A method for producing and purifying recombinant proteins based on ion-responsive controllable phase separation

Abstract

The application discloses a recombinant protein production and purification method based on ion response controllable phase separation, constructs an engineering cell expressing a fusion protein, the fusion protein comprising a phase separation polypeptide tag and a target protein; the fusion protein is induced to be expressed; the engineering cell is collected, and the cell is crushed in a crushing liquid containing anions, so that the fusion protein is subjected to phase separation and enters an enrichment phase; a supernatant containing host impurities is removed, and a precipitate containing the fusion protein is collected; the concentration of the anions in the precipitate is reduced, so that the fusion protein is redissolved and returns to the supernatant; the supernatant is collected, and a crude extract of the target protein is obtained; wherein the phase separation polypeptide tag comprises at least one continuous five-amino-acid core sequence: X1-X2-X3-X4-X5. The corresponding phase separation peptide, fusion protein and expression vector are also disclosed. The method can reduce the traditional chromatography step, reduce the purification cost, improve the recovery rate and purity of the target protein, and is suitable for the large-scale production of recombinant proteins.

Description

Technical Field

[0001] This invention relates to a method for the production and purification of recombinant proteins based on ion-responsive controllable phase separation, belonging to the field of protein expression technology. Background Technology

[0002] Recombinant proteins, especially recombinant human proteins and humanized proteins, have wide applications in biomedicine, functional skincare products, tissue engineering, cell culture, enzyme engineering, and synthetic biology. With the increasing demand for industrial-scale recombinant protein production, obtaining high-purity target proteins with lower costs, higher recovery rates, and more stable processes has become a key issue in the large-scale production of recombinant proteins. Downstream separation and purification is typically one of the most costly and complex steps in recombinant protein production, accounting for 60%-70% of the total production cost. Therefore, developing low-cost, high-efficiency, and easily scalable recombinant protein purification processes is of great significance.

[0003] Currently, the production of recombinant proteins typically involves steps such as engineered cell construction, culture or fermentation, cell collection, cell disruption, separation of the target protein, purification, concentration, and medium exchange. After cell disruption, the target protein usually coexists with a large amount of host proteins, nucleic acids, cell debris, endotoxins, and other impurities in a complex lysis system. Therefore, subsequent separation and purification processes usually require multiple steps, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, precipitation, membrane filtration, or ultrafiltration.

[0004] Affinity chromatography, with its high selectivity, is one of the commonly used methods for purifying recombinant proteins. However, affinity chromatography packing materials are expensive, have limited throughput, and require sophisticated equipment and process scale-up. Furthermore, some affinity tags may affect the structure, solubility, bioactivity, or subsequent applications of the target protein. If tag removal is required, additional proteases and corresponding removal steps are needed, increasing process complexity, production costs, and potential sources of impurities.

[0005] In recent years, the technology of using phase separation tags to assist in the expression and separation of recombinant proteins has gradually attracted attention. This type of technology typically involves fusing short peptides or protein tags with specific interaction capabilities to the target protein, causing the fusion protein to aggregate, precipitate, or form an enriched phase under specific salt concentrations, pH, temperature, or other stimuli, thereby achieving the separation of the target protein from some impurities. However, existing phase separation purification systems still have certain shortcomings: some tags have large molecular weights, which may affect the expression, folding, or function of the target protein; some phase separation processes are irreversible or have low reconstitution efficiency, leading to a decrease in the recovery rate of the target protein; and some systems have strict requirements for buffer conditions, which is not conducive to stable scale-up production.

[0006] Therefore, developing a purification method capable of selectively capturing the target protein during the cell disruption stage and reversibly resolving the target protein under mild conditions has significant industrial application value. An ideal purification system should possess the following characteristics: First, a short tag sequence with minimal impact on target protein expression and subsequent applications; second, the ability to induce the target protein into the precipitation phase during disruption using simple ionic conditions, thus achieving initial separation from host impurities; third, a reversible sedimentation process, allowing the target protein to return to the supernatant by adjusting the ionic state within the system; and fourth, the ability to further refine, concentrate, and exchange the crude target protein extract through membrane filtration to obtain a high-purity refined target protein extract. Summary of the Invention

[0007] The purpose of this invention is to provide a method for the production and purification of recombinant proteins based on ion-responsive controllable phase separation.

[0008] The technical solution adopted in this invention is as follows: A method for the production and preliminary purification of recombinant proteins based on ion-responsive controllable phase separation, comprising the following steps: (1) Constructing engineered cells expressing a fusion protein, wherein the fusion protein comprises a phase-separated polypeptide tag and a target protein; (2) Culture the engineered cells and induce expression of the fusion protein; (3) Collect engineered cells and break them in a disruption solution containing anions to cause phase separation of the fusion protein and allow it to enter the enriched phase, where the enriched phase refers to precipitation. (4) Remove the supernatant containing host proteins and collect the precipitate containing the fusion protein; (5) Reduce the concentration of anions in the precipitate to allow the fusion protein to redissolve and return to the supernatant; (6) Collect the supernatant to obtain the crude extract of the target protein; The phase-separating polypeptide tag is an anion-responsive phase-separating polypeptide tag containing at least one continuous five-amino acid core sequence: X1-X2-X3-X4-X5; X1 is a basic amino acid residue; X2 and X3 are each an aliphatic hydrophobic amino acid residue; X4 and X5 are each an aromatic amino acid residue; The anion-responsive phase-separating polypeptide tag enables the fusion protein to undergo phase separation in the presence of the anion, and at least partially reverses the phase separation after reducing the effective concentration of free anion.

[0009] Preferably, the sequence of the anion-responsive phase-separated polypeptide tag is as shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.4.

[0010] Preferably, the structure of the fusion protein is a phase-separated polypeptide tag-target protein, a target protein-phase-separated polypeptide tag, a phase-separated polypeptide tag-linker peptide-target protein, or a target protein-linker peptide-phase-separated polypeptide tag, wherein the sequence of the linker peptide is as shown in any one of SEQ ID NO. 9, 11-13. More preferably, it is SEQ ID NO. 9, 12, or 13.

[0011] Preferably, one or more of the anions sulfate, phosphate, citrate, carbonate, and tartrate are used in step (5) to reduce the anion concentration in the precipitate by adding a phase reversal agent.

[0012] Preferably, the anion is sulfate, and the concentration of the anion in the breaking solution in step (3) is 0.05 mol / L-2 mol / L, more preferably 0.1 mol / L-1.5 mol / L.

