration-absorbance standard curve.

[0080] The linear regression equation obtained by fitting the standard curve is: y = 0.0041x – 0.13271, R0 2 = 0.99535. Substituting the measured absorbance value into this equation gives the NADH concentration.

[0081] (2) Enzyme activity The substrate NADH exhibits an absorption peak at 340 nm, but the substrate NAD+ does not. The rate of NOX enzymatic reaction was determined by monitoring the rate of decrease in absorbance at 340 nm over time.

[0082] The sample contained 1 μM enzyme, 500 μM substrate NADH, 100 μM cofactor FAD (Shanghai Yuanye Biotechnology Co., Ltd.), and low-salt buffer. When investigating enzyme activity in high-salt buffer, the sample contained 1 μM enzyme, 500 μM substrate NADH, 100 μM cofactor FAD, and high-salt buffer. After adding buffer, FAD, NADH, and enzyme to a cuvette and mixing well, the absorbance at 340 nm was immediately monitored using a spectrophotometer.

[0083] The liquid-liquid phase separation microscopy characterization method is as follows: Phase separation was induced by diluting the protein solution to appropriate protein and NaCl concentrations using a low-salt buffer. The buffer and protein were mixed in a PCR tube to a final protein concentration of 50 μM, and incubated for 10 min. The sample was then dropped onto a coverslip in the chamber and observed and photographed under a confocal microscope.

[0084] The fluorescent bleach recovery (FRAP) method is as follows: FRAP experiments were performed using a Leica TCS SP8 inverted confocal microscope system. Phase separation was induced using the same method as in 2.11. The sample was dropped onto the chamber coverslip, and the phase separation image was previewed. A drop of appropriate size was selected, and an original image was taken before bleaching. A circular area was selected in the center of the droplet as the fluorescence bleaching area.

[0085] The bleached areas were photobleached for 1 second using a 488 nm laser at 100% laser power. After bleaching, one image was captured every 2 seconds, for a total of 50 images. The fluorescence intensity data of the regions of interest (ROIs) were corrected by comparison with the unbleached control areas and normalized to the fluorescence intensity level before bleaching.

[0086] The experimental method for substrate recruitment of NOX enzymes is as follows: Dilute the protein concentration to 100 μM with low-salt buffer, and the substrate NADH and cofactor FAD concentration to 900 μM. After mixing in the PCR tube, drop the sample onto the chamber coverslip, observe the sample and record the results using a confocal microscope.

[0087] To avoid the influence of rapid reactions, the partitioning of NADH and FAD was measured using a Leica TCS SP8 inverted confocal microscope. NADH was excited with 405 nm excitation light, and fluorescence signals were received in the 410-440 nm range; FAD was excited with 458 nm excitation light, and fluorescence signals were received in the 510-530 nm range. The partition coefficient (Kp) of the two molecules in the droplet was represented by the ratio of fluorescence intensity inside and outside the droplet.

[0088] The following are protein purification techniques based on phase separation: The precipitate from the induction culture in Method 2.6 was resuspended in low-salt buffer. 50 mL of the bacterial culture was poured into a beaker and placed on ice. Sonication lysis was performed using a cycle of 3 s disruption followed by 3 s pause, at a total power of 260 W for 30 min. The lysis buffer (CL) was centrifuged at 14000 rpm and 4°C for 25 min. The supernatant (S1) was discarded. The precipitate (P1) was resuspended in high-salt buffer and centrifuged again at 14000 rpm and 4°C for 25 min to separate the supernatant (S2) and the precipitate (P2). The supernatant (S2) was the purified protein product, which was flash-frozen in liquid nitrogen and stored at -80°C. Samples of the precipitate and supernatant from both centrifugations were taken for SDS-PAGE analysis of protein purification.

[0089] Example 1. Bioinformatics Analysis of eIDR The 36th-147th amino acids of the Phytophthora capsici effector NLP869 (PHYCA_107869) constitute the intrinsic disorder region (hereinafter referred to as eIDR). eIDR can significantly promote the soluble expression of its own protein. Based on this, it is expected that eIDR can be used as a solubilizing tag for the regulation of the soluble expression of other proteins.

[0090] Bioinformatics analysis of eIDR showed that the gene length of eIDR is 336 bp (SEQ ID NO:4), encoding 112 amino acids (SEQ ID NO:1), with a predicted protein molecular weight of 11.9 kDa and a predicted isoelectric point of 9.52.

[0091] Example 2. eIDR promotes soluble protein expression This embodiment selects a biological enzyme as the fusion expression target for eIDR. By fusing eIDR with the enzyme protein, it is hoped to improve the soluble expression effect of the enzyme, increase production efficiency, and study the effect of eIDR fusion on enzyme activity. In this embodiment, six commonly used enzymes, enterokinase, chitinase, tanninase, TEV protease, NADH oxidase (NOX), and HRV 3Cp, were selected for research.

[0092] 1. Enterokinase Enterokinase (EC 3.4.21.9, EK), also known as intestinal peptidase, is a serine protease with extremely high substrate specificity. It can precisely recognize the pentapeptide sequence Asp-Asp-Asp-Asp-Lys and cleave the amide bond at the carboxyl terminus of the lysine residue.

