Bioengineering-based elastin-like protein purification method

Abstract

The invention discloses a bioengineering-based elastin-like protein purification method, which comprises: 1) obtaining an engineering bacterium thallus expressing elastin-like protein, and re-suspending the thallus by using a buffer solution to obtain a thallus re-suspension; 2) adding lysozyme and DNA enzyme into the thallus resuspension, heating to 35-55 DEG C, and stirring for 0.5-2 hours to obtain a thallus lysate; 3) rapidly cooling to 2-10 DEG C, and centrifuging to obtain clear supernate; 4) purifying the supernate through cation exchange chromatography to obtain a protein solution; 5) performing ultrafiltration desalting treatment on the protein liquid to obtain a pure protein aqueous solution; and 6) freeze-drying to obtain the protein. According to the method, the pure protein can be obtained by purifying the protein twice in a thermal cracking mode, the protein recovery efficiency is improved, the production cost is low, meanwhile, efficient purification of the K protein independent of His tag peptide is achieved, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of elastin-like protein purification, in particular to a method for purifying elastin-like protein based on bioengineering. Background Art

[0002] Elastin-like proteins are derived from the characteristic repeating sequence (VPGXG) in the hydrophobic region of elastin in the human extracellular matrix. When X is replaced by any amino acid, functional proteins with different physical characteristics can be obtained, namely, elastin-like polypeptides (ELPs). ELPs have typical flexible segment characteristics, biocompatibility and temperature sensitivity, and are widely used in the fields of flexible protein linkers and thermosensitive protein functional materials in scientific research. When the X amino acid residue is mutated to lysine (K protein), the isoelectric point of the ELPs polypeptide with continuous repeating sequences is greater than 9.50, and it has a transcellular membrane delivery effect. It has been verified that it has good transmembrane delivery effect and biocompatibility, and also provides abundant protein coupling sites (-NH), which can be used to provide a cross-linked network of adhesives.

[0003] The team of Changchun Institute of Applied Chemistry of Chinese Academy of Sciences disclosed in the invention patent ZL202010111619.5 that the ELP protein was obtained by fermentation of Escherichia coli cell chassis and carried the His tag end (HHHHHH), and after high-pressure homogenization and cell disruption, the elastin-like protein ELP was obtained through nickel affinity chromatography medium, solution desalination and liquid exchange, SP ion exchange chromatography medium, solution desalination and liquid exchange, etc., with a protein purity of 99%. Beijing Kangpai Medical Equipment Co., Ltd. also disclosed in the invention patent ZL202410077756.X that the cell fragment mixture was broken by high-pressure homogenization, and after centrifugation and separation of the supernatant, the His tag peptide (HHHHHH) at the end of the protein was adsorbed under the action of nickel affinity chromatography medium, and a relatively pure protein was obtained by gradient elution with imidazole buffer; and then through membrane package ultrafiltration, cation exchange chromatography, and membrane package ultrafiltration; a protein with high purity was obtained.

[0004] It should be noted that when the tag peptide His is used as a cross-linking adhesive component, the histidine residue terminal does not participate in the cross-linking reaction; and when K protein acts as a protein delivery carrier, it mainly provides affinity with the cell membrane through the lysine residue to exert a transmembrane effect, and histidine does not participate in this process. Proteins containing the tag peptide His that have no actual function are usually difficult to be approved for use in biological products. That is, it is necessary to develop an efficient purification method for K protein that is independent of the His tag peptide. Summary of the invention

[0005] The purpose of the present invention is to provide a purification method of elastin-like protein based on bioengineering, so as to solve the problem that the existing technology overly relies on the efficient purification method of K protein of His tag peptide, resulting in the protein product being difficult to be approved for use in biological products.

[0006] In order to solve the above problems, the present invention adopts the following technical solutions:

[0007] According to the present invention, a method for purifying elastin-like protein based on bioengineering is provided, comprising the following steps: 1) obtaining engineered bacterial cells expressing elastin-like protein, resuspending the cells with a buffer solution, and obtaining a bacterial resuspension; 2) adding lysozyme and DNA enzyme to the bacterial resuspension, heating to 35-55°C, stirring for 0.5-2h, and obtaining a bacterial lysis solution; 3) rapidly cooling to 2-10°C and centrifuging to obtain a clarified supernatant; 4) purifying the supernatant by cation exchange chromatography to obtain a protein solution; 5) subjecting the protein solution to ultrafiltration and desalination treatment to obtain a pure protein aqueous solution; and 6) freeze-drying to obtain the protein.