[0013] Preferably, the phase separation reversal agent includes one or more of calcium salts, magnesium salts, zinc salts, barium salts, anion exchange materials, dialysate, and ultrafiltration fluid systems.

[0014] Preferably, the phase separation reversal agent comprises a calcium salt, wherein the calcium salt is one or more of calcium chloride, calcium acetate, calcium lactate, and calcium gluconate.

[0015] Preferably, the final concentration of calcium ions in the phase separation reversal agent is 1 mmol / L-1 mol / L, more preferably 5 mmol / L-200 mmol / L.

[0016] Preferably, the method further includes step (7): refining the crude extract of the target protein to obtain the refined target protein.

[0017] Preferably, the refining in step (7) refers to passing the crude extract of the target protein through a high molecular weight filter membrane to remove impurities with a molecular weight greater than that of the target protein, and then concentrating the target protein using a low molecular weight filter membrane to obtain the refined extract of the target protein. The high molecular weight filter membrane is a filter membrane that retains a molecular weight greater than that of the target protein, with the molecular weight cutoff selected in the range of 5-1000 kDa. The low molecular weight filter membrane is a filter membrane that retains a molecular weight less than that of the target protein, with the molecular weight cutoff selected in the range of 1-30 kDa.

[0018] The present invention also discloses an ion-responsive controllable phase separation peptide, the amino acid sequence of which is shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.4.

[0019] And the application of the aforementioned phase-separated peptides in the production of recombinant proteins based on ion-responsive controllable phase separation.

[0020] The present invention also discloses a fusion protein comprising an ion-responsive controllable phase-separation peptide and a target protein, wherein the amino acid sequence of the phase-separation peptide is as shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.4, and the target protein is located at the N-terminus or C-terminus of the phase-separation peptide.

[0021] Preferably, it further comprises a linker peptide, and the structure of the fusion protein is: phase-separated polypeptide tag-linker peptide-target protein or target protein-linker peptide-phase-separated polypeptide tag.

[0022] Preferably, the amino acid sequence of the linker peptide is as shown in any one of SEQ ID NO. 9, 11-13.

[0023] The present invention also discloses an expression vector for expressing the above-mentioned fusion protein.

[0024] The present invention also discloses a host cell that expresses the above-described fusion protein.

[0025] This invention proposes a reversible ion-responsive phase separation purification method based on anion-responsive peptide tags. The method utilizes the responsive characteristics of peptide tags to anionic environments, causing the target protein fused to the tag to undergo controlled precipitation in the presence of anions such as sulfate. During cell disruption, the protein accumulates in the precipitate phase, while most host impurities remain in the supernatant. Subsequently, by adding a phase reversal agent or adjusting the effective concentration of free anions, the target protein is redissolved and returned to the supernatant, yielding a crude extract. Large molecular weight impurities are then removed using a high molecular weight filter membrane, and the target protein is concentrated using a low molecular weight filter membrane, ultimately yielding a refined extract. This method reduces traditional chromatography steps, lowers purification costs, and improves the recovery rate and purity of the target protein, making it suitable for large-scale production of recombinant proteins.

[0026] Compared with the prior art, the present invention has the following beneficial effects: First, this invention utilizes the responsive characteristics of polypeptide tags to anions, enabling the target protein to undergo controlled sedimentation directly during cell disruption, thereby achieving early separation of the target protein from host impurities.

[0027] Secondly, the phase separation process of the present invention is reversible. By adding a phase separation reversal agent or adjusting the effective concentration of free anions in the system, the target protein can be redissolved and returned to the supernatant, reducing the problem of the target protein being difficult to redissolve or having a low recovery rate in traditional precipitation methods.

[0028] Third, the present invention can reduce or replace some chromatographic purification steps, reduce the cost of packing materials, buffers, equipment and operation, and facilitate the large-scale production of recombinant proteins.

[0029] Fourth, the polypeptide tag sequence used in this invention is short, which has little impact on the expression, folding and subsequent application of the target protein, making it suitable for use as a controllable phase separation tag. Attached Figure Description

[0030] Figure 1 Flowchart of the efficient recombinant protein extraction method of the present invention.

[0031] Figure 2 Colony PCR agarose gel electrophoresis results. 1: DL2000 Marker; 2: X1; 3: X2; 4: X3; 5: X4.

[0032] Figure 3 SDS-PAGE results of X1-X4 recombinant protein extraction. 1: Marker; 2: X1 lysate supernatant; 3: X1 lysate precipitate; 4: X1 extract supernatant; 5: X1 extract precipitate; 6: X2 lysate supernatant; 7: X2 lysate precipitate; 8: X2 extract supernatant; 9: X2 extract precipitate; 10: 0.5 mg / mL BSA; 11: X3 lysate supernatant; 12: X3 lysate precipitate; 13: X3 extract supernatant; 14: X3 extract precipitate; 15: Marker; 16: 0.5 mg / mL BSA.

[0033] 17: Supernatant of X4 lysate; 18: Precipitate of X4 lysate; 19: Supernatant of X4 extract; 20: Precipitate of X4 extract; 21: Marker; 22: 0.5 mg / mL BSA.

[0034] Figure 4 SDS-PAGE results of recombinant protein lysis. 1: Lysis supernatant (1M sodium citrate); 2: Lysis supernatant (1M disodium hydrogen phosphate); 3: Lysis supernatant (1M sodium carbonate); 4: Lysis supernatant (1M sodium dihydrogen phosphate); 5: Lysis supernatant (1M sodium sulfate); 6: 0.5 mg / mL BSA; 7: Marker; 8: Lysis precipitate (1M sodium citrate); 9: Lysis precipitate (1M disodium hydrogen phosphate); 10: Lysis precipitate (1M sodium carbonate); 11: Lysis precipitate (1M sodium dihydrogen phosphate); 12: Lysis precipitate (1M sodium sulfate).

[0035] Figure 5 SDS-PAGE results of recombinant protein extraction. 1: Extract supernatant (1M sodium citrate); 2: Extract supernatant (1M disodium hydrogen phosphate); 3: Extract supernatant (1M sodium carbonate); 4: Extract supernatant (1M sodium dihydrogen phosphate); 5: Extract supernatant (1M sodium sulfate); 6: 0.5 mg / mL BSA; 7: Marker; 8: Extract precipitate (1M sodium citrate); 9: Extract precipitate (1M disodium hydrogen phosphate); 10: Extract precipitate (1M sodium carbonate); 11: Extract precipitate (1M sodium dihydrogen phosphate); 12: Extract precipitate (1M sodium sulfate).