[0093] The solubility of EK was studied using pET39b (Sangon Biotech (Shanghai) Co., Ltd.) as the prokaryotic expression vector, with BamHI and XhoI as restriction enzyme sites. Overlap PCR was employed, and primers eIDR-EK-F1 (SEQ ID NO:5), eIDR-EK-R1 (SEQ ID NO:6), eIDR-EK-F2 (SEQ ID NO:7), and eIDR-EK-R2 (SEQ ID NO:8) were used to fuse the DNA fragment encoding the eIDR polypeptide (SEQ ID NO:4) to the N-terminus of the EK gene, successfully constructing the eIDR-EK fusion protein expression vector. Figure 1(a) Using HindⅢ as the restriction site, the DNA fragment encoding the eIDR polypeptide (SEQ ID NO:4) was fused to the C-terminus of the EK gene using primers EK-eIDR-F (SEQ ID NO:9) and EK-eIDR-R (SEQ ID NO:10), successfully constructing the EK-eIDR fusion protein expression vector. Figure 1 (a) in the middle.

[0094] The successfully constructed expression vector was transformed into competent E. coli Rosetta (DE3) cells for expression. The expression results are as follows: Figure 1 As shown in b, compared with the control group culture without IPTG induction, all three target proteins were successfully induced to express in Rosetta (DE3).

[0095] The optimal expression strain was selected based on SDS-PAGE results and cultured in 1 L LB medium. 1 g of wet bacterial pellet was collected, resuspended in Tris buffer (30 mM Tris, 300 mM NaCl, pH 8.0), and the cells were sonicated. The supernatant and pellet were separated by centrifugation. Solubility analysis was performed by comparing the proportions of insoluble and soluble proteins. Figure 2 ).

[0096] Depend on Figure 2 It can be seen that when EK is expressed alone, the content of soluble protein is only 8.9%; after fusing with eIDR, the content of soluble protein in the N-terminal fusion protein eIDR-EK increases to 45.7%, and the content of soluble protein in the C-terminal fusion protein EK-eIDR increases to 84.0%. eIDR, regardless of its location at the N-terminus or C-terminus of EK, can promote the soluble expression of enterokinase. From the perspective of soluble protein expression levels, the soluble protein expression levels of eIDR-EK and EK-eIDR fusion proteins are increased by 6.5 times and 6.8 times, respectively, compared to EK expressed alone, which is beneficial for improving purification efficiency.

[0097] 2. Chitinase Chitinase (EC 3.2.1.14, CHI) is a class of enzymes that can specifically hydrolyze the β-1,4 glycosidic bonds in chitin. Its hydrolysis products are mainly chitin oligosaccharides or monosaccharides.

[0098] The study of CHI used pET32a (Sangon Biotech (Shanghai) Co., Ltd.) as the prokaryotic expression vector, and selected NdeI and EcoRI as restriction enzyme sites. The experiment employed overlap PCR technology, using primers eIDR-CHI-F1 (SEQ ID NO:27), eIDR-CHI-R1 (SEQ ID NO:28), eIDR-CHI-F2 (SEQ ID NO:29), eIDR-CHI-R2 (SEQ ID NO:30), CHI-eIDR-F1 (SEQ ID NO:31), CHI-eIDR-R1 (SEQ ID NO:32), CHI-eIDR-F2 (SEQ ID NO:33), and CHI-eIDR-R2 (SEQ ID NO:34) to fuse the DNA fragment encoding the eIDR polypeptide (SEQ ID NO:4) to the N-terminus and C-terminus of the CHI gene, respectively, successfully constructing two fusion protein expression vectors: eIDR-CHI and CHI-eIDR. Figure 3 (a) Subsequently, the three expression vectors CHI, eIDR-CHI, and CHI-eIDR were transformed into Rosetta-gamiB for expression. Expression results ( Figure 3 b) shows that, compared with the control group culture without IPTG induction, all three target proteins could be successfully induced to express in Rosetta-gamiB.

[0099] The optimal expression strain was selected based on SDS-PAGE results and cultured in 1 L LB medium. After induction of expression, 1 g of wet bacterial pellet was collected, resuspended in Tris buffer (30 mM Tris, 300 mM NaCl, pH 8.0), and the bacterial cells were lysed. The supernatant and pellet were separated by centrifugation, and solubility analysis was performed by comparing the proportions of insoluble and soluble proteins. Figure 4 The content of unfused CHI in the supernatant was only 46.3%; after fusing the eIDR tag, the content of the N-terminal fusion protein eIDR-CHI in the supernatant increased to 87.5%, and the content of the C-terminal fusion protein CHI-eIDR in the supernatant increased to 86.5%.

[0100] From the perspective of protein expression levels, although the overall protein expression levels of eIDR-CHI and CHI-eIDR in E. coli decreased compared to CHI expression alone, purification revealed that the soluble protein yield of the N-terminal fusion protein eIDR-CHI increased by 1.5 times, and the soluble protein yield of the C-terminal fusion protein CHI-eIDR increased by 1.1 times. This is beneficial for large-scale protein purification.

