Recombinant protein purification method

Abstract

The invention discloses a method for purifying recombinant protein. The framework of the fusion protein is characterized in that a series tag consisting of elastin-like protein (ELP) and SUMO is connected with a target protein. The fusion protein is purified by utilizing the reversible phase change cycle (ITC) property of ELP, and the separation and purification of the recombinant protein can be realized only through simple centrifugal operation, so that the fusion protein with higher purity is obtained, and the required recombinant target protein can be obtained subsequently through enzyme digestion and ITC. The technology is wide in application range, simple and convenient in purification method, low in cost and suitable for industrial amplification, and has a good commercial prospect.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to the application of a tandem tag composed of ELP and SUMO to express and purify recombinant proteins. Background Art

[0002] ELP is a synthetically designed peptide sequence whose sequence composition and structural properties mimic those of natural elastin. Its key characteristic is the repeating pentapeptide structural unit "Val-Pro-Gly-Xaa-Gly," where Xaa is any amino acid other than proline (Pro, P), most commonly valine (Val, V), alanine (Ala, A), glycine (Gly, G), leucine (Leu, L), isoleucine (Ile, I), lysine (Lys, K), phenylalanine (Phe, F), and histidine (His, H). ELP peptides exhibit remarkable temperature-responsive properties, accumulating at specific temperatures (usually above their phase transition temperature) and redissolving upon cooling. ELP also exhibits different phase transition temperatures depending on the composition and number of the repeating pentapeptide units. Fusion proteins containing ELP also exhibit this property, preventing denaturation and precipitation of the target protein during aggregation. Therefore, by utilizing this property of ELP, the aggregation of ELP-containing fusion proteins can be triggered by changing temperature or salt ion concentration. The fusion protein can then be separated by centrifugation. The aggregated fusion protein can then be re-dissolved under low-salt and low-temperature conditions and centrifuged again to remove insoluble impurities. This purification process of centrifugal precipitation and re-dissolution of the precipitate is called Inverse Transition Cycling (ITC).

[0003] The non-chromatographic (column-free) purification method is a separation and purification technology that does not rely on traditional chromatography columns. The target protein can be separated from the crude extract using simple means such as centrifugation, precipitation, and filtration. At present, the core of this method is the polymerization tag in the target protein, which mainly consists of two parts, namely, a sequence that is easy to aggregate and a cleavable site. The cleavable site can use the enzymatic cleavage site of the protease to remove the sequence that is easy to aggregate. Commonly used proteases include enterokinase, SUMO protease, grass etch virus protease (TEV), rhinovirus 3C, etc. Among them, SUMO protein can not only promote the correct folding of the target protein, increase the stability and solubility of the protein, but also reduce the impact of certain toxic proteins on host cells. In addition, SUMO protease specifically recognizes the tertiary structure of SUMO and cuts at the N-terminus of the fusion protein to obtain a target protein with a natural structure.

[0004] In summary, there is still room for improvement in the process of expressing and purifying recombinant proteins using tandem fusion tags of ELP and SUMO, so as to obtain the target protein more simply and efficiently. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for expressing and purifying a fusion protein comprising an aggregation tag and a cleavage tag and a tag-free recombinant protein thereof.

[0006] In a first aspect of the present invention, a fusion protein is provided, which comprises a target protein portion and an aggregation tag ELP and a SUMO cleavage tag combination portion, wherein the target protein portion is connected to the aggregation tag ELP portion via a spacer, and wherein the spacer comprises the cleavage tag SUMO, wherein the aggregation tag ELP is composed of repeated tandem (GVGXG)n, wherein n is 36, 48, 96 or 80, and the X position can be a single or a combination of several amino acids: lysine (K), valine (V), phenylalanine (F), isoleucine (I), serine (S), alanine (A) or glycine (G).

[0007] Preferably, the ELP tandem repeating unit (GVGXG)n is one of ELP[KV7F]36 (SEQ ID NO.1), ELP[V]48 (SEQ ID NO.2), ELP[I]48 (SEQ ID NO.3), ELP[SI]96 (SEQ ID NO.4), and ELP[V5A2G3]80 (SEQ ID NO.5).