[0036] Figure 6 SDS-PAGE results of recombinant protein extraction using different reversal systems. 1: Marker; 2: Plutonium lysate (1M sodium sulfate); 3: Extract supernatant (barium chloride reversal solution); 4: Extract supernatant (calcium chloride reversal solution); 5: Extract supernatant (anion exchange material reversal system); 6: 0.5 mg / mL BSA; Figure 7 SDS-PAGE results of recombinant proteins with different expression structures. 1: Marker; 2: lysate (Y1); 3: supernatant (Y1); 4: lysate (Y2); 5: supernatant (Y2); 6: lysate (Y3); 7: supernatant (Y3); 8: 0.5 mg / mL BSA.

[0037] Figure 8 SDS-PAGE results of recombinant protein extraction mediated by different linkers. 1: K1 lysis buffer supernatant; 2: K1 lysis buffer precipitate; 3: K1 extract supernatant; 4: K1 extract precipitate; 5: K2 lysis buffer supernatant; 6: K2 lysis buffer precipitate; 7: K2 extract supernatant; 8: K2 extract precipitate; 9: K3 lysis buffer supernatant; 10: K3 lysis buffer precipitate; 11: K3 extract supernatant; 12: K3 extract precipitate; 13: Marker; 14: 0.5 mg / mL BSA; 15: Marker; 16: X4 lysis buffer supernatant; 17: X4 lysis buffer precipitate; 18: X4 extract supernatant; 19: X4 extract precipitate; 20: 0.5 mg / mL BSA.

[0038] Figure 9 SDS-PAGE results of recombinant protein extraction. 1: 0.5 mg / mL; 2: Marker; 3: crude extract stock solution; 4: high molecular weight membrane concentrate; 5: high molecular weight membrane filtrate; 6: low molecular weight membrane concentrate (protein extract) diluted 5 times; 7: low molecular weight membrane concentrate filtrate; Figure 10 Image of SDS-PAGE results for recombinant protein (COL-III). 1: Marker; 2: Supernatant of lysate; 3: Precipitate of lysate; 4: Supernatant of extract; 5: Precipitate of extract; 6: 0.5 mg / mL BSA. Detailed Implementation

[0039] The present invention is further illustrated below by way of examples, but is not intended to limit the invention. Specific materials used in the embodiments of the present invention and their sources are provided below. However, it should be understood that these are merely exemplary and not intended to limit the invention. Materials of the same or similar type, model, quality, properties, or function as the reagents and instruments described below can be used to implement the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, etc., used in the following examples are commercially available.

[0040] The principle of this invention is as follows Figure 1 As shown.

[0041] Example 1: Construction of expression vector and its engineered strain (1) Construction of recombinant expression vector Taking the construction of recombinant expression vectors for the fusion proteins S1-AP2-FLG, S2-AP2-FLG, S3-AP2-FLG, and S4-AP2-FLG as an example, S1-S4 are reversibly ion-responsive phase-separated short peptides with amino acid sequences of SEQ ID No. 1-4 and nucleotide sequences of SEQ ID No. 5-8; AP2 is a rigid linker peptide with amino acid sequence of SEQ ID No. 9 and nucleotide sequence of SEQ ID No. 10; and FLG is the target protein (its sequence is shown in ProFLG1 in CN118344460A). Taking S1 as an example, the structure of the fusion protein is: S1-AP2-FLG. S1 is located at the N-terminus of the fusion protein and is used to promote the controlled phase separation of the fusion protein and its entry into the precipitation phase in the presence of anions such as sulfate. AP2 is located between S1 and FLG and is used to spatially separate S1 and FLG, so that the short peptide of S1 is fully exposed and the spatial obstruction of the phase separation function of FLG on S1 is reduced. FLG is the target protein to be expressed and purified.

[0042] Based on the amino acid sequences of the S1-S4 short peptides, the AP2 rigid linker peptide, and the FLG target protein, nucleotide sequences encoding S1-AP2-FLG, S2-AP2-FLG, S3-AP2-FLG, and S4-AP2-FLG fusion proteins were designed and synthesized. Preferably, the nucleotide sequences were codon-optimized according to the codon preference of the host bacteria to improve the expression efficiency of the fusion proteins in the host bacteria. The designed encoding DNAs of S1-AP2-FLG, S2-AP2-FLG, S3-AP2-FLG, and S4-AP2-FLG were sent for synthesis, and PCR amplification was performed using the biosynthesized gene fragments as templates. The AP2-FLG encoding gene and the pET30a expression vector backbone were ligated using Gibson assembly to obtain the corresponding expression plasmids.

[0043] Table 1 PCR Primers

[0044] Table 2 PCR amplification system

[0045] After preparing the PCR system, mix well and centrifuge. The PCR amplification conditions are as follows: Stage 1: 98℃ pre-denaturation for 30 s; Stage 2: 98℃ denaturation for 10 s, 50-72℃ annealing for 30 s, 72℃ extension for 30 s / kb, 32 cycles; Stage 3: 72℃ final extension for 2 min. The vectors and gene fragments were recovered using a universal DNA purification kit (Tiangen Biotech Co., Ltd.), following the instructions in the product manual.

[0046] Table 3 Gibson Assembly System

[0047] After mixing the above components on ice, place them in a 37°C heat bath for 60 minutes. Store the resulting ligation product on ice or at -20°C for subsequent competent cell transformation.

[0048] The ligation product was transformed into the host bacterium *E. coli* DH5α using a heat shock method, plated onto LB agar plates, and incubated overnight at 37°C. Single colonies were picked for colony PCR, and the results are as follows: Figure 2 As shown in the figure. Positive clones with confirmed sequencing results were transferred to LB liquid medium and cultured overnight in a shaker at 37°C and 220 rpm. Plasmids were then extracted using a rapid plasmid extraction kit. Other plasmids were constructed using the same method, yielding plasmids S1-AP2-FLG, S2-AP2-FLG, S3-AP2-FLG, and S4-AP2-FLG.