[0101] To investigate the effect of eIDR fusion on CHI activity, the proteins (CHI, eIDR-CHI, and CHI-eIDR) were purified using affinity chromatography and gel filtration chromatography. The purified enzyme solutions were diluted to 100 μM and reacted at 55 °C for 20 min. Color development was performed using DNS solution. The enzyme activities of the three CHIs were measured as follows: Figure 5 As shown in b, the enzyme activity of CHI was 0.29 U / mL, the enzyme activity of eIDR-CHI (with eIDR at the N-terminus of CHI) was 0.39 U / mL, and the enzyme activity of CHI-eIDR (with eIDR at the C-terminus of CHI) was 0.41 U / mL. Compared with unlabeled CHI, the enzyme activity of eIDR-CHI increased by 23.3%, and the enzyme activity of CHI-eIDR increased by 36.7%, indicating that eIDR can enhance the enzyme activity of CHI.

[0102] 3. Tanninase Tanninase (EC 3.1.1.20), also known as tannin acyl hydrolase (TAH), can specifically hydrolyze the ester bonds and phenolic acid bonds in hydrolyzed tannins, converting substrates such as gallic acid tannin, gallic acid ester, and epicatechin gallic acid ester into gallic acid and polyols.

[0103] The study on TAH used pET30a (Sangon Biotech (Shanghai) Co., Ltd.) as the prokaryotic expression vector, with EcoRI and NotI as restriction enzyme sites. The experiment employed overlap PCR technology, using primers eIDR-TAH-F1 (SEQ ID NO:11), eIDR-TAH-R1 (SEQ ID NO:12), eIDR-TAH-F2 (SEQ ID NO:13), eIDR-TAH-R2 (SEQ ID NO:14), TAH-eIDR-F1 (SEQ ID NO:15), TAH-eIDR-R1 (SEQ ID NO:16), TAH-eIDR-F2 (SEQ ID NO:17), and TAH-eIDR-R2 (SEQ ID NO:18) to fuse the DNA fragment encoding the eIDR polypeptide (SEQ ID NO:4) to the N-terminus and C-terminus of the TAH gene, respectively. This successfully constructed two fusion protein expression vectors: eIDR-TAH and TAH-eIDR. Figure 6 (a) in the middle.

[0104] The three expression vectors, TAH, eIDR-TAH, and TAH-eIDR, were transformed into Rosetta (DE3). The expression results of the three TAH proteins are shown below. Figure 6As shown in b, all three target proteins were expressed in Rosetta (DE3) compared to bacterial cultures without IPTG induction.

[0105] The optimal expression strain was selected based on SDS-PAGE results and cultured in 1 L LB medium. After induction, 1 g of wet bacterial pellet was collected, resuspended in Tris buffer (30 mM Tris, 600 mM NaCl, pH 8.5), the bacterial cells were lysed, and the supernatant and pellet were separated by high-speed centrifugation. The solubility of the target protein was analyzed by comparing the proportions in the supernatant and pellet. Figure 7 ).

[0106] The results showed that the proportion of soluble target protein expressed alone by TAH was only 19.0%; when the eIDR tag was fused, the soluble expression level of the target protein was significantly improved, with the proportion of soluble protein in the N-terminal fusion protein eIDR-TAH increasing to 56.6%, and the proportion of soluble protein in the C-terminal fusion protein TAH-eIDR increasing to 70.1%. eIDR, regardless of whether it is located at the N-terminus or C-terminus of TAH, can promote the soluble expression of TAH.

[0107] From the perspective of protein expression levels, although the overall protein expression levels of eIDR-TAH and TAH-eIDR in E. coli decreased compared to TAH expression alone, purification revealed that the soluble protein yield of the N-terminal fusion protein eIDR-TAH increased by 2.5 times, and the soluble protein yield of the C-terminal fusion protein TAH-eIDR increased by 2.3 times. The increase in soluble protein is beneficial to subsequent protein purification work.

[0108] 4. TEV protease TEV protease (tobacco etch virus protease, EC 3.4.22.44, TEVp) is a 27 kDa catalytic region in the NIa protein encoded by tobacco spot virus. TEVp can specifically recognize a 7-amino acid polypeptide sequence Glu-Asn-Leu-Tyr-Phe-Gln-Gly, precisely cleaving it between Gln (glutamine) and Gly (glycine).

[0109] The TEVp study used pET28a (Sangon Biotech (Shanghai) Co., Ltd.) as the prokaryotic expression vector, and NdeI and SalI as restriction enzyme sites. The experiment employed overlap PCR technology, using primers eIDR-TEVp-F1 (SEQ ID NO:19), eIDR-TEVp-R1 (SEQ ID NO:20), eIDR-TEVp-F2 (SEQ ID NO:21), eIDR-TEVp-R2 (SEQ ID NO:22), TEVp-eIDR-F1 (SEQ ID NO:23), TEVp-eIDR-R1 (SEQ ID NO:24), TEVp-eIDR-F2 (SEQ ID NO:25), and TEVp-eIDR-R2 (SEQ ID NO:26) to fuse a DNA fragment encoding the eIDR polypeptide (SEQ ID NO:4) to the N-terminus and C-terminus of the TEVp gene, respectively, successfully constructing two fusion protein expression vectors: eIDR-TEVp and TEVp-eIDR. Figure 8 (a) in the middle.

[0110] The three expression vectors, TEVp, eIDR-TEVp, and TEVp-eIDR, were transformed into Rosetta (DE3) for expression. The expression results of TEVp are as follows: Figure 8 As shown in b, compared with the bacterial culture without IPTG induction, all three target proteins can be induced to be expressed in Rosetta (DE3).