[0008] Preferably, the aggregation-tagged ELP comprises a linker between its C-terminus and the N-terminus of the cleavage tag.

[0009] Preferably, the linker is selected from the GS-type linker, ie, GGGGS (SEQ ID NO: 9).

[0010] Preferably, the cleaved tag is attached to said target protein portion.

[0011] Preferably, the target protein portion is located at the C-terminus of the fusion protein, and the cleavage tag is connected to the N-terminus of the target protein portion.

[0012] Preferably, the cleavage tag, ie, the cleavage site, is selected from a temperature-dependent cleavage site, a pH-dependent cleavage site, an ion-dependent cleavage site, an enzyme cleavage site, or a self-cleavage site.

[0013] Preferably, the cleavage site comprises a protease cleavage site; the enzyme used in the cleavage site is selected from enterokinase, SUMO protease, grass etch virus protease (TEV), and rhinovirus 3C.

[0014] Preferably, the enzyme cleavage site is SUMO, which comprises the amino acid sequence shown in SEQ ID NO:6.

[0015] In a second aspect of the present invention, a polynucleotide is provided, which comprises a nucleotide sequence encoding the fusion protein of the present invention or a complementary sequence thereof.

[0016] Preferably, the fusion protein comprises an aggregation tag ELP, a cleavage tag SUMO and a gene sequence of a target protein, wherein the ELP has an amino acid sequence as shown in SEQ ID NO: 1-5, and the SUMO has an amino acid sequence as shown in SEQ ID NO: 6.

[0017] In the third aspect of the present invention, an expression vector is provided, which comprises the above-mentioned polynucleotide.

[0018] In a fourth aspect of the present invention, a host cell is provided, comprising the above-mentioned polynucleotide or the above-mentioned expression vector, wherein the host cell is capable of expressing the fusion protein.

[0019] Preferably, the host cell is selected from the prokaryotic Escherichia coli (E. coli).

[0020] In a fifth aspect of the present invention, a method for expressing and purifying a target protein is provided, comprising the following steps:

[0021] (1) Connecting the fusion partner gene containing ELP and SUMO to the target protein gene to form a plasmid, and then transferring it into the host cell to express the fusion protein;

[0022] (2) changing at least one of the temperature and salt ion concentration of the fusion protein obtained in step (1), and centrifuging after insoluble aggregates appear to obtain the aggregated fusion protein;

[0023] (3) recovering the aggregates obtained in step (2);

[0024] (4) cleavage of SUMO by the addition of Ulp1 protease to release the target protein;

[0025] (5) Adjusting the temperature or salt ion concentration of the solution after the enzyme cleavage reaction, centrifuging after aggregates appear, and recovering the soluble portion containing the target protein.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] 1) This purification method utilizes the tandem tags of the aggregation tag ELP and the cleavage tag SUMO to improve the expression effect of the target protein, increase the stability and solubility of the protein, and thus the yield and quality of the target protein; 2) This purification method utilizes the reversible phase transition property of the aggregation tag ELP, and can obtain a high-purity target protein through simple centrifugation, which is simple to operate and has low production cost; 3) The cleavage tag SUMO in this purification method can be specifically recognized by the tertiary structure of the SUMO protease and cleaved at the N-terminus of the fusion protein, thereby obtaining a target protein with a native structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Shown is a protein purification strategy based on the aggregation tag ELP and the cleavage tag SUMO.

[0029] Figure 2 A map showing the protein purification method based on the aggregation tag ELP and the cleavage tag SUMO, which relies on the pET30a-ELP-SUMO-POI expression vector.

[0030] Figure 3 The graph shows the results of SDS-PAGE analysis of ITC of ELP fusion protein and ITC after Ulp1 digestion in an environment of 0.7 M (NH4)2SO4 at 40°C.

[0031] Figure 4 The figure shows the results of SDS-PAGE analysis of the red fluorescent protein RFP purified by inducing aggregation of the aggregable tag ELP by 1M NaCl, 1.5M NaCl, 2M NaCl, 0.4M (NH4)2SO4, 0.7M (NH4)2SO4, and 1M (NH4)2SO4 at 37°C.