[0008] Preferably, in step 1), every 1 g of engineered bacterial cells is resuspended in 10-20 mL of lysis buffer.

[0009] Preferably, in step 2), the mixture is heated to 35-50° C. and stirred for 0.5-1 h.

[0010] Preferably, in step 2), the amount of lysozyme added is 0.4-1 mg / L, and the amount of DNase added is 1-5 μg / mL.

[0011] Preferably, in step 3), the bacterial lysis solution is rapidly cooled to 2-4°C.

[0012] Preferably, step 4) comprises: using buffer A to balance the ion exchange column, then loading the sample, and after loading, using buffer A to balance until the chromatograph shows that the A280 absorption peak returns to the baseline value before loading ~ baseline + 20 value, and then using buffer B1 and buffer B2 of different concentrations for elution in turn to obtain an eluate, i.e., a protein solution.

[0013] Preferably, the A buffer comprises 2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, 20-80mM NaCl, and its pH is 7.8-8.0; the B1 buffer comprises 2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, 0-200mM NaCl, and its pH is 7.8-8.0; the B2 buffer comprises 2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, 200-800mM NaCl, and its pH is 7.8-8.0.

[0014] More preferably, the A buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 50 mM NaCl, and its pH is 7.8-8.0; the B1 buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 100 mM NaCl, and its pH is 7.8-8.0; the B2 buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 500 mM NaCl, and its pH is 7.8-8.0.

[0015] Preferably, step 5) comprises: placing the protein solution in a barrel, performing tangential flow filtration on the protein solution using an ultrafiltration membrane package with a molecular weight cutoff of 3-10 kDa, or a hollow fiber with a molecular weight cutoff of 3-10 kDa, or a spiral membrane material with a molecular weight cutoff of 3-10 kDa, during which purified water, water for injection, or sterile water is added to the barrel, and the desalination is terminated when the conductivity value of the concentrated protein liquid decreases to ≤5.0 μS / cm, or the conductivity value of the concentrated protein liquid remains unchanged after three consecutive water replenishments, and the desalination is deemed to be completed.

[0016] Preferably, the amino acid sequence of the elastin-like protein is the sequence shown in SEQ ID NO.1, or multiple copies of the sequence shown in SEQ ID NO.1 connected by amino acid linkers, such as 8 copies of the protein molecule, and the sequence is shown in SEQ ID NO.2.

[0017] It should be understood that the method of the present invention is applicable to all sequences containing the repeating unit. The reason is that the ratio of lysine to total protein of the protein shown in SEQ ID NO.1 and its various copies remains unchanged, and its isoelectric point does not change much. Cation exchange technology uses the different charged properties of proteins in solution to exchange proteins through competitive ions, which is directly related to the isoelectric point of proteins.

[0018] According to the elastin-like protein purification method provided by the present invention, bacteria are lysed and broken under the action of enzymes, and intracellular substances are released to form bacterial lysate; a large amount of protein in the bacterial lysate changes structure, loses activity and forms precipitation under high temperature heating, and the target protein K protein has good heat resistance and can resist high temperature treatment, so it remains in the supernatant of the lysate, and it can be separated by centrifugation to obtain the supernatant and remove a large amount of impurities. Before centrifugation, the heated solution is rapidly cooled at low temperature to promote the yellow pigment in the lysate to enter the precipitation component, and this pigment cannot be removed by filter in the dissolved state. The supernatant obtained by centrifugation is washed by cation exchange chromatography, and then the target protein is eluted with 200-500mM sodium chloride buffer to obtain K protein salt solution. Subsequently, it is treated by ultrafiltration to obtain a pure protein aqueous solution for protein freeze-drying operation.

[0019] According to a preferred embodiment of the present invention, the purification method comprises:

[0020] Step 1), the bacterial cells and the lysis solution are mixed at a mass volume ratio of 1:10 to 1:20, that is, 10 to 20 mL of lysis solution is used to resuspend 1 g of bacterial cells to obtain a bacterial resuspension. According to tests, proteins below this ratio cannot be completely dissolved in the supernatant, forming a precipitate that is removed during the centrifugation stage; the volume of the bacterial lysis solution above this ratio is too large and is not suitable for industrial production.

