A purification method for humanized elastin expressed in Pichia pastoris

Abstract

The present invention discloses a method for purifying humanized elastin expressed in Pichia pastoris. Sodium chloride is added to the fermentation broth of engineered Pichia pastoris, stirred at room temperature, and then centrifuged. Impurities in the precipitate are cleaned and redissolved in a urea solution. Clarification by filtration, ultrafiltration concentration, and freeze-drying are performed to obtain humanized elastin with a purity greater than 95%. The yield of the pure product reaches over 0.8 g per liter of fermentation broth, with a yield greater than 70%. This method solves the problems of high production equipment requirements, lengthy purification processes, and low yields in the fermentation production of humanized elastin.

Description

Technical Field

[0001] The invention belongs to the technical field of protein engineering, and relates to an industrial separation and purification method for a crude humanized elastin product liquid obtained by fermentation. Background Art

[0002] Elastin is the primary component of elastic fibers in hide tissue. Its peptide chain contains over 713 amino acid residues. Elastin extracted from bovine cervical ligament has been shown to exhibit superoxide radical scavenging activity, whereas human elastin expressed through in vitro recombinant expression has not been reported to exhibit such biological activity. Human elastin has a relatively high molecular weight, and humanized elastin typically consists of a repeating structure composed of a portion of the complete human elastin sequence (providing proper folding). It is typically used as a scaffold material in the manufacture of cosmetics and medical aesthetic products.

[0003] The expression products of recombinant Escherichia coli and Pichia pastoris are fundamentally different, and obtaining the target protein (i.e., humanized elastin) using the latter offers greater safety during fermentation and purification processes. Current methods for purifying humanized elastin primarily utilize the thermal phase transition of elastin, a property of the protein that changes its solubility (from dissolved to insoluble and precipitated) when a heated dissolution system reaches a certain temperature. However, in practice, multiple thermal phase transitions are required (i.e., reversible temperature cycling: precipitation at temperatures exceeding 40°C followed by resolubilization at temperatures below 10°C; see "Optimization of Expression Conditions and Purification of Human-like Elastin Polypeptide Genes"). This results in significant equipment and energy consumption during operation. Furthermore, the thermal phase transition purification process requires strict temperature control to achieve optimal purification results. However, even with a purity exceeding 95%, this process often results in low yields and other indicators due to the multiple purification steps. Summary of the Invention

[0004] The purpose of the present invention is to provide a purification method for humanized elastin expressed in Pichia pastoris, which solves the problems of high functional requirements for production equipment, long purification process, low yield and pure product output caused by complicated temperature control of the thermal phase-change purification process during the fermentation production of humanized elastin.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The method for purifying humanized elastin comprises the following steps:

[0007] 1) Sodium salt is added to the fermentation broth of the engineered Pichia pastoris, followed by stirring at an ambient temperature of 20-30° C., followed by solid-liquid separation using a high-speed centrifuge, and the resulting precipitate is collected and recorded as solid phase A. The purpose of mixing the sodium salt with the fermentation broth of the engineered Pichia pastoris in this step is not to cause salting out, but to lower the temperature at which the humanized elastin in the fermentation broth undergoes phase transformation, thereby causing the solubility state of the target protein (i.e., humanized elastin) to change during stirring at room temperature, ultimately forming solid phase A composed of components such as the target protein precipitated by the phase transformation and bacterial cells in the fermentation broth.

[0008] 2) The precipitate collected in step 1 (i.e., solid phase A) is mixed with a solvent containing a buffer salt system and stirred at an ambient temperature of 20-30°C. The mixture is then subjected to solid-liquid separation in a high-speed centrifuge to remove soluble impurities (soluble impurities enter the supernatant). The precipitate obtained by centrifugation is collected and recorded as solid phase B. The purpose of this step is to clean the soluble impurities in solid phase A.

[0009] 3) The precipitate collected in step 2 (i.e., solid phase B) is mixed with a solvent containing urea to redissolve the target protein, and then the insoluble impurities are removed by solid-liquid separation using a high-speed centrifuge. The supernatant obtained by centrifugation is collected and recorded as the target protein extract;

[0010] 4) filtering the supernatant collected in step 3 through a microfiltration system to clarify the supernatant and collecting the filtrate;

[0011] 5) The filtrate collected in step 4) is concentrated by an ultrafiltration system to collect the retentate, and the retentate is freeze-dried to obtain a freeze-dried humanized elastin powder with a purity greater than 95%.

[0012] Preferably, in step 1, the fermentation broth of the Pichia pastoris engineered bacteria is a crude humanized elastin feed liquid, that is, the feed liquid containing the target protein and impurities (including bacteria and other impurity proteins) formed at the end of fermentation (fermentation temperature is about 30°C) of the fermentation system of the Pichia pastoris engineered bacteria for secreting and expressing humanized elastin. The feed liquid temperature for purification should generally be controlled at 28-35°C, so purification can generally be carried out directly after the fermentation is completed and the material is discharged.