[0032] Figure 5 A graph shows the results of SDS-PAGE analysis of the purified red fluorescent protein RFP by inducing aggregation of the aggregable peptide ELP at 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, and 60°C.

[0033] Figure 6 The figure shows the results of SDS-PAGE analysis of the purified protein CPL-711 by inducing aggregation of the aggregable peptide ELP under 0.7 M (NH4)2SO4 and room temperature conditions. DETAILED DESCRIPTION

[0034] Below in conjunction with example, specific implementation of the present invention is further described, but implementation and protection of the present invention are not limited thereto.It should be pointed out that below if there is the process that is not particularly described in detail, all those skilled in the art can realize or understand with reference to prior art.In the following examples, method therefor is conventional method unless otherwise specified, and specific steps can be referring to, for example, Molecular Cloning:A Laboratory Manual (Sambrook J. etc., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).Primer used is all synthesized by Shanghai Tianyi Huiyuan.

[0035] In one embodiment, the target protein is red fluorescent protein (RFP).In one embodiment, the target protein portion comprises the DNA sequence shown in SEQ ID NO:8.

[0036] In the notes of Examples 1-8, a The expression level of the fusion protein in the lysis supernatant (the volume is calculated per liter of LB medium); b Soluble percentage = 100% × expression level of fusion protein in lysis supernatant / (expression level of fusion protein in lysis supernatant + expression level of fusion protein in lysis precipitate); c Aggregation efficiency = 100% × the amount of fusion protein in the precipitate after salt addition / (the amount of fusion protein in the precipitate after salt addition + the amount of fusion protein in the supernatant after salt addition); d Redissolution efficiency = 100% × amount of fusion protein in the supernatant after pellet resolubilization and centrifugation / (amount of fusion protein in the supernatant after pellet resolubilization and centrifugation + amount of fusion protein in the pellet after pellet resolubilization and centrifugation); e Yield of target protein after Ulp1 protease cleavage (volume per liter of LB medium); f The purification effect was poor and no corresponding data were available.

[0037] In the figures of Examples 1-6, ES: supernatant of cell lysate; EP: precipitate of cell lysate; ES1: supernatant after ES was aggregated by adding salt; EP1: precipitate after ES was aggregated by adding salt; ES2: supernatant after EP1 was re-dissolved; EP2: precipitate after EP1 was re-dissolved; ES3: supernatant after ES2 was aggregated by adding salt after cutting; among them, ES1 to EP2 were all diluted 10 times, and ES3 was diluted 5 times (some were not diluted); M: protein marker; BSA: bovine serum albumin standard; 1: 1 mg / mL; 2: 0.5 mg / mL; 3: 0.25 mg / mL; 4: 0.125 mg / mL; 5: 0.0625 mg / mL; 6: 0.03125 mg / mL; 7: 0.0015625 mg / mL.

[0038] Example 1: Construction of expression vectors containing five different ELP-SUMO-RFP fusion proteins

[0039] The first aggregable tag used in this example is ELP[KV7F]36, which is 36 repeats of VPGXG, wherein the amino acid ratio of X is K:V:F=1:7:1, and its amino acid sequence is shown in SEQ ID NO:1; the second is ELP[V]48, wherein the amino acid at position X is V, and the pentapeptide unit is repeated 48 times, and its amino acid sequence is shown in SEQ ID NO:2; the third is ELP[I]48, wherein the amino acid at position X is I, and the pentapeptide unit is repeated 48 times, and its amino acid sequence is shown in SEQ ID NO:3; the fourth is ELP[S48I48], wherein the amino acids at position X are S and I, and VPGSG is repeated 48 times, followed by VPGIG, and its amino acid sequence is shown in SEQ ID NO:4; the fifth is ELP[V5A2G3]110, wherein the amino acid ratio of X is V:A:G=5:2:3, and the pentapeptide unit is repeated 80 times, and its amino acid sequence is shown in SEQ ID NO: NO:5; the cleavage tag protein is SUMO protein, whose amino acid sequence is shown in SEQ ID NO:6; the target protein is red fluorescent protein RFP, whose amino acid sequences are shown in SEQ ID NO:7. The expression vectors used are pET30a-ELP36-SUMO-RFP, pET30a-ELPV48-SUMO-RFP, pET30a-ELPI48-SUMO-RFP, pET30a-ELP80-SUMO-RFP, and pET30a-ELPS48I48-SUMO-RFP. The primers required for plasmid construction were designed using oligo 6 and synthesized by Tianyi Huiyuan, as shown in Table 1.