[0021] Step 2), during the lysis process, the bacterial cells are dissolved in the lysis solution, the temperature is raised to 30°C, lysozyme and DNA enzyme are added, mixed evenly and kept warm for half an hour to obtain a mixture. The mixture is heated to 35-55°C, kept warm for 0.5-2h, and quickly cooled to 2-10°C to obtain a bacterial lysis solution.

[0022] Step 3), the lysate is centrifuged and filtered to collect the supernatant to obtain a clarified supernatant. At this stage, the amount of lysozyme added is 0.4-1 mg / L, and the amount of DNA enzyme added is 1-5 μg / mL. In some studies, magnesium salts need to be supplemented while adding DNA enzyme to lyse bacteria. The present invention finds that when the buffer pH is ≥7.4, magnesium salts will form magnesium hydroxide precipitates in the lysate environment and form white precipitates, reducing the lysis efficiency; adding 1-5 μg / mL DNA enzyme and heating at the same time can also increase the enzyme efficiency. Therefore, under the treatment system of the present invention, there is no need to add magnesium ions.

[0023] Step 4), the clarified supernatant is subjected to cation exchange chromatography to obtain a protein solution. Based on the molecular properties of K protein, a strong cation exchange medium is preferably used for the separation of the target protein from impurities in the protein in the alkaline buffer, so an SP chromatography medium is used. The specific operation steps are: the ion exchange column is balanced with A buffer, and then the sample is loaded. After the loading is completed, the A buffer is used for balance until the chromatograph shows that the A280 absorption peak returns to the baseline value before loading ~ baseline + 20 value, and then different concentrations of B1 buffer and B2 buffer are used for elution to obtain the eluate, i.e., the protein solution. Preferably, the equilibrium endpoint after loading is the baseline value before loading ~ baseline + 10 value. Among them, buffer A includes 2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, and 50mM NaCl, and its pH is 7.8-8.0; buffer B1 includes 2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, and 100mM NaCl, and its pH is 7.8-8.0; buffer B2 includes 2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, and 500mM NaCl, and its pH is 7.8-8.0.

[0024] By adopting the above technical scheme, the chloride ions in the B1 buffer and the B2 buffer can produce a concentration-dependent ion exchange reaction with the K protein adsorbed on the chromatographic medium. At low concentrations, the impurity proteins that are not tightly bound to the medium can be eluted, and at high elution concentrations, a large amount of the target protein can be eluted, thereby achieving the separation of the target protein.

[0025] By adopting the above technical scheme, sodium dihydrogen phosphate and disodium hydrogen phosphate buffer pairs are used as the basic components of the buffer solution, it is unnecessary to adjust the pH during the solution preparation process to introduce chloride ions, and the configuration stability of each batch of buffer solution can be controlled; the two buffer salts in this ratio can adjust the system pH to 7.8-8.0, and the pH is not reduced by the influence of high concentration salt (sodium chloride); the cost of the buffer salt is much lower than that of trisodium phosphate, and it is suitable for industrial production and preparation processes.

[0026] Step 5), the mixture obtained in step 4) is subjected to ultrafiltration desalination treatment. When the ultrafiltration medium is pure water, the salt component in the protein can be removed so that the salt residue does not affect its use in the fields of biology and chemistry, that is, this step obtains a pure protein aqueous solution. The specific operation is: the protein solution is placed in a bucket, and the protein solution is subjected to tangential flow filtration treatment with an ultrafiltration membrane bag with a molecular weight cutoff of 3-10kDa, or a hollow fiber with a molecular weight cutoff of 3-10kDa, or a roll membrane material with a molecular weight cutoff of 3-10kDa, during which purified water or water for injection (cooling) or sterile water is added to the bucket. The water replenishment time point is when the protein solution is concentrated to 5 times the initial volume, and the water is replenished to the original volume. During this period, the conductivity value of the solution in the bucket is detected, and when the value is reduced to ≤5.0μS / cm, or when the conductivity value of the concentrated protein liquid concentrated after 3 consecutive water replenishments remains unchanged, it is determined that the desalination ends. Preferably, the tangential flow filtration medium adopts an ultrafiltration membrane bag.

