A method for protein purification based on coacervates

Abstract

The application belongs to the technical field of protein purification, and discloses a protein purification method based on condensate, which comprises the following steps: firstly, constructing a target expression vector; secondly, expressing a fusion protein with an LplA label in E. coli; thirdly, ultrasonically crushing the E. coli, centrifuging the crushing liquid, and taking supernatant; fourthly, performing first temperature change, heating the supernatant, centrifuging and collecting the fusion protein after the fusion protein forms condensate, discarding the supernatant, and taking the precipitate; and fifthly, performing second temperature change, redissolving the precipitate obtained in the fourth step in a buffer at low temperature, removing insoluble proteins by centrifugation at low temperature, and taking the supernatant as the fusion protein with the LplA label after purification. The application connects the temperature-responsive purification label LplA which can form condensate to the target protein, and only needs to perform two temperature change treatments to quickly obtain the target protein with high purity, without the need of complex chromatography equipment and expensive fillers, and the application is suitable for large-scale production and purification of functional proteins.

Description

Technical Field

[0001] This invention belongs to the field of protein purification technology, and more specifically, relates to a protein purification method based on condensates. Background Technology

[0002] Proteins are essential functional molecules for life activities. Their structure and function research, industrial enzyme development, biopharmaceutical production, and biosensor construction all rely on efficient, stable, and scalable protein purification technologies. In recombinant expression systems, especially in prokaryotic systems represented by *E. coli*, target proteins often coexist with a large number of host proteins. Achieving high purity and high recovery rates while maintaining activity is a key challenge in bioengineering.

[0003] Currently, the most widely used methods are affinity chromatography, such as His-tag, GST-tag, and MBP-tag systems. These methods have high specificity, but require expensive packing materials and specialized equipment (such as His-tag / Ni²). + Methods such as NTA, GST-tag / glutathione, and MBP-tag / starch resin have many steps and high costs, making them unsuitable for large-scale production. Furthermore, the elution process often requires the addition of reagents such as imidazole, which may affect protein stability and increase post-processing steps. Other methods, such as salting out, isoelectric point precipitation, and hydrophobic chromatography, suffer from insufficient selectivity or demanding conditions, making them unsuitable for rapid and convenient purification.

[0004] To overcome the limitations of traditional chromatographic purification, researchers have recently begun exploring non-chromatographic protein purification strategies, among which stimulus-responsive protein tags have gained increasing attention. These tags can undergo reversible conformational changes, aggregation, or phase transitions in response to external stimuli (such as temperature, pH, ionic strength, or small molecule ligands), thereby achieving the separation of the target protein from contaminating proteins. Temperature-responsive purification tags are an important branch, with typical examples including reversible phase transition cycling (ITC) purification systems based on elastic peptide-like peptides (ELPs). ELP tags can undergo reversible precipitation under heating and salting conditions, achieving protein recovery without chromatographic media. However, these systems typically require multiple repeated heating-cooling cycles at high temperatures and high salt concentrations to promote precipitation, significantly impacting the structure and activity of the target protein. Furthermore, ELP tags require increased sequence length to improve separation efficiency, potentially affecting the expression level and function of the target protein. These limitations restrict their practical application.

[0005] Taking the Chinese patent application (CN101955960A) as an example, the ELP used in this method is 480 amino acids long. The protein must be separated at a final concentration of (NH4)2SO4 of 1.2M and a phase transition temperature of 37°C, and must go through 5 ITC cycles.

[0006] Taking the Chinese patent application CN102127156A, which describes a fusion tag protein capable of non-chromatographic separation of target proteins, its encoding gene, and preparation method, as an example, this method involves adding NaCl solution at a volume ratio of 1:2 (NaCl solution: supernatant), separating the protein at 45°C, and requiring two ITC cycles.

[0007] Taking a recombinant protein purification method (CN120441715A) from Chinese patent application as an example, the temperature range used in this method is 30°C. At 60℃, the salt ions are those produced by NaCl and (NH4)2SO4, wherein the concentration of (NH4)2SO4 is in the range of 0.4%. 1M, NaCl concentration range is 1 2M.