[0111] The optimal expression level was selected based on SDS-PAGE results and cultured in 1 L LB medium. After IPTG induction, 1 g of wet cell pellet was collected, resuspended in buffer (30 mM Hepes, 300 mM NaCl, pH 7.5), and the cells were lysed. The supernatant and pellet were separated by centrifugation, and solubility analysis was performed by comparing the proportions of insoluble and soluble proteins. Figure 9 The soluble protein percentages of TEVp, eIDR-TEVp, and TEVp-eIDR were 82.1%, 87.3%, and 88.2%, respectively. Since TEVp itself is a readily soluble protein when expressed in E. coli, the increase in solubility after fusing with eIDR was relatively small. However, from the perspective of expression levels, compared with the results of expressing TEVp alone, the soluble protein expression level of the N-terminal fusion protein eIDR-TEVp increased by 1.2 times; the soluble protein expression level of the C-terminal fusion protein TEVp-eIDR increased by 1.1 times.

[0112] 5. Human rhinovirus 3C protease Human rhinovirus 3C protease (EC 3.4.22.28, HRV 3Cp, referred to as 3Cp in this application) exhibits high specificity for the octapeptide sequence Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro, accurately recognizing and cleaving it between glutamine and glycine, with low requirements for the amino acid composition surrounding the sequence.

[0113] The study of HRV 3Cp used pET21b vector (Sangon Biotech (Shanghai) Co., Ltd.) as the prokaryotic expression vector, and selected NdeI and HindIII as cloning sites. The experiment employed overlap PCR technology, using primers eIDR-3Cp-F1 (SEQ ID NO:35), eIDR-3Cp-R1 (SEQ ID NO:36), eIDR-3Cp-F2 (SEQ ID NO:37), eIDR-3Cp-R2 (SEQ ID NO:38), 3Cp-eIDR-F1 (SEQ ID NO:39), 3Cp-eIDR-R1 (SEQ ID NO:40), 3Cp-eIDR-F2 (SEQ ID NO:41), and 3Cp-eIDR-R2 (SEQ ID NO:42) to fuse the DNA fragment encoding the eIDR polypeptide (SEQ ID NO:4) to the N-terminus and C-terminus of the 3Cp gene, respectively, successfully constructing two fusion protein expression vectors: eIDR-3Cp and 3Cp-eIDR. Figure 10 (a) in the middle.

[0114] Three expression vectors, 3Cp, eIDR-3Cp, and 3Cp-eIDR, were heat-transformed into E. coli Rosetta(DE3) for expression. The expression results are as follows: Figure 10 As shown in b, compared with the control group culture without IPTG induction, all three target proteins were successfully induced to express in Rosetta (DE3).

[0115] The optimal expression strain was selected based on SDS-PAGE results and cultured in 1 L of medium. After induction, 1 g of wet cell pellet was collected, resuspended in buffer (30 mM Hepes, 300 mM NaCl, pH 7.5), and the cells were lysed. The supernatant and pellet were separated by centrifugation. Solubility analysis was performed by comparing the proportions of insoluble and soluble proteins. Figure 11Protein electrophoresis showed that when 3Cp was expressed alone, the content of the target protein in the supernatant was only 8.7%; after fusing the eIDR tag, the content of the N-terminal fusion protein eIDR-3Cp in the supernatant increased to 69.2%, and the content of the C-terminal fusion protein 3Cp-eIDR in the supernatant increased to 86.0%. Whether eIDR is fused to the N-terminus or C-terminus of HRV 3Cp, it can significantly improve the soluble expression of HRV 3Cp.

[0116] Analysis of soluble protein expression levels showed that, compared with unlabeled HRV 3Cp, the soluble protein expression level of the N-terminal fusion protein eIDR-3Cp increased by 11.8 times, while the soluble protein expression level of the C-terminal fusion protein 3Cp-eIDR increased by 13.8 times. C-terminal fusion eIDR is more beneficial for improving the purification yield of HRV 3Cp.

[0117] 6. NADH oxidase NADH oxidase (EC 1.6.99.3, NOX) is an oxidoreductase that catalyzes the oxidation of NADH. In the mediated process of the cofactor FAD, NOX utilizes oxygen to catalyze the oxidation of NADH to NAD. + At the same time, H2O2 is generated.

[0118] The NOX study used pET15b (Sangon Biotech (Shanghai) Co., Ltd.) as the protein expression vector, with NdeI and BamHI as restriction enzyme sites. The experiment employed overlap PCR technology, using primers eIDR-NOX-F1 (SEQ ID NO:43), eIDR-NOX-R1 (SEQ ID NO:44), eIDR-NOX-F2 (SEQ ID NO:45), eIDR-NOX-R2 (SEQ ID NO:46), NOX-eIDR-F1 (SEQ ID NO:47), NOX-eIDR-R1 (SEQ ID NO:48), NOX-eIDR-F2 (SEQ ID NO:49), and NOX-eIDR-R2 (SEQ ID NO:50) to fuse the DNA fragment encoding the eIDR polypeptide (SEQ ID NO:4) to the N-terminus and C-terminus of the NOX gene, successfully constructing two fusion protein expression vectors: eIDR-NOX and NOX-eIDR. Figure 12 (a) in the middle.