[0027] The main inventive point of the present invention is that, on the one hand, the bacterial cells are treated by thermal lysis means. According to the characteristics of K protein, a thermal lysis scheme is adopted. During the heating process, some impurities can be denatured, and the impurities in the protein lysate can be efficiently removed, thereby reducing the pressure of subsequent protein purification. That is, the number of chromatography operations is reduced, thereby improving the protein yield; on the other hand, through the "heating + rapid cooling" method, the precipitation of impurities such as pigments in the cell lysate can be promoted, and the protein can be separated from the aqueous phase where the protein is located by centrifugation, thereby improving the protein purity. According to the protein purification process provided by the present invention, the limit of introducing affinity chromatography tags for purification of K protein has been broken, and the charge characteristics of K protein itself have been used to optimize the protein purification process, reduce the chromatography steps, and improve the protein yield.

[0028] The bioengineering-based purification method of elastin-like protein provided by the present invention has the following beneficial effects compared with the prior art:

[0029] (1) Use thermal lysis to treat bacteria instead of high-pressure homogenization. High-pressure homogenization is usually carried out at 2-8°C, which greatly protects the stability of all proteins. However, it introduces a large amount of non-target proteins into the chromatography medium, which puts pressure on the chromatography stage and reduces the purity of the first chromatography, thereby increasing the number of chromatography times and reducing the yield. The present invention uses an enzymatic thermal lysis scheme for K protein. K protein is thermally stable and can denature some impurities during the heating process, thereby obtaining a purer protein lysate, improving the purity of the first chromatography protein, and thus improving the purification yield.

[0030] (2) According to the present invention, by rapidly cooling the solution after lysis in the lysis stage, the precipitation of pigments and pigment-protein complexes can be promoted, and the pigments and pigment-protein complexes can be removed by centrifugation, thereby improving protein purity and reducing costs. The bacterial lysate obtained by the high-pressure homogenization method contains pigment components. The pigments usually require a long period of equilibrium to be washed out from the gaps in the column material during the chromatography process, which increases the amount of materials used in the purification stage, thereby increasing the preparation cost.

[0031] In summary, the present invention improves and optimizes the reported protein purification process, which not only reduces the purification steps but also improves the purification efficiency. On the basis of ensuring protein purity, it further reduces the use of solution and reduces the purification cost. At the same time, it also realizes the efficient purification of K protein that is independent of His tag peptide, avoiding the problem of difficulty in being approved for use in biological products due to the addition of tag peptide His that has no actual function. Therefore, it has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The eluates of various components obtained by ion exchange chromatography of the protein lysate in step (4) of Example 1 are shown;

[0033] Figure 2 The SDS-PAGE electrophoresis diagrams of the supernatant and precipitate samples of the protein lysate in step (3) of Examples 1 to 5 are shown, comparing the differences in the components of the protein lysate treated at different temperatures (30 to 50° C.);

[0034] Figure 3 The SDS-PAGE electrophoresis diagrams of the supernatant and precipitate samples of the protein lysate in step (3) of Examples 6 to 8 are shown, and the differences in the components of the protein lysate treated at different dilution ratios of bacteria to lysate (1:10, 1:15, 1:20) are compared;

[0035] Figure 4 The SDS-PAGE electrophoresis diagrams of the protein lysate supernatant and precipitate samples in step (3) of Examples 9 to 12 are shown, comparing the differences in the composition of the protein lysate under different lysis times (0.5 h, 1.0 h, 1.5 h, 2.0 h);

[0036] Figure 5 The SDS-PAGE electrophoresis diagrams of the supernatant samples of the protein lysate in step (3) of Example 1 and Example 13 to Example 19 are shown, comparing the differences in the composition of the protein lysate under treatment with different lysozyme concentrations. DETAILED DESCRIPTION

[0037] The present application is further described in detail below in conjunction with the examples. The raw materials used in the examples and comparative examples can all be obtained from commercial sources.

[0038] Example 1

[0039] The method for purifying the K protein solution of SEQ ID NO.2 comprises the following steps:

[0040] (1) Fermenting and centrifuging Escherichia coli, taking the solid and mixing it with the lysate to prepare a primary mixed solution. The mass ratio of the solid to the lysate is 1:10;

[0041] (2) heating the initial mixed solution obtained in step (1) to 30° C., adding lysozyme and DNase, mixing well and keeping warm for 30 min to obtain a mixture, wherein the amount of lysozyme added is 0.6 g / L and the amount of DNase added is 5 μg / ml;

[0042] (3) The mixture obtained in step (2) was heated to 50° C., kept warm for 30 min, rapidly cooled to 2-10° C., centrifuged, and filtered to obtain a clear supernatant.