[0040] Table 1 Oligonucleotide primers used in this example

[0041]

[0042] First, the ELP36 polynucleotide sequence was amplified by PCR using the synthetic pUC57-ELP36 template and primers ELP1-F and ELP1-R. The RFP-Backbone and Backbone-1 polynucleotide fragments were amplified by PCR using the synthetic pET30a-RFP (Beijing Qingke Biotechnology Co., Ltd.) template using primer pairs RFP-F / Backbone-R1 and Backbone-F1 / Backbone-R2, respectively. Overlapping PCR was then performed using the ELP36 polynucleotide sequence and Backbone-1 polynucleotide fragment as templates to generate the Backbone-1-ELP36 fragment. The SUMO polynucleotide sequence was amplified by PCR using the commercially available pET28a-SUMO template and primers SUMO-F and SUMO-R1. This polynucleotide was then combined with the RFP-Backbone polynucleotide sequence to generate the SUMO-RFP-Backbone fragment. The two purified polynucleotide fragments were assembled by Gibson assembly. The assembled products were transformed into Escherichia coli DH5α competent cells and plated on LB plates containing 50 μg / mL kanamycin to screen positive clones. The plasmid was extracted using a plasmid extraction kit and sequenced to obtain the pET30a-ELP36-SUMO-RFP plasmid. Its structure is shown in the figure. Figure 2 shown.

[0043] Using the constructed plasmid pET30a-ELP36-SUMO-RFP as a template, the primer pair SUMO-F2 and Backbone-R1 were used to amplify the SUMO-RFP-Backbone polynucleotide fragment by PCR. Then, using the purchased plasmid V48 ELP (Plasmid#68395) as a template, the primers Backbone-F and ELP2-R were used to amplify the Backbone-1-ELP[V]48 polynucleotide fragment by PCR. The purified polynucleotide fragments were assembled by Gibson assembly, and the assembly products were transformed into Escherichia coli DH5α competent cells. Positive clones were screened, and the plasmid was extracted and sequenced to obtain the pET30a-ELP[V]48-SUMO-RFP plasmid. The construction methods of plasmids pET30a-ELP[I]48-SUMO-RFP, pET30a-ELP[S48I48]-SUMO-RFP, and pET30a-ELP[V5A2G3]80-SUMO-RFP were similar to the construction method of the above-mentioned pET30a-ELP[V]48-SUMO-RFP.

[0044] Example 2: Expression of five different ELP-SUMO-RFP fusion proteins

[0045] The five plasmids constructed in Example 1 were transformed into Escherichia coli BL21 (DE3) competent cells to obtain five fusion protein expression strains. The expression strains were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured in a shaking incubator at 37°C until the logarithmic phase (OD 600 =0.4-0.6), 0.4 mM IPTG was added and induced at 18°C for 24 hours. The cells were harvested by centrifugation at 4°C, 4,000 rpm for 20 minutes (hereinafter, 1 mL of OD 600 The cell mass of 1 is called 1OD).

[0046] The cells were resuspended in phosphate buffered saline (PBS) (10 mM, pH 7.4) to 50 OD / mL and ultrasonically disrupted (disruption conditions: horn Φ5, 30% power, ultrasonic time 3 seconds, interval time 3 seconds, running for 25-30 minutes). Centrifuged at 4°C, 12,000 g for 30 minutes, and the supernatant and precipitate were collected for sample preparation. The expression of the fusion protein in the supernatant and precipitate was detected by SDS-PAGE. The results are shown in Figure 2. Figure 3 shown.