[0119] Three recombinant vectors, NOX, eIDR-NOX, and NOX-eIDR, were thermally transformed into Rosetta (DE3) for expression. The expression results are as follows: Figure 12As shown in b, compared with the control group culture without IPTG induction, all three target proteins could be induced to be expressed in E. coli.

[0120] The optimal expression strain was selected based on SDS-PAGE results and cultured in 1 L LB medium. After induction, 1 g of wet bacterial pellet was collected, resuspended in high-salt buffer, and the bacterial cells were lysed. The supernatant and pellet were separated by centrifugation, and equal volumes were loaded onto the sample. Solubility analysis was performed by comparing the proportions of insoluble and soluble proteins. Figure 13 ).

[0121] Figure 13 Figure b shows that when NOX protein was expressed alone, the soluble percentage of the target protein in the supernatant was 58.8%. When the eIDR tag was fused, the soluble expression rate of the target protein significantly increased—the N-terminal fusion protein eIDR-NOX increased to 89.9% in the supernatant, and the C-terminal fusion protein NOX-eIDR increased to 87.6%. This indicates that eIDR, regardless of whether it is located at the N-terminus or C-terminus of NOX, can improve its soluble expression. Compared with the expression level of soluble NOX protein alone, the N-terminal fusion protein eIDR-NOX increased by 1.5 times, and the C-terminal fusion protein NOX-eIDR increased by 1.4 times, demonstrating a beneficial effect on increasing NOX production.

[0122] NOX, eIDR-NOX, and NOX-eIDR proteins were first subjected to Ni column affinity chromatography ( Figure 14 The protein was purified using the AKTA purification system (a) to obtain high-purity protein. To compare the enzyme activities of the three proteins, the purified protein was diluted to 100 μM with high-salt buffer before the experiment. Low-salt buffer was used for enzyme activity assays to ensure the stability of the assay environment. The concentrations of each key component in the system were strictly controlled: 1 μM of the target enzyme, 500 μM of the substrate NADH, and 100 μM of the cofactor FAD. NADH, as a characteristic reducing substrate, has an oxidation-decomposition process directly related to enzyme activity, while FAD provides the necessary electron transfer support for the enzyme-catalyzed reaction.

[0123] The detection experiment was conducted at room temperature, and the absorbance change at 340 nm was continuously monitored using a spectrophotometer. Since NADH has a characteristic absorption peak at 340 nm, after it is oxidized to NAD⁺, the absorbance at this wavelength gradually decreases as the reaction proceeds. Therefore, the rate of absorbance decrease directly reflects the reaction rate of enzyme-catalyzed substrate oxidation, i.e., the strength of enzyme activity. Compared with NOX, the absorbance decrease rate of both eIDR-NOX and NOX-eIDR was accelerated, with the N-terminal fusion protein eIDR-NOX showing the fastest absorbance decrease rate. Figure 14As shown in Figure c, compared to unlabeled NOX, the initial velocities of eIDR-NOX and NOX-eIDR were increased by 2.42 times and 1.34 times, respectively. This clearly demonstrates that eIDR-NOX, with N-terminal eIDR fusion, can significantly improve NOX activity.

[0124] Example 3. Enhancing enzyme activity based on phase separation Examples show that eIDR can induce phase separation in NLP869 in low-salt buffer, and NOX fusion with eIDR can drive phase separation of the fusion protein, and the enzyme activity is improved by relying on phase separation.

[0125] 1. Fusion eIDR enhances NOx enzyme activity through phase separation Given that previous studies have shown that fluctuations in the external environment (such as salt ion concentration and pH value) and changes in the intracellular state can regulate the phase separation behavior of proteins (Boeynaems, S., Alberti, S., Fawzi, NL, Mittag, T., Polymenidou, M., Rousseau, F., Schymkowitz, J., Shorter, J., Wolozin, B., Van Den Bosch, L., Tompa, P., & Fuxreiter, M., 2018. Protein Phase Separation: A New Phase in Cell Biology. Trends in cell biology, 28(6), 420-435), the optimal pH for eIDR-NOX to undergo phase separation is 7.5. Deviations from this value will inhibit the phase separation of eIDR-NOX. Phase separation of eIDR-NOX requires a low salt concentration environment (approximately 30 mM), and excessively high salt ion concentrations will interfere with its phase separation process.

[0126] To eliminate the direct impact of eIDR-NOX binding on enzyme activity, the reaction rate of eIDR-NOX with NOX in high-salt buffer was measured, and the results are as follows: Figure 15 As shown in the figure, combined with the experimental data, it can be seen that in the low-salt buffer system, the reaction rate of eIDR-NOX is significantly higher than that of NOX alone; while in the high-salt buffer system, the reaction rates of the two tend to be comparable. This result clearly confirms that eIDR does not change the inherent activity of enzyme molecules by directly binding to them, but rather regulates the enzyme activity level of NOX through phase separation.

[0127] Enzyme activity is regulated by multiple factors, including not only enzyme concentration but also substrate and cofactor concentrations. eIDR-driven phase separation increases the local concentration of NOX. To investigate whether eIDR-NOX can increase the local concentrations of substrate and cofactor, confocal microscopy was used to record the intrinsic fluorescence signals of NADH and FAD at 410-440 nm and 510-530 nm. To eliminate the influence of rapid reactions, the substrate NADH and the cofactor FAD were added to the system, respectively. Figure 16 As shown, eIDR-NOX can recruit NADH and FAD into LLPS droplets, thereby increasing their local concentrations and increasing the effective collision frequency between the enzyme and substrate.