[0049] (2) Construction of engineered bacteria The recombinant expression plasmid obtained above was introduced into *E. coli* BL21(DE3) competent cells via chemical transformation, and positive engineered strains were obtained by screening with antibiotic plates. The specific procedure was as follows: 4 μL of the recombinant expression plasmid was added to 100 μL of *E. coli* BL21(DE3) competent cells, gently mixed, and incubated on ice for 30 min; then, the mixture was heat-shocked in a 42℃ water bath for 90 s, and quickly transferred to ice for 2 min; 900 μL of antibiotic-free LB liquid medium (containing 10 g / L peptone, 5 g / L yeast extract, and 10 g / L sodium chloride) was added to the heat-shocked mixture, and the mixture was then incubated at 37℃ and 220 rpm with shaking for 1 h; 200 μL of the recovered bacterial culture was evenly spread onto LB solid medium plates containing kanamycin (containing 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L sodium chloride). The plasmids S1-AP2-FLG and S2-AP2-FLG were prepared with 100 μg / mL agar and 100 μg / mL kanamycin. The plates were then inverted and incubated at 37°C for approximately 16 h. After clearly visible single colonies formed on the plates, positive clones were picked to obtain the corresponding recombinant engineered strains. The recombinant engineered strains corresponding to plasmids S1-AP2-FLG, S2-AP2-FLG, S3-AP2-FLG, and S4-AP2-FLG were named X1, X2, X3, and X4, respectively.

[0050] Example 2: Induction of engineered bacteria expression and extraction of recombinant proteins containing polypeptide tags with different phase separations Single colonies of the engineered strains X1, X2, X3, and X4 obtained in Example 1 were picked and inoculated into LB liquid medium containing kanamycin. The cultures were incubated overnight at 37°C and 220 rpm with shaking to obtain primary seed culture. The primary seed culture was then transferred at a 1% inoculation rate to fresh LB liquid medium containing kanamycin and cultured at 37°C and 220 rpm. When the OD600 of the bacterial culture reached 0.6-0.8, IPTG was added for induction, bringing the final IPTG concentration to 0.5 mmol / L. The culture temperature was adjusted to 18°C, and induction culture was continued for 16 h to induce the expression of the fusion proteins S1-AP2-FLG, S2-AP2-FLG, S3-AP2-FLG, and S4-AP2-FLG by the engineered strains X1, X2, X3, and X4, respectively.

[0051] After induction culture, the fermentation broth of each group was centrifuged at 4℃ and 8000 rpm for 10 min, the supernatant was discarded and the bacterial cells were collected. The bacterial cells were resuspended in physiological saline, washed, and centrifuged again, repeating the washing process 2-3 times to remove residual components of the culture medium. To ensure comparability between different engineered strains, the washed bacterial cells were sampled according to the same wet cell mass, or resuspended in buffer and the bacterial concentration was uniformly adjusted to OD. 600 =20.

[0052] The bacterial cells of each group were resuspended in an anion-induced disruption buffer containing 1 mol / L sodium sulfate and then subjected to ultrasonic disruption under ice bath conditions or high-pressure homogenization. After disruption, the disruption buffer was centrifuged at 12,000 rpm for 20 min, and the supernatant and precipitate were collected separately. The ability of different candidate peptide tags to mediate phase separation and sedimentation of fusion proteins under sulfate conditions was evaluated by comparing the distribution of the target protein in the supernatant and precipitate.

[0053] For samples that undergo phase separation and accumulate in the precipitate phase under sulfate ion conditions, the resulting precipitate is resuspended thoroughly in a phase reversal buffer containing calcium chloride (0.04 mol / L) to obtain the extract. The phase reversal buffer is Tris-HCl buffer or PBS buffer with a pH of 6.5-8.0. After resuspending, the sample is centrifuged at 12000 rpm for 10 min to remove calcium sulfate precipitate and other insoluble impurities. The supernatant is collected to obtain the crude extract of the target protein. SDS-PAGE analysis is performed on the supernatant, precipitate, and extract supernatant of samples X1, X2, X3, and X4, respectively. The results are shown in the figure. Figure 3 The target protein bands were quantitatively analyzed using grayscale analysis software and BSA standards.

[0054] Table 4. Results of target protein extraction mediated by different candidate peptide tags

[0055] Depend on Figure 3 As shown in Table 4, under the same expression, fragmentation, and ion treatment conditions, the ability of different candidate peptide tags to mediate sulfate-responsive phase separation of fusion proteins differed significantly. The X1 fusion protein was mainly enriched in the fragmentation precipitate under 1 mol / L sodium sulfate conditions, and could re-enter the supernatant of the extraction solution after calcium chloride reversal treatment, with a target protein extraction yield of 85.1 mg / L. The X3 fusion protein also underwent phase separation in the presence of sulfate and could be reconstituted and recovered by calcium chloride treatment, with a target protein extraction yield of 69.5 mg / L. The X4 target protein extraction yield was 98.5 mg / L. Among them, S4 mediated sulfate-responsive phase separation and reversal reconstitution showed the best results.

[0056] The peptide tags X1, X3, and X4 each contain at least one continuous five-amino acid core sequence X1-X2-X3-X4-X5, where X1 is a basic amino acid residue, X2 and X3 are each independently aliphatic hydrophobic amino acid residues, and X4 and X5 are each independently aromatic amino acid residues. Experimental results show that S1, S3, and S4, designed according to the above site composition, can promote the transformation of the fusion protein from a dispersed state to an enriched state in the presence of sulfate, and can at least partially reverse the phase separation after calcium chloride reduces the effective concentration of free sulfate in the system. This result verifies that the five-amino acid core structure of "basic amino acid-aliphatic hydrophobic amino acid-aliphatic hydrophobic amino acid-aromatic amino acid-aromatic amino acid" can serve as an effective design principle for screening anion-responsive phase-separating peptide tags. In contrast, no significant enrichment of the target protein into the precipitate phase was observed after the X2 fusion protein was disrupted; the target protein was mainly distributed in the supernatant of the disruption liquid, indicating that S2 did not exhibit observable sulfate-induced phase separation behavior under the conditions of this experiment. Therefore, the amount of target protein extracted based on the reverse recovery of the precipitate phase was not calculated. X2 can be used as a comparative example of candidate sequences to illustrate how the amino acid sequence composition affects the anion-responsive phase separation performance of peptide tags.

[0057] The above results indicate that the polypeptide tags contained in X1, X3, and X4 can all serve as specific implementation methods for anion-responsive phase separation of polypeptide tag P, mediating the entry of the fusion protein into the enrichment phase in the presence of sulfate, and partially reversing phase separation after reducing the effective concentration of free sulfate, with high purity. Furthermore, the recovery rate can be improved through multiple extraction cycles; among them, X4 exhibits the best overall extraction effect. Therefore, engineered strain X4 is preferred for further verification in subsequent examples.