[0047] According to the protein quantitative standard, the optical density of the target band was analyzed using ImageJ gel quantitative analysis software to calculate the yield of the fusion protein in the lysis supernatant. The results are shown in Table 2.

[0048] Table 2 Expression of five different ELP-SUMO-RFP fusion proteins

[0049]

[0050] Example 3: 0.7M (NH4)2SO4-mediated ELP-SUMO-RFP phase transition and protein purification by Ulp1 cleavage

[0051] After adding an equal volume of 1.4M (NH4)2SO4 solution to the lysis supernatant obtained in Example 2, the suspension was placed at 40°C for 15 to 20 minutes to allow the fusion protein to fully aggregate. The suspension was then centrifuged at room temperature and 12,000g for 20 minutes. The precipitate after centrifugation was fully resuspended with an equal volume of PBS buffer (after an ice bath), incubated on ice for 1 hour, and mixed every 20 minutes to allow the target protein to fully dissolve in the solution. The suspension was then centrifuged at 4°C and 12,000g for 30 minutes to separate the supernatant and precipitate. The supernatant after cell lysis, the supernatant and precipitate after salt aggregation, the supernatant and precipitate after redissolution, and the supernatant and precipitate after Ulp1 enzyme digestion and salt aggregation were subjected to SDS-PAGE detection together. The results are as follows. Figure 3 According to the protein quantitative standard, ImageJ gel quantitative analysis software was used to perform optical density analysis on the target bands to calculate the aggregation efficiency of the fusion protein, the yield and purity of the target protein. The results are shown in Table 4.

[0052] Table 3 Protein purification of ELP-SUMO-RFP phase transition and Ulp1 cleavage mediated by 0.7M (NH4)2SO4

[0053]

[0054] Example 4: NaCl-mediated phase transition of three ELP-SUMO-RFP and protein purification of Ulp1 cleavage

[0055] Add equal volumes of NaCl solutions of different concentrations to the lysis supernatant obtained in Example 2, and place at 37°C for 15 to 20 minutes to allow the fusion protein to fully aggregate. Centrifuge the suspension at room temperature and 12,000g for 20 minutes, resuspend the precipitate after centrifugation with an equal volume of PBS buffer (after ice bath), incubate on ice for 1 hour, and mix every 20 minutes to allow the target protein to fully dissolve in the solution. Centrifuge the suspension at 4°C and 12,000g for 30 minutes to separate the supernatant and precipitate. The supernatant after cell lysis, the supernatant and precipitate after salt aggregation, the supernatant and precipitate after redissolution, and the supernatant and precipitate after Ulp1 enzyme digestion and salt aggregation were subjected to SDS-PAGE detection together. The results are as follows. Figure 4 According to the protein quantitative standard, ImageJ gel quantitative analysis software was used to perform optical density analysis on the target bands to calculate the aggregation efficiency of the fusion protein, the yield and purity of the target protein. The results are shown in Table 4.

[0056] Table 4 Protein purification results of ELP-SUMO-RFP phase transition and Ulp1 cleavage mediated by different concentrations of NaCl

[0057]

[0058] The experimental results showed that the aggregation effect of all fusion proteins increased with the increase of NaCl concentration; among them, the ELP36-SUMO-RFP fusion protein was not very sensitive to NaCl and the purification effect was poor, while the other two fusion proteins responded better to NaCl and the purity of the obtained target proteins was higher.

[0059] Example 5: (NH4)2SO4-mediated phase transitions of three ELP-SUMO-RFPs and protein purification by Ulp1 cleavage

[0060] Add equal volumes of (NH4)2SO4 solutions of different concentrations to the lysis supernatant obtained in Example 2, and place at 37°C for 15 to 20 minutes to allow the fusion protein to fully aggregate. Centrifuge the suspension at room temperature and 12,000g for 20 minutes, resuspend the precipitate after centrifugation with an equal volume of PBS buffer (after ice bath), incubate on ice for 1 hour, and mix every 20 minutes to allow the target protein to fully dissolve in the solution. Centrifuge the suspension at 4°C and 15,000g for 30 minutes to separate the supernatant and precipitate. The supernatant after cell lysis, the supernatant and precipitate after salt aggregation, the supernatant and precipitate after redissolution, and the supernatant and precipitate after Ulp1 enzyme digestion and salt aggregation were subjected to SDS-PAGE detection together. The results are as follows. Figure 4 According to the protein quantitative standard, ImageJ gel quantitative analysis software was used to perform optical density analysis on the target bands to calculate the aggregation efficiency of the fusion protein, the yield and purity of the target protein. The results are shown in Table 5.