[0128] 2. Increase the isoelectric point of eIDR to enhance eIDR-NOX enzyme activity. The eIDR-NOX phase-separated condensate can recruit NADH and FAD, thereby increasing their local concentrations. Could increasing the recruitment ratio further enhance their local concentrations, thus improving enzyme activity? Since electrostatic interactions are the primary driving force for substrate partitioning, and both the substrate NADH and the cofactor FAD carry two negative charges at pH 7.5, a high isoelectric point eIDR mutant (eIDR...) was designed. H (SEQ ID NO:2) to verify the above hypothesis. Specifically, as... Figure 17 As shown, the acidic amino acids in eIDR—aspartic acid (Asp, D) and glutamic acid (Glu, E)—were mutated into the basic amino acid—lysine (Lys, K).

[0129] The isoelectric point of the protein was predicted using the online tool ExPASy-ProtParam (https: / / web.expasy.org / protparam / ). The eIDR-NOX was 9.44, while the eIDR... H -NOX increased to 10.45, confirming that the mutation of acidic amino acids into basic amino acids can increase the isoelectric point of the protein. Using the online tool NovoPro (https: / / www.novopro.cn / ), the net charge of the protein was predicted. At pH 7.5, the net charge of eIDR-NOX was +6.3, while eIDR... H The net charge of NOx increased to +26.3.

[0130] Using overlap PCR technology, eIDR was constructed using primers eIDR-NOX-F1 (SEQ ID NO:43), H-NOX-R1 (SEQ ID NO:51), H-NOX-F2 (SEQ ID NO:52), and eIDR-NOX-R2 (SEQ ID NO:46). H The NOX vector was transformed into E. coli Rosetta (DE3) for expression, and the protein was purified using a Ni column. The protein concentration was diluted to 50 μM with low-salt buffer, and the sample was added to a coverslip in the chamber for observation under a confocal microscope. The results are as follows: Figure 18 As shown, in low-salt buffer, the mutant eIDR H It can still drive the fusion protein eIDR H -NOx undergoes phase separation.

[0131] To explore eIDR H To investigate whether eIDR-NOX could further increase the local concentrations of NADH and FAD, the intrinsic fluorescence signals of NADH and FAD at 410-440 nm and 510-530 nm were recorded using confocal microscopy under the same conditions as eIDR-NOX. The partition coefficients (Kp) of NADH and FAD were determined by calculating the ratio of fluorescence intensity inside to outside the phase-separated condensate.

[0132] For the substrate NADH, the measured eIDR-NOX and eIDR H The partition coefficients (Kp) for -NOX were 6.9 ± 0.8 and 11.3 ± 1.1, respectively, and these values ​​increased with increasing net charge of the fusion protein. Figure 19 This also confirms eIDR H -NOX further increases the local concentration of the substrate NADH. For the cofactor FAD, the measured eIDR-NOX and eIDR H The partition coefficients (Kp) for NOX were 1.5 ± 0.1 and 2.1 ± 0.1. Although the partition coefficients (Kp) for cofactors were lower than those for substrates, FAD also showed the same trend as NADH.

[0133] To explore eIDR H Can -NOX further enhance the enzyme activity of eIDR-NOX? Using the same conditions as eIDR-NOX, the absorbance change at 340 nm was continuously monitored using a spectrophotometer. Figure 20 The results showed that in low-salt buffer, the initial reaction rate of the 1 μM enzyme solution decreased from 6.1 μM / s with eIDR-NOX to that with eIDR-NOX. H The NOx concentration was 2.1 μM / s, which is inconsistent with the prior assumptions.

[0134] The underlying cause is speculated to be a change in droplet viscosity due to mutation, which in turn reduces the rate of material exchange between the inside and outside of the droplet. This leads to a decrease in the rate at which substrate enters the droplet and the rate at which product leaves the droplet, ultimately affecting the enzymatic reaction. To verify this hypothesis, primers eIDR-NOX-F1 (SEQ ID NO:43), eIDR-NOX-G-R1 (SEQ ID NO:53), eIDR-NOX-G-F2 (SEQ ID NO:54), and eIDR-NOX-G-R2 (SEQ ID NO:55) were used to construct eIDR-NOX-GFP and eIDR-NOX-G-R1 carrying the green fluorescent tag GFP. H -NOX-GFP expression vector, FRAP was performed.

[0135] Under a microscope, the fluorescence intensity of droplets treated with laser bleaching gradually recovers within a short period of time. Figure 21 (a) Through quantitative analysis of droplet fluorescence intensity, the recovery curve of fluorescence intensity over time was obtained ( Figure 21 (b and c in the text). It was found that droplets formed by the eIDR-NOX-GFP protein recovered to 50% in approximately 60 seconds, while eIDR... H The droplets formed by the -NOX-GFP protein recover to 50% in approximately 90 seconds, and these results indicate that eIDR H -NOX droplets have higher viscosity than eIDR-NOX droplets.

[0136] 3. Reduce eIDR H Increased droplet viscosity eIDR H -NOX enzyme activity Since increasing the isoelectric point leads to increased droplet viscosity and inhibits enzyme activity, we are considering modifying eIDR. H While maintaining high concentrations of substrate and cofactor, the droplet viscosity was reduced to achieve higher enzyme activity.