[0008] In recent years, protein liquid-liquid phase separation and the resulting protein condensates have attracted widespread attention in the life sciences. Studies have shown that certain proteins can undergo spontaneous phase transitions under specific environmental conditions (such as changes in temperature, concentration, or ionic conditions), transforming from a homogeneous solution state into a protein-enriched condensate phase. This process is typically reversible, condition-dependent, and exhibits high molecular selectivity, thus holding potential applications in biological regulation, materials science, and bioengineering. Introducing the protein condensate formation mechanism into the protein purification process could potentially enable novel purification strategies that achieve rapid, single-round ITC separation without the need for chromatographic media, relying solely on relatively mild changes in physical conditions.

[0009] Lipoic acid protein ligase A (LplA), responsible for lipid acylation in *E. coli*, is a well-folded, monomeric globular protein with a molecular weight of 38 kDa, and its amino acid sequence is shown in SEQ ID No. 1. It exhibits structural self-assembly and a rare lowest leaching temperature (LCST) phase behavior in vitro, and forms small, tunable orthogonal condensates in *E. coli* and human cells. Applying the LCST phenomenon to protein purification would undoubtedly revolutionize the field. Summary of the Invention

[0010] To address the aforementioned deficiencies or improvement needs of existing technologies, the present invention aims to provide a protein purification method based on aggregates. This method utilizes a temperature-responsive purification tag, LplA, capable of forming aggregates, which is linked to the target protein (correspondingly, an expression vector capable of expressing the LplA-tagged fusion protein needs to be constructed). Upon heating, the fusion protein forms aggregates and separates from the solution. Cooling then allows it to dissolve in a buffer solution. High-purity target protein can be rapidly obtained through only two temperature variations and one ITC cycle (without repeated phase transition cycles). The protein purification method of this invention eliminates the need for complex chromatography equipment and expensive packing materials, achieving separation under mild conditions. It maximizes the preservation of the protein's natural structure, function, and biological activity, offering advantages such as high efficiency, economy, and convenience, and is suitable for the large-scale production and purification of functional proteins.

[0011] To achieve the above objectives, according to one aspect of the present invention, a method for constructing an expression vector is provided, characterized in that the expression vector is capable of expressing a fusion protein with an LplA tag, wherein the LplA-tagged fusion protein comprises an LplA tag and a target protein whose amino acid sequence meets pre-defined requirements; the amino acid sequence of the LplA tag, as shown in SEQ ID No. 1, is attached to the N-terminus of the target protein; The construction method involves constructing a gene expression sequence linking the LplA tag and the gene expression sequence of the target protein within an initial expression vector, thereby obtaining the target expression vector.

[0012] As a further preferred embodiment of the present invention, the target protein is either GFP protein or EglS protein.

[0013] As a further preferred embodiment of the present invention, the initial expression vector is pET-28a.

[0014] According to another aspect of the present invention, the present invention provides a target expression vector constructed by the above-described construction method.

[0015] According to another aspect of the present invention, the present invention provides a protein purification method based on condensates, characterized by comprising the following steps: (1) The target expression vector is constructed according to the above construction method; (2) Escherichia coli expressed a fusion protein with an LplA tag, collected E. coli cells by centrifugation, then suspended in PBS buffer, followed by sonication to disrupt the E. coli, centrifuged the disruption liquid, and collected the supernatant. (3) Perform the first temperature change to raise the temperature of the supernatant. When the fusion protein forms aggregates, centrifuge and collect the precipitate, discard the supernatant and take the precipitate. (4) Perform a second temperature change. At low temperature, redissolve the precipitate obtained in step (3) in PBS buffer, remove insoluble protein by low-temperature centrifugation, and the supernatant is the purified fusion protein with LplA tag. In step (3), the temperature corresponding to the heating is higher than the minimum co-solution temperature (LCST) of the LplA tag and lower than the denaturation temperature of the LplA tag and the target protein; in step (4), the temperature corresponding to the low temperature is lower than the minimum co-solution temperature (LCST) of the LplA tag. The protein purification method based on condensates has one and only one reversible phase transition cycle (ITC), which is achieved by the combination of the first temperature change in step (3) and the second temperature change in step (4).

[0016] As a further preferred embodiment of the present invention, in step (3), the temperature corresponding to the heating is 25~32℃; In step (4), the temperature corresponding to the low temperature is 0~10℃.