[0061] Table 5 Protein purification results of ELP-SUMO-RFP phase transition and Ulp1 cleavage mediated by different concentrations of (NH4)2SO4

[0062]

[0063] The experimental results show that all three fusion proteins have a good salt response effect on (NH4)2SO4. As the concentration of (NH4)2SO4 gradually increases, the aggregation efficiency improves. Moreover, under the condition of 0.7M (NH4)2SO4, the aggregation effect and re-dissolution effect of the three fusion proteins are all good.

[0064] Example 6: Temperature-mediated phase transition of ELP-SUMO-RFP

[0065] An equal volume of 1.4M (NH4)2SO4 solution was added to the lysis supernatant obtained in Example 2, and the suspension was placed at different temperatures for 15 to 20 minutes to allow the fusion protein to fully aggregate. The suspension was then centrifuged at room temperature and 12,000g for 20 minutes. The precipitate after centrifugation was fully resuspended with an equal volume of PBS buffer (after an ice bath), incubated on ice for 1 hour, and mixed every 20 minutes to allow the target protein to fully dissolve in the solution. The suspension was then centrifuged at 4°C and 12,000g for 30 minutes to separate the supernatant and precipitate. The supernatant after cell lysis, the supernatant and precipitate after salt aggregation, the supernatant and precipitate after redissolution, and the supernatant and precipitate after Ulp1 enzyme digestion and salt aggregation were subjected to SDS-PAGE detection together. The results are as follows. Figure 5 According to the protein quantitative standard, ImageJ gel quantitative analysis software was used to perform optical density analysis on the target bands to calculate the aggregation efficiency of the fusion protein, the yield and purity of the target protein. The results are shown in Table 6.

[0066] Table 6 ITC protein purification of ELP-SUMO-RFP mediated by different temperatures

[0067]

[0068] The results showed that as the temperature increased, the aggregation efficiency of the fusion protein continued to increase. Above 40°C, the aggregation efficiency of the fusion protein was higher than 90%. However, after exceeding its phase transition temperature, the resolubilization efficiency of the fusion protein decreased because the protein underwent an irreversible phase transition, thus reducing the resolubilization efficiency of the fusion protein.

[0069] Example 7: Expression of ELP[V]48-SUMO-CPL-711 fusion protein

[0070] In this example, the expression vector used was ELP[V]48-SUMO-CPL-711, and the target protein was CPL-711, an engineered chimeric antibacterial lytic enzyme. Primers required for plasmid construction were designed using oligo 6 and synthesized by Tianyi Huiyuan, as shown in Table 7-1.

[0071] Table 7-1 Oligonucleotide primers used in this example

[0072]

[0073] Using the constructed plasmid pET30a-ELP[V]48-SUMO-RFP as a template, the primer pair Backbone-F and SUMO-R2 were used to amplify the Backbone-ELP[V]48-SUMO polynucleotide fragment by PCR; using pET30a-Sc-CPL-711 (preserved in the laboratory) as a template, the primer pair CPL-711-F and Backbone-R3 were used to amplify the CPL-711-Backbone polynucleotide fragment by PCR. The purified polynucleotide fragments were assembled by Gibson assembly, and the assembly products were transformed into Escherichia coli DH5α competent cells. Positive clones were screened, and the plasmids were extracted and sequenced to obtain the ELP[V]48-SUMO-CPL-711 plasmid.

[0074] The same method as in Example 2 was used to transform the above-constructed expression vector into Escherichia coli BL21 (DE3) competent cells. After expressing the corresponding fusion protein, the cells were disrupted and the expression of the fusion protein in the lysis supernatant was detected by SDA-PAGE. The results are shown in Table 7-2.