[0058] Example 3: Extraction of recombinant proteins under different anions Selected engineered bacterial strain X4 was used for induction culture. After induction culture, the fermentation broth was centrifuged at 4℃ and 8000 rpm for 20 min, the supernatant was discarded, and the bacterial cells were collected. The collected bacterial cells were resuspended and washed with physiological saline, then centrifuged again to collect the bacterial cells. This washing process was repeated 2-3 times to remove residual components of the culture medium. The washed bacterial cells were divided into 5 equal groups, and each group was resuspended with a different anion induction lysis buffer. The final concentration of the anion salt in each group was 1 mol / L. The anion induction lysis buffer settings for each group are as follows: Group 1: 1 mol / L sodium citrate; Group 2: 1 mol / L disodium hydrogen phosphate; Group 3: 1 mol / L sodium carbonate; Group 4: 1 mol / L sodium dihydrogen phosphate; Group 5: 1 mol / L sodium sulfate.

[0059] The resuspended bacterial cells were subjected to ultrasonic disruption under ice bath conditions or high-pressure homogenization. After disruption, the lysate was centrifuged at 12,000 rpm for 20 min, and the supernatant and precipitate were collected separately. The supernatant was used as the supernatant sample of the extract under the corresponding anion condition, and the precipitate was used as the precipitate sample of the extract under the corresponding anion condition. By comparing the distribution of the fusion protein in the supernatant and precipitate under different anion conditions, the induction effect of different anions on the S4-mediated phase separation and sedimentation of the fusion protein was evaluated.

[0060] To further evaluate the recovery efficiency of the target protein under different anion conditions, the precipitates from each group were resuspended in a phase reversal buffer. The phase reversal buffer was preferably a Tris-HCl buffer or PBS buffer containing calcium ions, with a pH of 6.5-8.0. Upon addition of the phase reversal buffer, calcium ions react with anions in the system to form anion-capturing products, such as calcium sulfate, calcium phosphate, calcium citrate, or calcium carbonate precipitates or low-soluble products. This reduces the effective concentration of free anions in the system, disrupts the S4-mediated phase separation, and allows the fusion protein X4 to redissolve and return to the supernatant.

[0061] After resuspending the precipitates in the phase-reversal buffer, each precipitate was centrifuged at 12,000 rpm for 10 min to remove anion-captured products and other insoluble impurities. The supernatant was collected to obtain the crude extract of the target protein for each group. SDS-PAGE analysis was performed on the crude extracts and reconstituted precipitates of each group, and the target protein bands were quantitatively analyzed using grayscale analysis software to calculate the extraction yield and recovery of the target protein under different anion conditions.

[0062] Table 5 Target Protein Yield

[0063] Depend on Figure 4 As shown in Tables 5 and 5, different anion inducers exhibited significant differences in their effects on phase separation, sedimentation, and reconstitution recovery of the X4 fusion protein. Under the same concentration conditions, when 1 mol / L sodium sulfate was used as the anion inducer, the target protein extraction yield reached 107.5 mg / L, significantly higher than that of the disodium hydrogen phosphate group, sodium dihydrogen phosphate group, sodium citrate group, and sodium carbonate group.

[0064] Specifically, the extraction yield of the target protein in the sodium sulfate group was approximately 2.42 times that of the disodium hydrogen phosphate group, 2.12 times that of the sodium dihydrogen phosphate group, 1.79 times that of the sodium citrate group, and 3.21 times that of the sodium carbonate group. This result indicates that, under the conditions described in this study, sulfate ions can more effectively induce S1-mediated controlled phase separation of the fusion protein, allowing the target protein to be more fully enriched in the precipitate phase during cell disruption. Simultaneously, after treatment with calcium chloride, sulfate ions can react with calcium ions to form calcium sulfate precipitate, thereby reducing the effective concentration of free sulfate ions in the system and allowing the fusion protein to be returned to the supernatant, achieving a high-efficiency reconstitution and recovery.

[0065] Therefore, sodium sulfate exhibits the best extraction effect of the target protein when used as an anion inducer, indicating that sulfate is more suitable for the S4-mediated reversible ion-responsive phase separation and purification system described in this invention. In subsequent embodiments, sodium sulfate is preferably used as the anion inducer.

[0066] Example 4: Extraction of recombinant proteins under different phase separation reversal solutions To verify that reducing the effective concentration of free sulfate in the system through different methods can reverse anion-responsive phase separation mediated by peptide tags, this embodiment uses calcium chloride reversal solution, barium chloride reversal solution, and magnesium chloride reversal solution to reconstitute and extract the sulfate-induced X4 fusion protein precipitate.

[0067] The engineered strain X4 was induced to express its culture according to the method described in Example 2. After the induction culture was completed, the bacterial cells were collected by centrifugation, washed 2-3 times with physiological saline, and samples from each group of bacterial cells were taken according to the same wet cell mass, or resuspended in buffer and uniformly adjusted to OD600=20.

[0068] The bacterial cells were resuspended in an anion-induced disruption buffer containing 1 mol / L sodium sulfate and then sonicated under ice bath conditions, or homogenized under high pressure. After disruption, the disruption buffer was centrifuged at 12,000 rpm for 20 min, the supernatant containing host proteins was discarded, and the precipitate containing the X1 fusion protein was collected.

[0069] After thoroughly mixing the precipitate, it was divided into three equal groups, and each group was treated using the phase separation reversal system described below.

[0070] Group 1: Barium chloride reversal solution Resuspend the first precipitate by adding barium chloride reversal solution. The barium chloride reversal solution is a 20 mmol / L Tris-HCl buffer solution containing 0.01–0.50 mol / L barium chloride, with a pH of 7.5; preferably, the barium chloride concentration is 0.04 mol / L.

[0071] After thorough resuspension, allow the mixture to stand at room temperature or 4°C for 10–30 min to allow barium ions to combine with sulfate ions in the system to form insoluble barium sulfate precipitate, thereby reducing the effective concentration of free sulfate ions in the system, disrupting the S4-mediated phase separation state, and allowing the X1 fusion protein to at least partially redissolve and enter the liquid phase.

[0072] Subsequently, the sample was centrifuged at 12,000 rpm for 10 min to remove barium sulfate precipitate and other insoluble impurities. The supernatant was collected to obtain the first group of crude extracts of the target protein.

[0073] Group 2: Magnesium chloride reversal solution Resuspend the second precipitate by adding magnesium chloride reversal buffer. The magnesium chloride reversal buffer is a 20 mmol / L Tris-HCl buffer containing 0.01–0.50 mol / L magnesium chloride, with a pH of 7.5; alternatively, a PBS buffer containing the same concentration of magnesium chloride can be used. Preferably, the magnesium chloride concentration is 0.04 mol / L.