[0017] As a further preferred embodiment of the present invention, the PBS buffer uses water as a solvent and contains the following solutes and concentrations: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, and 1.8mM KH2PO4. In step (4), the precipitate obtained in step (3) is redissolved in PBS buffer using an equal volume of PBS buffer.

[0018] As a further preferred embodiment of the present invention, in step (2), the ultrasonic disruption of Escherichia coli is carried out at a temperature of 0°C; The centrifugation of the broken liquid was carried out at a temperature of 4°C.

[0019] Addressing the challenge of existing technologies lacking protein tags capable of controllable, reversible, and selective aggregation under mild conditions, which hinders the application of stimulus-responsive protein purification methods, this invention utilizes the rare minimum co-solution temperature (LCST) behavior exhibited by LplA. By discovering and leveraging the controllable aggregation characteristics of LplA protein at specific temperatures, LplA is used as a temperature-responsive purification tag. By raising the temperature above the LCST but below the denaturation temperatures of both LplA and the target protein, the separation of the target protein from solution is successfully achieved. Due to the low LCST of LplA, the heating temperature is not high, and high salt concentrations are not required to promote aggregation. This environment allows most proteins to maintain their normal structure and function, maximizing the preservation of the target protein's activity and function. This enables efficient separation of the target protein under mild conditions, overcoming the aforementioned technical difficulties and developing a method for purifying the fermentation broth of engineered E. coli to obtain high-yield, high-purity, and high-activity proteins.

[0020] This invention uses LplA as a temperature-responsive purification tag, enabling the target protein to form aggregates at elevated temperatures (e.g., 30°C as used in the following examples, where aggregate formation efficiency is high and the impact on protein structure and function is minimal; of course, other temperatures higher than the LCST of LplA but lower than the denaturation temperatures of LplA and the target protein, i.e., temperatures that will not cause destructive effects on the native structure, function, and biological activity of LplA and the target protein) and be separated from the solution by centrifugation (e.g., centrifuging at 10,000 rpm for 20 min can be used to collect aggregates to the maximum extent). Then, a second temperature change is performed, and the resulting precipitate is redissolved in buffer at low temperature. Low-temperature centrifugation removes insoluble proteins, obtaining the purified supernatant (as illustrated in the following examples, the aggregates can be redissolved at 4°C, and finally centrifuged at 12,000 rpm for 20 min at 4°C to remove insoluble contaminants, obtaining the purified protein). This method offers mild purification conditions, eliminates the need for complex ion exchange chromatography, achieves high protein purity, preserves activity well, minimizes protein loss, and requires only one phase transition cycle (i.e., one ITC cycle), making the process simple and rapid. The purification process and associated risks are controllable, making it a simple and efficient protein purification method.

[0021] This invention utilizes a temperature-responsive purification tag to achieve high-purity protein purification through a simple method of temperature variation and centrifugation. Based on this invention, using the temperature-responsive purification tag LplA, when purifying fusion proteins from cell lysate supernatant, only two temperature variations and two centrifugations are needed to obtain the target protein with high purity. The operation is simple and rapid, and it ensures the function and activity of the protein, making it suitable for the production and purification of functional proteins.

[0022] Specifically, the present invention can achieve the following beneficial effects: (1) LplA temperature-responsive purification tags rely solely on temperature changes to induce aggregate formation, requiring no chromatography media or specialized equipment (such as His-tag / Ni²). + -NTA, GST-tag / glutathione, MBP-tag / starch resin, etc.) significantly reduce reagent and consumable costs, making them particularly suitable for pilot-scale and large-scale production.

[0023] (2) The protein purification method provided by the present invention only requires two temperature changes and two centrifugations to purify the fusion protein from the cell lysate supernatant to achieve the enrichment and separation of the target protein. The process is short and the number of steps is small, which significantly shortens the purification cycle and improves experimental and production efficiency.

[0024] (3) The protein purification process provided by the present invention does not require the addition of any chemical eluent. The protein state is controlled only by the physical parameter of temperature, which is more conducive to maintaining the native conformation and biological activity of the target protein. Attached Figure Description

[0025] Figure 1 This is the carrier spectrum obtained in Example 1.