[0043] (4) The clarified supernatant is chromatographed on an ion exchange chromatography column. Use A buffer (2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, 50mM NaCl (pH 7.8-8.0)) to balance the ion exchange column, then load the sample. After loading, use A buffer to balance until the chromatograph shows that the A280 absorption peak returns to the baseline value before loading ~ baseline + 20 value; then use B1 buffer (2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, 100mM NaCl (pH 7.8-8.0)) and B2 buffer (2.65mM sodium dihydrogen phosphate, 47.35mM sodium dihydrogen phosphate, 500mM NaCl (pH 7.8-8.0)) to elute in turn, collect the target protein peak, and obtain the protein eluate;

[0044] (5) The protein eluate obtained in step (4) is subjected to desalination treatment by an ultrafiltration membrane package, wherein the ultrafiltration medium is pure water, and the ultrafiltration is terminated when the conductivity value of the protein solution decreases to ≤5.0 μS / cm, or when the conductivity value of the concentrated protein liquid remains unchanged after three consecutive water replenishments, thereby obtaining a protein solution.

[0045] The protein solution can be freeze-dried or used directly.

[0046] Example 2

[0047] The difference from Example 1 is that in step (3), the mixture is heated to 35° C. and then kept at a constant temperature for 30 minutes.

[0048] Example 3

[0049] The difference from Example 1 is that in step (3), the mixture is heated to 40° C. and then kept at a constant temperature for 30 minutes.

[0050] Example 4

[0051] The difference from Example 1 is that in step (3), the mixture is heated to 45° C. and then kept at a constant temperature for 30 minutes.

[0052] Example 5

[0053] The difference from Example 1 is that in step (3), the mixture is heated to 50° C. and then kept at a constant temperature for 30 minutes.

[0054] Example 6

[0055] The difference from Example 1 is that the ratio of the mixed material bacteria to the lysate in step (1) is 1:10; and the amount of lysozyme added in step (2) is 1.0 g / L.

[0056] Example 7

[0057] The difference from Example 1 is that the ratio of the mixed material bacteria to the lysate in step (1) is 1:15; and the amount of lysozyme added in step (2) is 1.0 g / L.

[0058] Example 8

[0059] The difference from Example 1 is that the ratio of the mixed material bacteria to the lysate in step (1) is 1:20; and the amount of lysozyme added in step (2) is 1.0 g / L.

[0060] Example 9

[0061] The difference from Example 1 is that in step (3), the mixture is heated to 55° C. and then kept at a constant temperature for 30 minutes.

[0062] Example 10

[0063] The difference from Example 1 is that in step (3), the mixture is heated to 55° C. and then kept at a constant temperature for 60 minutes.

[0064] Embodiment 11

[0065] The difference from Example 1 is that in step (3), the mixture is heated to 55° C. and then kept at a constant temperature for 1.5 hours.

[0066] Example 12

[0067] The difference from Example 1 is that in step (3), the mixture is heated to 55° C. and then kept at a constant temperature for 2.0 h.

[0068] Embodiment 13

[0069] The difference from Example 1 is that the mass ratio of solid to lysate in step (1) is 1:5; and the amount of lysozyme added in step (2) is 0.4 g / L.

[0070] Embodiment 14

[0071] The difference from Example 1 is that: in step (1), the mass ratio of solid to lysate is 1:5; and in step (2), the amount of lysozyme added is 0.6 g / L.

[0072] Embodiment 15

[0073] The difference from Example 1 is that: in step (1), the mass ratio of solid to lysate is 1:5; and in step (2), the amount of lysozyme added is 0.8 g / L.

[0074] Example 16

[0075] The difference from Example 1 is that the mass ratio of the solid to the lysate in step (1) is 1:5; and the amount of lysozyme added in step (2) is 1.0 g / L.

[0076] Embodiment 17

[0077] The difference from Example 1 is that the amount of lysozyme added in step (2) is 0.4 g / L.

[0078] Embodiment 18

[0079] The difference from Example 1 is that the amount of lysozyme added in step (2) is 0.8 g / L.

[0080] Embodiment 19

[0081] The difference from Example 1 is that the amount of lysozyme added in step (2) is 1.0 g / L.