[0074] After thorough resuspension, allow the mixture to stand at room temperature or 4°C for 10–30 min to allow magnesium ions to combine with sulfate ions in the system to form magnesium sulfate precipitate, thereby reducing the effective concentration of free sulfate ions, disrupting the S4-mediated phase separation state, and allowing the X1 fusion protein to redissolve and return to the liquid phase.

[0075] Subsequently, the sample was centrifuged at 12,000 rpm for 10 min to remove magnesium sulfate precipitate and other insoluble impurities. The supernatant was collected to obtain the second group of crude extracts of the target protein.

[0076] Group 3: Calcium chloride reversal solution Resuspend the third precipitate by adding calcium chloride reversal buffer. The calcium chloride reversal buffer is a 20 mmol / L Tris-HCl buffer containing 0.01–0.50 mol / L calcium chloride, with a pH of 7.5; alternatively, a PBS buffer containing the same concentration of calcium chloride can be used. Preferably, the calcium chloride concentration is 0.04 mol / L.

[0077] After thorough resuspension, allow the mixture to stand at room temperature or 4°C for 10–30 min to allow calcium ions to combine with sulfate ions in the system to form calcium sulfate precipitate, thereby reducing the effective concentration of free sulfate ions, disrupting the S4-mediated phase separation state, and allowing the X1 fusion protein to redissolve and return to the liquid phase.

[0078] Subsequently, the sample was centrifuged at 12,000 rpm for 10 min to remove calcium sulfate precipitate and other insoluble impurities. The supernatant was collected to obtain the crude extract of the target protein in group 3.

[0079] The supernatant and precipitate after phase separation and reversal treatment of each group were analyzed by SDS-PAGE, and the target protein band was quantified by grayscale analysis software combined with BSA standard. The extraction amount, recovery rate and purity of crude extract of target protein were calculated.

[0080] Table 6. Results of target protein extraction under different phase separation reversal systems

[0081] Depend on Figure 6 As shown in Table 6, phase reversal is not limited to calcium ion treatment. Both calcium chloride and barium chloride can reduce the effective concentration of free sulfate in the system by forming poorly soluble products with sulfate ions; anion exchange materials can achieve the same purpose by adsorbing and removing sulfate ions. All of the above treatment methods can be used to disrupt the anion-responsive phase separation peptide tag-mediated phase separation state and promote the reentry of fusion proteins from the precipitate phase into the liquid phase.

[0082] This result demonstrates that the core of the phase separation reversal described in this invention lies in reducing the effective concentration of free anions in the system, and is not limited to a specific reversal agent or a single chemical reaction. Therefore, in addition to calcium salts, other substances or treatment systems capable of capturing, precipitating, adsorbing, exchanging, or removing free anions in the system can also be used for the phase separation reversal and reconstitution recovery of the fusion protein described in this invention.

[0083] Taking into account the target protein recovery effect, reagent safety, difficulty of subsequent desalting, and convenience of industrial operation, calcium chloride reversal solution can be regarded as the preferred phase separation reversal system; barium chloride reversal solution and other reversal systems can be regarded as other optional implementation methods.

[0084] Example 5: Extraction of recombinant proteins under different expression frameworks To verify the effect of the position of the phase-separation polypeptide tag in the fusion protein and the setting of the linker peptide on the phase separation effect, this embodiment used S4 as an anion-responsive phase-separation polypeptide tag, FLG as the target protein, and AP2 as the linker peptide to construct three expression frameworks: FLG-S4, S4-FLG, and FLG-AP2-S4, respectively. The corresponding engineered strains were named Y1, Y2, and Y3, respectively. The amino acid sequence of S4 is shown in SEQ ID NO.4, and the amino acid sequence of AP2 is shown in SEQ ID NO.9.

[0085] Recombinant expression plasmids encoding the above-mentioned fusion proteins were constructed according to the method described in Example 1 and transformed into Escherichia coli BL21(DE3). Single colonies of positive engineered strains were picked and inoculated into LB medium containing kanamycin and cultured at 37°C and 220 rpm until the OD600 reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mmol / L and induced to culture at 18°C ​​for 16 h.

[0086] After induction, bacterial cells were collected by centrifugation at 4℃ and 8000 rpm. After washing with physiological saline, the bacterial concentration of different engineered strains was uniformly adjusted to OD600=20. The bacterial cells were resuspended in lysis buffer containing 1 mol / L sodium sulfate and sonicated under ice bath conditions. The lysis buffer was centrifuged at 12000 rpm for 20 min, and the supernatant and precipitate were collected to evaluate the phase separation of fusion proteins under different expression frameworks.

[0087] Each group of fusion protein precipitates was resuspended in a phase reversal buffer containing 0.04 mol / L calcium chloride and allowed to stand at room temperature or 4°C for 10–30 min to allow calcium ions to bind with sulfate ions in the system, reducing the effective concentration of free sulfate ions and disrupting the S4-mediated phase separation. The precipitates were then centrifuged at 12000 rpm for 10 min to remove calcium sulfate precipitates and other insoluble impurities. The supernatant was collected to obtain the corresponding crude extract of the target protein.

[0088] SDS-PAGE analysis was performed on the precipitate and supernatant of the lysate from Y1, Y2, and Y3, respectively, and grayscale analysis was used to evaluate the phase separation, reconstitution, and extraction effects of the target proteins under different expression frameworks.

[0089] like Figure 7 The results show that S4, whether located at the N-terminus or C-terminus of the target protein, can mediate phase separation of the fusion protein in the presence of sulfate. Even after adding the AP2 linker peptide between the target protein and S4, the anion-responsive phase separation function of S4 is still maintained. These results demonstrate that the anion-responsive phase-separation polypeptide tag described in this invention can be located at either the N-terminus or C-terminus of the target protein, and can be directly linked to the target protein or linked via a linker peptide.

[0090] Example 6: Extraction of recombinant proteins mediated by different linkers To verify the effect of the linker peptide between the phase separation polypeptide tag and the target protein on the phase separation effect, this embodiment used S4 as the anion-responsive phase separation polypeptide tag, FLG as the target protein, and AP2, L1, L2, and L3 as linker peptides to construct three expression frameworks: S4-AP2-FLG, S4-L1-FLG, S4-L2-FLG, and S4-L2-FLG, respectively. The corresponding engineered strains were named X4, K1, K2, and K3, respectively. The amino acid sequence of S4 is shown in SEQ ID NO.4, the amino acid sequence of AP2 is shown in SEQ ID NO.9, the amino acid sequences of L1-L3 are shown in SEQ ID NO.11-13, and the nucleotide sequences are shown in SEQ ID NO.14-16.