[0013] Preferably, in step 1, the sodium salt is selected from any one of sodium ion inorganic salts such as sodium chloride that is not likely to cause salting out of impurities in the fermentation broth of the Pichia pastoris engineered bacteria within a certain addition concentration range, and the amount of sodium salt (such as sodium chloride) in the fermentation broth is 90 g / L to 117 g / L, the stirring time is 1 to 3 hours, and the stirring speed is 180 to 200 rpm.

[0014] Preferably, in step 2, the solvent is composed of a sodium salt (e.g., sodium chloride) and a buffer solution, and has a pH of 4 to 6. In this step, the solubility and dissolution efficiency of impurities in the buffer solution are increased by adding a sodium salt (e.g., sodium chloride), so that any buffer salt system capable of controlling the pH within the above-mentioned range is feasible as a solvent component, thereby dissolving impurities contained in the solid phase A using the corresponding buffer solution while adding a small amount of sodium salt (e.g., sodium chloride).

[0015] Preferably, in step 2, 1 kg of solid phase A is mixed with ≥ 3 L of 0.01-0.03 mol / L citric acid-trisodium citrate buffer containing 9.8 g / L-11.7 g / L sodium chloride and a pH of 4-6; the stirring time is 1-3 hours, and the stirring speed is 180-200 rpm. In this step of dissolving and cleaning impurities, the addition of a small amount of sodium chloride can increase the solubility and dissolution efficiency of the impurity protein in the buffer, that is, promote the dissolution of the impurity protein. However, if the amount of sodium chloride added is too large, the impurity protein may not be dissolved.

[0016] Preferably, in step 3, the redissolution specifically includes the following steps: using ≥3L of 300g / L~360g / L urea aqueous solution to dissolve 1Kg of solid phase B, mixing the solid phase B with the urea aqueous solution, and then stirring at an ambient temperature of 20~30°C, wherein the stirring time is 1.5~2h and the stirring speed is 180~200rpm.

[0017] Preferably, in steps 1, 2, and 3, the conditions for solid-liquid separation are: centrifugation at 20 to 30° C. and a speed of not less than 4000 r / min for 10 to 15 minutes.

[0018] Preferably, in step 4, the conditions for filtration and clarification include: a microfiltration pore size of 0.22 to 1 μm and a temperature of 20 to 30°C.

[0019] Preferably, in step 5, the concentration conditions include: a molecular weight cut-off of 1 to 6 kD and a temperature of 20 to 30°C.

[0020] The beneficial effects of the present invention are embodied in:

[0021] The present invention adopts a treatment process of adding sodium salt and stirring to achieve phase change of the target protein (i.e., humanized elastin) in the fermentation broth of Pichia pastoris engineered bacteria under room temperature and promote the precipitation of the target protein, thereby providing a method for purifying humanized elastin that can be operated at room temperature throughout the entire process, has a simple process flow (no need for multiple temperature change processes, and purification of the target protein can be achieved at room temperature), is stable (has good process reproducibility), and is low in cost.

[0022] Furthermore, in the purification process of the present invention, the temperature at which humanized elastin undergoes phase transformation is lowered by controlling the amount of sodium salt (e.g., sodium chloride) added, enabling rapid phase transformation and precipitation under mild conditions (including temperature and stirring speed). Purified humanized elastin (greater than 95% purity) is then obtained through impurity removal, redissolution, filtration clarification, ultrafiltration concentration, and freeze-drying. The yield of the pure product reaches over 0.8 g / L, with a yield exceeding 70%.

[0023] Furthermore, in the impurity removal process, the present invention adds an appropriate concentration of sodium salt (such as sodium chloride) to the buffer solution, which not only keeps the target protein separated by phase change from redissolving at room temperature, but also avoids salting out of the impurity proteins remaining in the phase change of the target protein while dissolving and removing the impurity proteins, thereby improving the impurity removal efficiency.

[0024] Furthermore, during the re-dissolution process, the present invention controls the concentration of the urea solution so that the target protein is quickly and completely dissolved at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The amino acid sequence design, theoretical isoelectric point, and theoretical molecular weight of the humanized elastin in the examples are shown.

[0026] Figure 2 The following is an SDS-PAGE electrophoresis analysis of samples from different stages of the humanized elastin purification process in Example 1; wherein, lane 1: protein marker; lane 2: fermentation broth; lane 3: supernatant obtained by adding sodium chloride to the fermentation broth, stirring, and centrifuging; lane 4: supernatant obtained by adding buffer and sodium chloride, stirring, and centrifuging; lane 5: supernatant obtained by dissolving in urea solution and centrifuging; lane 6: 2 mg / mL solution prepared from lyophilized powder; the sample volume for lanes 2 to 6 is 10 μL.