[0082] In summary, the technical route of the present invention can effectively remove foreign proteins and pigment components in the solution through a combination of bacterial lysis and rapid cooling of the solution after heating. At the same time, through the comparison results of Examples 1 to 7, it can be found that within the temperature range of 30°C to 55°C, as the lysis temperature increases, the relative content of foreign proteins in the protein supernatant decreases, and the effect is comparable to the crushing effect of a high-pressure homogenizer. When the temperature is in the range of 30 to 50°C, the content of target protein in the precipitate is relatively low; when the temperature rises to 55°C, the content of target protein in the precipitate increases, resulting in a decrease in yield. When the treatment temperature is prolonged, the target protein will gradually enter the precipitate. In order to ensure the lysis effect, a lysis time of 30 minutes is optimal. Figure 3 The results show that the optimal ratio of bacteria to lysis solution is 1:15-1:20. The lysis ratio of 1:10 may be due to protein solubility problems, and the protein content in the supernatant is reduced because it is not fully released into the solution. When the solution volume increases by 15-20 times, there is no significant difference in the total content of the target protein, indicating that the protein has been fully released into the supernatant. The bacterial dilution ratio can be adjusted within this range according to the actual production requirements. Figure 4 , Figure 5 In the test of lysozyme addition concentration, it was found that under different bacterial dilution solid-liquid ratios, lysozyme addition concentrations of 0.6-1.0g / L had almost the same bacterial lysis effect.

[0083] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for purifying elastin-like protein based on bioengineering, characterized in that: The following steps are involved: 1) obtaining engineered bacterial cells expressing elastin-like protein, and resuspending the cells in a buffer to obtain a resuspended cell liquid; 2) adding lysozyme and DNase to the bacterial resuspension, heating to 30-50° C., and stirring for 0.5-2 h to obtain a bacterial lysis solution; 3) Rapidly cool to 2-10°C and centrifuge to obtain a clear supernatant; 4) purifying the supernatant by cation exchange chromatography to obtain a protein solution; 5) treating the protein solution by ultrafiltration and desalting to obtain a pure protein aqueous solution; 6) Freeze-dry to obtain protein.

2. The purification method according to claim 1, characterized in that In step 1), 10 to 20 mL of lysis buffer is used to resuspend 1 g of engineered bacterial cells.

3. The purification method according to claim 1, characterized in that In step 2), heat to 40-50° C. and stir for 0.5-1 h.

4. The purification method according to claim 1, characterized in that In step 2), the amount of lysozyme added is 0.4-1 mg / L, and the amount of DNase added is 1-5 μg / mL.

5. The purification method according to claim 1, characterized in that In step 3), the bacterial lysate is rapidly cooled to 2-4°C.

6. The purification method according to claim 1, characterized in that Step 4) comprises: using buffer A to balance the ion exchange column, and then loading the sample. After the loading is completed, using buffer A to balance until the chromatograph shows that the A280 absorption peak returns to the baseline value before loading ~ baseline + 20 value, and then using buffer B1 and buffer B2 of different concentrations to elute in turn to obtain an eluate, i.e., a protein solution.

7. The purification method according to claim 1, characterized in that The A buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 20-80 mM NaCl, and its pH is 7.8-8.0; the B1 buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 0-200 mM NaCl, and its pH is 7.8-8.0; the B2 buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 200-800 mM NaCl, and its pH is 7.8-8.

0.

8. The purification method according to claim 7, characterized in that The A buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 50 mM NaCl, and its pH is 7.8-8.0; the B1 buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 100 mM NaCl, and its pH is 7.8-8.0; the B2 buffer comprises 2.65 mM sodium dihydrogen phosphate, 47.35 mM sodium dihydrogen phosphate, and 500 mM NaCl, and its pH is 7.8-8.

0.

9. The purification method according to claim 1, characterized in that Step 5) comprises: placing the protein solution into a barrel, and subjecting the protein solution to tangential flow filtration using an ultrafiltration membrane package with a molecular weight cutoff of 3-10 kDa, or a hollow fiber with a molecular weight cutoff of 3-10 kDa, or a spiral membrane material with a molecular weight cutoff of 3-10 kDa, during which purified water, water for injection, or sterile water is added to the barrel, and the desalination process is terminated when the conductivity value of the concentrated protein liquid decreases to ≤5.0 μS / cm, or when the conductivity value of the concentrated protein liquid remains unchanged after three consecutive water additions.

10. The purification method according to claim 1, characterized in that: The amino acid sequence of the elastin-like protein is the sequence shown in SEQ ID NO.1, or multiple copies of the sequence shown in SEQ ID NO.1 connected by amino acid linkers.

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