[0091] Recombinant expression plasmids encoding the above-mentioned fusion proteins were constructed according to the method described in Example 1 and transformed into Escherichia coli BL21(DE3). Single colonies of positive engineered strains were picked and inoculated into LB medium containing kanamycin and cultured at 37°C and 220 rpm until the OD600 reached 0.6–0.8. IPTG was added to a final concentration of 0.5 mmol / L and induced to culture at 18°C ​​for 16 h.

[0092] After induction, bacterial cells were collected by centrifugation at 4℃ and 8000 rpm. After washing with physiological saline, the bacterial concentration of different engineered strains was uniformly adjusted to OD600=20. The bacterial cells were resuspended in lysis buffer containing 1 mol / L sodium sulfate and sonicated under ice bath conditions. The lysis buffer was centrifuged at 12000 rpm for 20 min, and the supernatant and precipitate were collected to evaluate the phase separation of fusion proteins under different expression frameworks.

[0093] Each group of fusion protein precipitates was resuspended in a phase reversal buffer containing 0.04 mol / L calcium chloride and allowed to stand at room temperature or 4°C for 10–30 min to allow calcium ions to bind with sulfate ions in the system, reducing the effective concentration of free sulfate ions and disrupting the S4-mediated phase separation. The precipitates were then centrifuged at 12000 rpm for 10 min to remove calcium sulfate precipitates and other insoluble impurities. The supernatant was collected to obtain the corresponding crude extract of the target protein.

[0094] SDS-PAGE analysis was performed on the supernatant, precipitate, extract supernatant, and extract precipitate of X4, K1, K2, and K3, respectively. The phase separation, reconstitution, and extraction effects of the target proteins under different expression frameworks were evaluated by combining grayscale analysis.

[0095] Table 7. Target protein yield mediated by different linkers

[0096] Depend on Figure 8 As shown in Table 7, the fusion proteins all underwent phase separation and accumulated in the precipitate phase in the presence of sulfate after introducing different linker peptides AP2, L1, L2, and L3 between S4 and FLG, respectively. After reversal treatment with calcium chloride, the precipitate could be returned to the supernatant. Among them, the L3 linker peptide yielded the highest target protein extraction amount, reaching 108.2 mg / L; AP2 was second, at 98.5 mg / L; and L1 yielded the worst, at 20.7 mg / L. These results indicate that the sequence composition of the linker peptide has a significant impact on the efficiency of anion-responsive phase separation peptide-mediated phase separation and the recovery of the target protein. By selecting appropriate linker peptides, the phase separation behavior and reconstitution recovery efficiency of the fusion protein can be further optimized, thereby improving the extraction yield of the target protein.

[0097] Example 7: Refined Extraction of Recombinant Protein To further verify the applicability of the method of the present invention in the purification of crude extract of target protein, this embodiment performs membrane filtration purification on the recovered crude extract of target protein.

[0098] The engineered strain X4 obtained in Example 1 was used as an example for induced expression. After induction culture, the bacterial cells were collected and lysed in an anionic lysing buffer containing sodium sulfate according to the methods described in Examples 2-4, so that the X4 fusion protein underwent phase separation in the presence of sulfate and entered the precipitation phase. After centrifugation, the supernatant containing host proteins was removed, and the precipitate containing the target protein was collected. Subsequently, the precipitate was resuspended and washed with sodium sulfate washing buffer to further remove entrained host proteins.

[0099] The washed precipitate was resuspended in 0.04 mol / L calcium chloride phase reversal buffer, allowing calcium ions to combine with sulfate ions in the system to form calcium sulfate precipitate. This reduces the effective concentration of free sulfate ions, disrupts the S4-mediated phase separation, and allows the target protein to redissolve and return to the supernatant. After resuspending, the sample was centrifuged at 12,000 rpm for 10 min, and the crude protein extract was collected.

[0100] Subsequently, the crude extract of the target protein was subjected to membrane filtration for further purification. First, the crude extract was filtered through a microporous membrane (0.22 μm) to remove residual insoluble particles, calcium sulfate precipitate, and other solid impurities. Then, the filtered crude extract was passed through a 30 kJ high molecular weight membrane to remove aggregates larger than the target protein, large host proteins, and other large molecular weight impurities. Finally, the target protein was concentrated using a 3 kJ low molecular weight membrane, with simultaneous liquid exchange to remove salt ions, small molecule impurities, and residual calcium chloride, yielding the purified target protein extract.

[0101] The results of the fine extraction are shown below. Figure 9 See Table 8.

[0102] Table 8 Effect of bacterial cell washing times on recombinant protein extraction

[0103] Depend on Figure 9 As shown in Table 7, after filtration and purification, the target protein crude extract showed further removal of impurity protein bands, resulting in a single target protein band with no obvious impurity protein bands. SDS-PAGE grayscale analysis showed that the target protein in the purified extract reached 100% purity, indicating that the filtration and purification step of this invention can effectively remove residual impurities and macromolecular impurities from the target protein crude extract.

[0104] Meanwhile, after impurity removal via a high-molecular-weight filter membrane and concentration via a low-molecular-weight filter membrane, the recovery rate of the final refined extract of the target protein was 83.8%. This result indicates that the "anion-induced sedimentation-calcium chloride recycling-filter membrane refinement" process described in this invention not only achieves efficient recovery of the target protein but also yields a high-purity refined extract of the target protein, with a purity of up to 100%, making it suitable for large-scale preparation of recombinant proteins.

[0105] Example 8: Expression and extraction of different target proteins in this system To verify the applicability of the reversible ion-responsive phase separation and purification system described in this invention to different target proteins, this embodiment uses type III collagen (COL-III, whose sequence can be found in Pro.C3a 1-132aa in CN117304306A) as another target protein for expression and extraction verification. A recombinant expression vector for the fusion protein was constructed according to the method described in Example 1.

[0106] Single colonies of the above-mentioned engineered strains were inoculated into LB broth containing the corresponding antibiotics and cultured overnight with shaking at 37°C and 220 rpm to obtain seed culture. The seed culture was then inoculated into fresh LB broth for small-flask fermentation. Once the bacterial culture reached the appropriate induction stage, IPTG was added to induce expression. After induction, the bacterial cells were collected by centrifugation.