[0075] Table 7-2 Expression of ELP[V]48-SUMO-CPL-711 fusion protein

[0076]

[0077] Example 8: Protein Purification by 0.7M (NH4)2SO4-Mediated ELP[V]48-SUMO-CPL-711 and Ulp1-Mediated Cleavage

[0078] After adding an equal volume of 0.4 M (NH₄)₂SO₄ solution to the lysate obtained in Example 7, the ELP[V]48-SUMO-CPL-711 was allowed to react at room temperature for 10-15 minutes to allow the fusion protein to fully aggregate. The suspension was then centrifuged at 12,000 g for 20 minutes at room temperature. The pellet was thoroughly resuspended in an equal volume of PBS buffer (after an ice bath) and incubated on ice for 1 hour, mixing every 20 minutes to allow the target protein to fully reconstitute with the solution. The suspension was then centrifuged at 15,000 g for 30 minutes at 4°C to separate the supernatant and pellet. The separated supernatant was then digested with Ulp1 at 30°C for 3 hours. (NH₄)₂SO₄ was then added to a final concentration of 0.7 M, and the pellet was allowed to stand at room temperature for 10-15 minutes before centrifugation. The supernatant after cell lysis, the supernatant and precipitate after salt aggregation, the supernatant and precipitate after resolubilization, and the supernatant after Ulp1 enzyme digestion and salt aggregation were subjected to SDS-PAGE detection. The results are as follows. Figure 6According to the protein quantitative standard, ImageJ gel quantitative analysis software was used to perform optical density analysis on the target bands to calculate the fusion protein aggregation efficiency, target protein yield and purity. The results are shown in Table 8.

[0079] Table 8 Protein purification of 0.2M (NH4)2SO4-mediated ELP[V]48-SUMO-CPL-711 phase transition and Ulp1 cleavage

[0080]

[0081] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

Claims

1. A method for purifying a target protein, characterized in that: The following steps are involved: (1) expressing a fusion protein comprising a target protein, an ELP tag, and a SUMO tag, wherein the ELP tag has an amino acid sequence as shown in SEQ ID NO: 1-5, and the SUMO tag has an amino acid sequence as shown in SEQ ID NO: 6; (2) Inducing the fusion protein to form reversible aggregates by adjusting the temperature or salt ion concentration, and then separating them by centrifugation; (3) Ulp1 protease cleaves the peptide bond between the SUMO tag and the target protein to release the target protein; (4) Adjust the temperature or salt concentration again to separate the target protein and the ELP tag.

2. The method for purifying a target protein according to claim 1, characterized in that: The ELP tag consists of a repeating unit (VPGXG)n, wherein X is K, V, F, I, S, A or G, and n is 36, 48, 96 or 80; the target proteins are red fluorescent protein RFP and CPL-711, the amino acid sequence of the red fluorescent protein RFP is SEQ ID NO: 7, and the DNA sequence of the CPL-711 is SEQ ID NO:

8.

3. The method for purifying a target protein according to claim 1, characterized in that: The SUMO tag and the target protein are connected via a linker.

4. The method for purifying a target protein according to claim 3, characterized in that: The linker is GGGGS.

5. The method for purifying a target protein according to claim 1, characterized in that: In step (2), the adjustment temperature is 30-60°C, and the salt ions are salt ions generated by NaCl or (NH4)2SO4; wherein the adjusted concentration of (NH4)2SO4 is 0.4-1M, and the adjusted concentration of NaCl is 1-2M.

6. The method for purifying a target protein according to claim 1, characterized in that: In step (4), the adjustment temperature is room temperature or 50° C., and the salt ion concentration is ≤0.7M (NH 4 ) 2 SO 4 .

7. A polynucleotide, characterized in that Encodes the fusion protein according to claim 1.

8. An expression vector, characterized in that Comprising the polynucleotide of claim 8.

9. A host cell, characterized in that The method comprises the polynucleotide according to claim 7 or the expression vector according to claim 8, and is capable of expressing the fusion protein.

10. The host cell according to claim 9, characterized in that It is Escherichia coli (E. coli).

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