[0137] Sequence analysis revealed that the eIDR sequence contains 10 TPAP tetrapeptide repeats; as the number of TPAP repeats increases, the viscosity of the LLPS droplets decreases. This was observed in the high isoelectric point mutant eIDR. H Five additional TPAP repeats were added to reduce droplet viscosity, resulting in a mutant eIDR with a total of 15 TPAP repeats. H-15R (SEQ ID NO:3); Simultaneously, a mutant eIDR without TPAP duplication was set up. H-0R As an experimental control ( Figure 22 ).

[0138] eIDR H-0R-NOX and eIDR H-15R The NOX plasmid was transformed into Rosetta (DE3) for expression. The optimal expression strain was selected for further large-scale induction of expression. The protein was purified using Ni-NTA (Yonglian Biotechnology (Shanghai) Co., Ltd.). The protein concentration was diluted to 50 μM using low-salt buffer. The sample was dropped onto a coverslip in the chamber and observed under a confocal microscope. The results are shown in the figure. Figure 23 mutated eIDR H-0R and eIDR H-15R It can still drive the fusion protein eIDR H-0R -NOX and eIDR H-15R -NOX undergoes phase separation in low-salt buffer solutions.

[0139] Similarly, to verify the mutant eIDR H-0R -NOX and eIDR H-15R By altering the viscosity of NOX droplets, and using primers eIDR-NOX-F1 (SEQ ID NO:43), eIDR-NOX-G-R1 (SEQ ID NO:53), eIDR-NOX-G-F2 (SEQ ID NO:54), and eIDR-NOX-G-R2 (SEQ ID NO:55), an eIDR-NOX droplet carrying the green fluorescent tag GFP was constructed. H-0R -NOX-GFP and eIDR H-15R -NOX-GFP plasmid was used to perform a fluorescence bleaching recovery experiment on the phase-separated droplets formed by its protein.

[0140] Under a microscope, the fluorescence intensity of droplets treated with laser bleaching gradually recovers within a short period of time. Figure 24 (a) Through quantitative analysis of droplet fluorescence intensity, the recovery curve of fluorescence intensity over time was obtained ( Figure 24 b) and c) found that eIDR H-0R The fluorescence intensity of droplets formed by the -NOX-GFP protein recovers to 50% in approximately >100 s, while eIDR H-15R It takes approximately 14 seconds for the fluorescence intensity of droplets formed by the -NOX-GFP protein to recover to 50%. These results demonstrate that a reduction in the number of TPAP repeats leads to a decrease in eIDR. H-0R -NOX increases droplet viscosity, and the number of TPAP repetitions increases eIDR. H-15R -NOX reduces the droplet viscosity.

[0141] To investigate whether reducing droplet viscosity could increase NOX enzyme activity, eIDR was used. H Under the same conditions as NOX, the absorbance change at a wavelength of 340 nm was continuously monitored using a spectrophotometer. Figure 25(a) in the middle. Figure 25 Figure b shows that when the substrate concentration is 500 μM, the eIDR at 1 μM is... H-15R The initial reaction rate of -NOx was 18.0 μM / s, and the eIDR was... H-0R The initial reaction rate for -NOx is 1.2 μM / s. eIDR H-0R -NOX compared to eIDR H -NOX enzyme activity decreases, eIDR H-15R -NOX compared to eIDR H The activity of NOX enzymes increased, which is consistent with the hypothesis.

[0142] Based on the combined NOX enzyme activity assay results, at a substrate concentration of 500 μM, the initial rate of 1 μM NOX was 2.5 μM / s. After fusion with eIDR, the initial rate increased to 6.1 μM / s, representing a 2.42-fold increase in the enzymatic reaction rate. After multiple mutations of eIDR, eIDR... H-15R The isoelectric point increased to 10.45, which led to an increase in the net charge of the fusion protein to +26.4 at pH 7.5, increasing the electrostatic interaction with the substrate NADH and cofactor FAD. Furthermore, the increased number of TPAP repeats decreased the viscosity of the LLPS droplets of the fusion protein, thereby accelerating the exchange of substrate NADH and cofactor FAD between the phase-separated concentrated and dilute phases. Ultimately, this further increased the initial rate of the enzymatic reaction to 18.0 μM / s, representing a 7.2-fold increase in the enzymatic reaction rate. Figure 26 ).

[0143] Example 4. Simple and efficient protein purification based on phase separation eIDRs can undergo liquid-liquid phase separation in low-salt buffers. Upon increasing the ion concentration, the phase-separated droplets disappear and can be redissolved in the buffer to form a homogeneous solution. This property provides a theoretical basis for developing eIDRs as protein purification tags.