[0026] Figure 2 The connection obtained in Example 2 gfp Recombinant plasmid map of genes.

[0027] Figure 3 The connection obtained in Example 2 eglS Recombinant plasmid map of genes.

[0028] Figure 4 This is an SDS-PAGE electrophoresis image of the purified fusion protein LplA-GFP.

[0029] Figure 5 This is an SDS-PAGE electrophoresis image of the purified fusion protein LplA-EglS.

[0030] Figure 6 This is the purification flowchart corresponding to the purification in Example 5. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0032] Based on the present invention, the protein purification method may include the following steps: (1) Production of tagged fusion proteins. Construct recombinant plasmids with the LplA tag attached to the N-terminus of the fusion protein.

[0033] (2) E. coli expressed a tagged fusion protein. After the protein expression was completed, the bacterial cells were collected by centrifugation, then suspended in PBS buffer, and then the E. coli were disrupted by sonication. The disrupted liquid was centrifuged and the supernatant was collected.

[0034] (3) Heat the supernatant to form aggregates of fusion protein, then centrifuge and collect the supernatant.

[0035] (4) The separated aggregates were redissolved in PBS buffer at low temperature, and insoluble proteins were removed by low-temperature centrifugation. The supernatant was the purified protein.

[0036] Specifically, you can follow these steps to perform the actual operation: (1) Design a prokaryotic expression vector, which, in addition to the basic components of the vector, includes the gene expression sequence of the temperature-responsive purification tag LplA and the gene expression sequence of the target protein whose amino acid sequence meets the pre-defined requirements. Based on the principle of recombinant protein expression, and through a prokaryotic expression system, produce various tagged fusion proteins.

[0037] (2) The fusion protein was expressed using E. coli. After the protein expression was completed, the bacterial cells were collected by centrifugation and then resuspended in PBS buffer. The E. coli were then sonicated to release the intracellular fusion protein. After the disruption was completed, the disruption solution was centrifuged, and the precipitate was cell debris. The fusion protein was in the supernatant.

[0038] (3) A first temperature change (this temperature change is an increase) is performed. Utilizing the LCST phase behavior of LplA, the molecules aggregate to form aggregates when the temperature is increased. These aggregates can be separated from the impurities in the solution by centrifugation (this centrifugation is the first centrifugation in the purification stage of the fusion protein from the cell lysate supernatant). The resulting centrifuged precipitate contains the separated aggregates. At 30°C, the LplA tag forms aggregates with high efficiency and has little impact on protein structure and function, making it suitable for protein separation. It is preferable to set the target temperature for the temperature increase to 30°C (as illustrated in Example 5 below).

[0039] (4) After centrifuging to separate the aggregates in step (3), a second temperature change (this temperature change is cooling) is performed. The aggregates are fully dissolved with an equal volume of PBS buffer and centrifuged again at low temperature (this centrifugation is the second centrifugation in the purification of fusion protein from cell lysate supernatant). This process aims to remove impurities that precipitate due to structural changes caused by temperature rise. The high-purity target protein is obtained by SDS-PAGE electrophoresis verification.

[0040] The PBS buffer used in the following examples all use the same formulation: water as the solvent, and the solutes and their concentrations are as follows: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4, without any other components.

[0041] The following are specific examples: Example 1: Carrier Modification Using pET-28a plasmid as the recombinant plasmid vector, the LplA gene sequence was amplified from the *E. coli* BL21(DE3) genome. Homologous recombination was used to insert the tag downstream of the T7 promoter, positioning it in the correct open reading frame. Figure 1 As shown.

[0042] Example 2: Construction of fusion protein expression plasmid Using homologous recombination, the target protein gene ( gfp , eglS Connected to lplA Following gene sequencing, the fusion protein expression plasmids pET28a-LplA-GFP and pET28a-LplA-EglS were obtained, respectively. See [link / reference needed]. Figure 2 , Figure 3 .