[0107] The collected bacterial cells were washed with physiological saline, resuspended in an anion-induced lysis buffer containing sodium sulfate, and then lysed. In the presence of sulfate, S1-mediated controlled phase separation of the fusion protein occurred, and the protein entered the precipitation phase, while some host proteins remained in the supernatant. After centrifugation, the supernatant was removed, and the precipitate containing the target protein was collected. The precipitate was then resuspended and washed with sodium sulfate washing buffer to further remove any host proteins entrained in the precipitate.

[0108] The washed precipitate was resuspended in calcium chloride phase reversal buffer, allowing calcium ions to combine with sulfate ions in the system to form calcium sulfate precipitate. This reduces the effective concentration of free sulfate ions, disrupts the S1-mediated phase separation, and allows the fusion protein to redissolve and return to the supernatant. After resuspending, the mixture was centrifuged at 12,000 rpm for 10 min to remove the calcium sulfate precipitate and other insoluble impurities. The supernatant was collected to obtain the initial extract of the target protein.

[0109] SDS-PAGE analysis was performed on the fragmentation supernatant, precipitate sample, primary extract of the target protein, and relevant control samples. Results are shown in the table below. Figure 10 .Depend on Figure 10 It is known that the metallothionein expressed using the system of the present invention can enter the precipitation phase under sulfate-induced conditions and can return to the supernatant under the action of calcium chloride, indicating that the target protein is also suitable for the reversible ion-responsive phase separation and extraction system described in the present invention.

[0110] Gray-scale analysis and protein quantification showed that the initial extraction yield of the target protein during the small-flask fermentation stage was 46.2 mg / L. This result indicates that the system described in this invention is not only applicable to the aforementioned target protein, but also to the expression and extraction of other target proteins such as metallothionein, demonstrating its good applicability and versatility for different target proteins.

[0111] Therefore, this invention enables the rapid extraction of different target proteins through anion-responsive phase-separated peptide-mediated anion-induced sedimentation and calcium chloride reversal resolution, providing a versatile technical solution for the low-cost, large-scale production of recombinant proteins.

Claims

1. An ion-responsive controllable phase-separation peptide, characterized in that... Its amino acid sequence is shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.

4.

2. The application of the phase-separated peptide of claim 1 in the production of recombinant proteins based on ion-responsive controllable phase separation.

3. A fusion protein comprising an ion-responsive controllable phase-separation peptide and a target protein, wherein the amino acid sequence of the phase-separation peptide is as shown in SEQ ID NO.1, SEQ ID NO.3 or SEQ ID NO.4, and the target protein is located at the N-terminus or C-terminus of the phase-separation peptide.

4. The fusion protein according to claim 3, characterized in that... It also contains a linker peptide, and the structure of the fusion protein is: phase-separated polypeptide tag-linker peptide-target protein or target protein-linker peptide-phase-separated polypeptide tag.

5. The fusion protein according to claim 4, characterized in that... The amino acid sequence of the linker peptide is shown in any one of SEQ ID NO. 9, 11-13.

6. An expression vector for expressing the fusion protein according to any one of claims 3-5.

7. A host cell expressing the fusion protein according to any one of claims 3-5.

8. A method for the production and preliminary purification of recombinant proteins based on ion-responsive controllable phase separation, characterized in that, The steps include: (1) Constructing engineered cells expressing a fusion protein, wherein the fusion protein comprises a phase-separated polypeptide tag and a target protein; (2) Culture the engineered cells and induce expression of the fusion protein; (3) Collect engineered cells and break them in a disruption solution containing anions to cause phase separation of the fusion protein and allow it to enter the enriched phase; (4) Remove the supernatant containing host proteins and collect the precipitate containing the fusion protein; (5) Reduce the concentration of anions in the precipitate to allow the fusion protein to redissolve and return to the supernatant; (6) Collect the supernatant to obtain the crude extract of the target protein; The phase-separating polypeptide tag is an anion-responsive phase-separating polypeptide tag containing at least one continuous five-amino acid core sequence: X1-X2-X3-X4-X5; X1 is a basic amino acid residue; X2 and X3 are each an aliphatic hydrophobic amino acid residue; X4 and X5 are each an aromatic amino acid residue; The anion-responsive phase-separating polypeptide tag enables the fusion protein to undergo phase separation in the presence of the anion, and at least partially reverses the phase separation after reducing the effective concentration of free anion.

9. The method for producing and initially purifying recombinant proteins according to claim 8, characterized in that, The structure of the fusion protein is phase-separated polypeptide tag-target protein, target protein-phase-separated polypeptide tag, phase-separated polypeptide tag-linking peptide-target protein, or target protein-linking peptide-phase-separated polypeptide tag.

10. The method for producing and initially purifying recombinant proteins according to claim 9, characterized in that, One or more of the anions sulfate, phosphate, citrate, carbonate, and tartrate are used in step (5) to reduce the anion concentration in the precipitate by adding a phase reversal agent.

11. The method for producing and initially purifying recombinant protein according to claim 10, characterized in that, The anion is sulfate, and the concentration of the anion in the breaking solution in step (3) is 0.05 mol / L-2 mol / L.

12. The method for producing and initially purifying recombinant protein according to claim 10, characterized in that, The phase separation reversal agent includes one or more of calcium salts, magnesium salts, and barium salts.

13. The method for producing and initially purifying recombinant protein according to claim 10, characterized in that, The phase separation reversal agent includes a calcium salt, which is one or more of calcium chloride, calcium acetate, calcium lactate, and calcium gluconate.

14. The method for producing and initially purifying recombinant protein according to claim 13, characterized in that, The final concentration of calcium ions in the phase reversal agent is 1 mmol / L-1 mol / L.

15. The method for producing and initially purifying recombinant protein according to claim 8, characterized in that, It also includes step (7): refining the crude extract of the target protein to obtain the refined target protein.

16. The method for producing and initially purifying recombinant protein according to claim 15, characterized in that, The refining process in step (7) refers to passing the crude extract of the target protein through a high molecular weight filter membrane to remove impurities with a molecular weight greater than that of the target protein, and then concentrating the target protein using a low molecular weight filter membrane to obtain the refined extract of the target protein. The high molecular weight filter membrane refers to a filter membrane that retains a molecular weight greater than that of the target protein, and the low molecular weight filter membrane refers to a filter membrane that retains a molecular weight less than that of the target protein.

17. The method for producing and initially purifying recombinant protein according to claim 9, characterized in that, The amino acid sequence of the linker peptide is shown in any one of SEQ ID NO. 9, 11-13.

18. The method for producing and initially purifying recombinant proteins according to any one of claims 8-17, characterized in that, Its features are, The phase-separated polypeptide tag is the phase-separated peptide as described in claim 1.

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