[0144] To verify the hypothesis and facilitate research, the GFP gene was constructed into the pET21b vector (Sangon Biotech (Shanghai) Co., Ltd.) using primers GFP-21b-F (SEQ ID NO:56) and GFP-21b-R (SEQ ID NO:57). Then, eIDR was constructed at the N-terminus of GFP using primers eIDR-GFP-F (SEQ ID NO:58) and eIDR-GFP-R (SEQ ID NO:59), resulting in the eIDR-GFP fusion expression vector, which was expressed using Rosetta (DE3). Figure 27The procedure shown is as follows for protein purification. First, the E. coli culture medium is centrifuged, and the bacterial pellet is resuspended in a low-salt buffer. After resuspending the bacteria, the bacterial resuspension is disrupted at 4°C. The eIDR-GFP protein enters the low-salt buffer and undergoes phase separation, forming aggregates. Centrifugation is then performed, and the eIDR-GFP protein aggregates, along with insoluble components, are centrifuged until a pellet (P1) is formed. The supernatant (S1) is discarded, and the pellet (P1) is resuspended in a high-salt buffer and centrifuged again at 4°C. As the salt concentration in the buffer increases, the phase separation of the eIDR-GFP protein is reversed. The pellet (P2) contains cell debris and insoluble host cell proteins, while the eIDR-GFP protein remains in the supernatant (S2).

[0145] During the purification process, the centrifuge tubes were photographed under both natural light (top) and ultraviolet light (bottom), as shown below. Figure 28 As shown, after the first centrifugation, the eIDR-GFP protein was mainly found in the precipitate (P1), while after the second centrifugation, the eIDR-GFP protein was mainly found in the supernatant (S2). This preliminarily proves that eIDR can purify proteins simply and efficiently based on phase separation.

[0146] During the purification process, samples of the precipitate and supernatant from both centrifugations were taken, and after equal volumes were loaded, SDS-PAGE analysis was performed. Figure 29 As shown in Figure a, a large amount of eIDR-GFP protein was found to be removed during the first centrifugation. This reflects that using low-salt buffer alone is insufficient to achieve phase separation of all eIDR-GFP protein, and other methods are needed to promote phase separation.

[0147] In in vitro phase separation experiments, 10% (v / v) of PEG 8000 is typically added as a molecular crowding agent to simulate the highly crowded physiological microenvironment within cells. Therefore, this study added PEG to the bacterial culture before lysis to promote phase separation of the target protein. PEG concentration gradients of 10%, 7%, and 3% were set up to observe the effect of different PEG concentrations on the purification of eIDR-GFP protein. Figure 29 As shown in b, as the PEG concentration increases, the amount of eIDR-GFP protein lost during the first centrifugation decreases. However, this also results in a higher amount of contaminating proteins in the final purified protein. A higher PEG concentration is not always better; a suitable PEG concentration needs to be selected for protein purification. In preliminary experiments, 7% PEG was a relatively suitable concentration, reducing both loss and contaminating protein content.

[0148] eIDR tags not only improve the solubility of target proteins but also serve as a novel purification tag, enabling the purification of target proteins through phase separation. Compared to other purification tags, eIDR tags only require centrifugation to purify proteins, eliminating the need for expensive resins and specific eluents. This reduces protein purification costs and simplifies the entire protein production process, making it suitable for large-scale protein purification and production.

[0149] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An eIDR polypeptide for promoting soluble expression of a target protein, enhancing enzyme catalytic activity, or for protein purification, characterized in that, The polypeptide is selected from the following (a) or (b): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO:1; (b) A derivative peptide having equivalent function formed by substitution, deletion or addition of one or more amino acid residues in the amino acid sequence shown in SEQ ID NO:

1.

2. The eIDR polypeptide according to claim 1, characterized in that, The derived peptide is selected from (i) or (ii): (i) The polypeptide shown in SEQ ID NO:1 was obtained by replacing all of the aspartic acid and glutamic acid in the polypeptide with lysine; (ii) The peptide obtained by adding at least one TPAP tetrapeptide repeat sequence to the polypeptide shown in SEQ ID NO:1 or the derivative peptide of (i).

3. The use of the eIDR polypeptide according to claim 1 or 2 in the following: 1) As a protein purification tag; 2) Improve the soluble expression of the target protein; 3) Enhance the catalytic activity of enzymes.

4. A fusion protein, characterized in that, The fusion protein comprises a target protein and an eIDR polypeptide as described in claim 1 or 2 fused to the target protein, wherein the target protein is an enzyme.

5. The fusion protein according to claim 4, characterized in that, The enzyme is selected from one of NADH oxidase, chitinase, TEV protease, tanninase, enterokinase, or human rhinovirus 3C protease.

6. The fusion protein according to claim 4 or 5, characterized in that, The eIDR polypeptide is fused to the N-terminus or C-terminus of the target protein.

7. A method for purifying a target protein using the eIDR polypeptide according to claim 1 or 2, characterized in that, The method includes the following steps: (1) Provide a cell lysis buffer containing the fusion protein; (2) The cell lysis buffer was placed in a low-salt buffer to induce the fusion protein to undergo liquid-liquid phase separation and form aggregates; (3) The aggregates formed in step (2) are separated and collected by centrifugation; (4) The collected aggregates are resuspended in a high-salt buffer solution to re-dissolve the aggregates, thereby obtaining a solution containing the purified target protein.

8. The method according to claim 7, characterized in that, Step (2) also includes adding a molecular crowding agent to the cell lysate to promote the liquid-liquid phase separation.

9. Use of the eIDR polypeptide of claim 1 or 2 or the fusion protein of any one of claims 4-6 in the preparation of compositions for improving enzyme catalytic activity.

10. A method for improving enzyme catalytic activity, characterized in that, The method includes placing the fusion protein of claim 4 in a reaction system capable of inducing liquid-liquid phase separation for an enzymatic reaction.

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