[0043] Example 3: Expression of fusion protein For each of pET28a-LplA-GFP and pET28a-LplA-EglS: The constructed recombinant plasmid was purified. 100 μL of *E. coli* BL21 competent cells were thawed on ice. In a laminar flow hood, 100 ng of plasmid DNA was added to the competent cells, gently pipetted to mix, and incubated on ice for 2 min. The tube was then placed on a 42°C metal bath and incubated for 90 s. The tube was quickly transferred to ice and incubated for 2 min. In a laminar flow hood, 800 μL of antibiotic-free LB medium was added to the tube and sealed. The tube was then incubated at 37°C for 1 h at 220 rpm in a shaker. In a laminar flow hood, 100 μL of the transformed competent cells were transferred to an LB agar plate containing 50 μg / mL kanamycin and spread evenly. The plate was inverted and incubated at 37°C for 12 h.

[0044] Single colonies from LB agar plates were picked and cultured in 5 mL LB broth vials at 37°C and 220 rpm for 12 h. The bacterial culture was then transferred to a larger vial at a 1% inoculation rate and cultured at 37°C and 220 rpm until the OD600 reached 0.6. 0.4 mM IPTG was then added, and expression was induced at 16°C and 200 rpm for 16 h. BL21-pET28a-LplA-GFP and BL21-pET28a-LplA-EglS cells were obtained after expression.

[0045] Example 4: Bacterial Disruption For each of BL21-pET28a-LplA-GFP and BL21-pET28a-LplA-EglS: After protein expression, bacterial cells were collected by centrifugation at 8000 rpm for 5 min at 4°C. The cells were washed once with PBS buffer, centrifuged at 8000 rpm for 5 min, and the supernatant was discarded. The cells were then resuspended in PBS buffer (5 mL PBS buffer per 1 g of cells). E. coli were sonicated at 0°C with a power setting of 220 W for 3 seconds followed by a 5-second pause. Typically, the solution became clear after 20 min of sonication, indicating complete cell lysis (however, sometimes the fusion protein forms a large number of aggregates, resulting in a lower concentration of the target protein in the solution, and the solution remains clear; in this case, the sonication time can be appropriately extended to break up the aggregates and release the protein into the supernatant). After lysis, the sonicated solution was centrifuged at 12000 rpm for 30 min at 4°C, and the supernatant was collected and labeled as cell lysis supernatant BL21-pET28a-LplA-GFP and cell lysis supernatant BL21-pET28a-LplA-EglS, respectively.

[0046] Meanwhile, the resulting precipitates were designated as cell lysis buffer precipitate BL21-pET28a-LplA-GFP and cell lysis buffer precipitate BL21-pET28a-LplA-EglS, respectively.

[0047] In addition, the cell lysis supernatant BL21-pET28a-LplA-GFP and the cell lysis precipitate BL21-pET28a-LplA-GFP were subjected to SDS-PAGE electrophoresis. The results are shown in [Figure number missing]. Figure 4 Lanes 3 and 4.

[0048] The cell lysis supernatant BL21-pET28a-LplA-EglS and the cell lysis precipitate BL21-pET28a-LplA-EglS were subjected to SDS-PAGE electrophoresis. The results are shown in the figure. Figure 5 Lanes 3 and 4.

[0049] Example 5: Purification of the fusion protein For each of the cell lysate supernatant BL21-pET28a-LplA-GFP and cell lysate supernatant BL21-pET28a-LplA-EglS, as follows Figure 6 As shown, the purification process is as follows: The collected supernatant was incubated at 30°C for 1 hour, centrifuged at 10,000 rpm for 20 minutes, and the supernatant was discarded. The aggregates were collected. The aggregates were redissolved with an equal volume of PBS buffer and incubated at 4°C for 1 hour to allow the fusion protein to fully rehydrate. The aggregates were centrifuged at 12,000 rpm for 20 minutes at 4°C to remove the precipitate of insoluble protein. The supernatant contained the purified target protein, labeled as purified LplA-GFP and purified LplA-EglS, respectively. The SDS-PAGE electrophoresis results are shown in [Figure number missing]. Figure 4 , Figure 5 Lane 5.

[0050] In comparison, the inventors also provided the following comparative examples: Comparative Example 1: The empty plasmid containing the unintegrated gene pET28a was transformed into E. coli BL21 competent cells according to the method in Example 3, and its expression was induced. The bacteria were then lysed according to the method in Example 4. BL21-pET28a was obtained as cell lysate supernatant and BL21-pET28a as cell lysate precipitate.

[0051] Two batches of cell lysis supernatant BL21-pET28a and cell lysis precipitate BL21-pET28a were subjected to SDS-PAGE electrophoresis. The results are shown in the figures below. Figure 4 Lanes 1 and 2, and Figure 5 Lanes 3 and 4 (Due to experimental fluctuations, the electrophoretic bands of the same sample from different batches may vary slightly, which is normal).

[0052] Comparative Example 2: The cell lysate supernatant BL21-pET28a-LplA-GFP obtained in Example 4 was treated at 12°C for 1 hour and centrifuged at 10,000 rpm for 20 minutes, but no aggregates were obtained, making further separation and purification impossible. This is because the minimum co-solubility temperature (LCST) of LplA is 13°C, while the treatment temperature of the cell lysate supernatant in the purification method of Comparative Example 2 is lower than the LCST of LplA, preventing the target protein from forming aggregates and thus hindering separation and purification.

[0053] The above embodiments are merely examples. For instance, the temperature corresponding to the first temperature change (heating) in the purification stage of fusion protein from cell lysate supernatant should be higher than the LCST of the LplA tag and lower than the denaturation temperature of the LplA tag and the target protein (e.g., other temperatures within the 25~32℃ range; of course, if the temperature used exceeds the temperature at which the aggregates are fully matured, the more effective the aggregation will be and the more conducive it will be to achieving high purification efficiency). The temperature corresponding to the second temperature change (cooling) should be lower than the LCST of the LplA tag (e.g., other temperatures within the 0~10℃ range).

[0054] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing an expression vector, characterized in that, This expression vector can express a fusion protein with an LplA tag, wherein the LplA-tagged fusion protein includes an LplA tag and a target protein whose amino acid sequence meets pre-defined requirements; the amino acid sequence of the LplA tag is shown in SEQ ID No.1 and is attached to the N-terminus of the target protein; The construction method involves constructing a gene expression sequence linking the LplA tag and the gene expression sequence of the target protein within an initial expression vector, thereby obtaining the target expression vector.

2. The construction method as described in claim 1, characterized in that, The target protein is either GFP protein or EglS protein.

3. The construction method as described in claim 1, characterized in that, The initial expression vector was pET-28a.

4. The target expression vector constructed by the construction method according to any one of claims 1-3.

5. A protein purification method based on condensates, characterized in that, Includes the following steps: (1) The target expression vector is constructed using the construction method according to any one of claims 1-3; (2) Escherichia coli expressed a fusion protein with an LplA tag, collected E. coli cells by centrifugation, then suspended in PBS buffer, followed by sonication to disrupt the E. coli, centrifuged the disruption liquid, and collected the supernatant. (3) Perform the first temperature change to raise the temperature of the supernatant. When the fusion protein forms aggregates, centrifuge and collect the precipitate, discard the supernatant and take the precipitate. (4) Perform a second temperature change. At low temperature, redissolve the precipitate obtained in step (3) in PBS buffer, remove insoluble protein by low-temperature centrifugation, and the supernatant is the purified fusion protein with LplA tag. In step (3), the temperature corresponding to the heating is higher than the minimum co-solution temperature (LCST) of the LplA tag and lower than the denaturation temperature of the LplA tag and the target protein; in step (4), the temperature corresponding to the low temperature is lower than the minimum co-solution temperature (LCST) of the LplA tag. The protein purification method based on condensates has one and only one reversible phase transition cycle (ITC), which is achieved by the combination of the first temperature change in step (3) and the second temperature change in step (4).

6. The protein purification method based on aggregates as described in claim 5, characterized in that, In step (3), the temperature corresponding to the heating is 25~32℃; In step (4), the temperature corresponding to the low temperature is 0~10℃.

7. The protein purification method based on condensates as described in claim 5, characterized in that, The PBS buffer uses water as a solvent and contains the following solutes and concentrations: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, and 1.8mM KH2PO4. In step (4), the precipitate obtained in step (3) is redissolved in PBS buffer using an equal volume of PBS buffer.

8. The protein purification method based on aggregates as described in claim 5, characterized in that, In step (2), the ultrasonic disruption of E. coli is performed at a temperature of 0°C; The centrifugation of the broken liquid was carried out at a temperature of 4°C.

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