[0027] Figure 3 The SDS-PAGE electrophoresis detection diagram of the lyophilized powders obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4; wherein, lane 1: protein marker; lane 2: 2 mg / mL solution prepared from the lyophilized powder in Example 1; lane 3: 2 mg / mL solution prepared from the lyophilized powder in Comparative Example 1; lane 4: 2 mg / mL solution prepared from the lyophilized powder in Comparative Example 2; lane 5: 2 mg / mL solution prepared from the lyophilized powder in Comparative Example 3; lane 6: 2 mg / mL solution prepared from the lyophilized powder in Comparative Example 4; the sample volume for lanes 2 to 6 is 7 μL. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and examples. The examples are only used to explain the present invention but not to limit the scope of protection of the present invention.

[0029] (1) Fermentation to obtain crude humanized elastin solution

[0030] The humanized elastin-producing Pichia pastoris was constructed. The humanized elastin expressed by the genetically engineered bacteria has 273 amino acid residues. The specific amino acid sequence is shown in Figure 1 Determine the gene sequence based on the amino acid sequence and the codon preference of Pichia pastoris. Construction and transformation of the expression vector (containing the above gene sequence), and screening of positive recombinant Pichia pastoris (i.e., humanized elastin-producing Pichia pastoris) are as follows: Refer to the Pichia Pastoris Expression Operation Manual.

[0031] The humanized elastin-producing Pichia pastoris genetically engineered bacteria were cultured as primary seeds in a 10 L fermenter for 18 h. The cultured bacteria were inoculated into a 100 L fermenter as secondary seeds. The humanized elastin was secreted and expressed during the fermentation process, and the fermentation liquid was released after the fermentation was completed.

[0032] For the culture medium and specific culture conditions for the above-mentioned first-level seed culture, and the culture medium and specific culture conditions for fermentation, please refer to the Pichia pastoris Fermentation Manual.

[0033] (2) Purification process of humanized elastin

[0034] In the present invention, there is no need to centrifuge the humanized elastin Pichia pastoris genetically engineered fermentation system that has completed the fermentation process. The target protein (humanized elastin) can be directly separated and purified from the fermentation broth (containing the bacteria), as described below.

[0035] Example 1

[0036] 1) To 60 L of fermentation broth (29.1°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 19 kg.

[0037] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring for 2 h at room temperature, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.5 kg.

[0038] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 55 L.

[0039] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 59 L of the clarified filtrate was collected.

[0040] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 52 g of humanized elastin freeze-dried powder with a purity of 97%, i.e., a yield of 0.866 g / L per liter of fermentation broth, and a yield of 74%.

[0041] In the above purification process, the fermentation broth, the supernatant in steps 1, 2, and 3, and the solution prepared by the lyophilized powder in step 5 were subjected to SDS-PAGE analysis. The results are as follows: Figure 2 As shown in lanes 2, 3, 4, 5, and 6, the humanized elastin in the fermentation broth was effectively purified.

[0042] Example 2

[0043] 1) To 60 L of fermentation broth (29.1°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 19 kg.

[0044] 2) Add 0.01 mol / L citric acid-trisodium citrate buffer at pH 4.7 to the precipitate collected in step 1 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 3 hours, and then centrifuge at 4000 r / min for 15 minutes. Discard the supernatant and collect the precipitate. The precipitate weighs 18.5 kg.

[0045] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 55 L.

[0046] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 59 L of the clarified filtrate was collected.

[0047] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 51 g of humanized elastin freeze-dried powder with a purity of 97.1%, i.e., a yield of 0.85 g / L per liter of fermentation broth, and a yield of 74.1%.

[0048] In the above purification process, only 0.01 mol / L citric acid-trisodium citrate buffer with a pH of 4.7 was added in step 2, without sodium chloride, and the same process effect as in Example 1 was achieved. However, the stirring and dissolving in this step required 3 h, indicating that sodium salt could be omitted in step 2. However, the omission of sodium salt resulted in low overall purification efficiency.

[0049] Example 3

[0050] 1) To 60 L of fermentation broth (29.2°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, followed by centrifugation at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.93 kg.

[0051] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:4 (Kg / L). The stirring speed was set to 200 rpm. After stirring at room temperature for 2 h, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.5 kg.

[0052] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:4 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 73.7 L.

[0053] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 74 L of the clarified filtrate was collected.

[0054] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 50 g of humanized elastin freeze-dried powder with a purity of 97.0%, i.e., a yield of 0.833 g / L per liter of fermentation broth, and a yield of 72.6%.

[0055] In the above purification process, the solvent ratio in steps 2 and 3 is increased to 4 times that of the precipitate, which can achieve the same process effect as Example 1, but the increase in solvent volume leads to an increase in process cost.

[0056] Comparative Example 1

[0057] 1) To 60 L of fermentation broth (29.2°C), 80 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.8 kg.

[0058] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring at room temperature for 1 hour, the mixture was centrifuged at 4000 r / min for 15 minutes. The supernatant was discarded and the precipitate was collected. The precipitate weighed 17.2 kg.

[0059] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 53 L.

[0060] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 56 L of the clarified filtrate was collected.

[0061] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain humanized elastin with a purity of 96.7% (see Figure 3 ) 45.2 g of freeze-dried powder, that is, the yield per liter of fermentation liquid is 0.766 g / L, and the yield is 64.6%.

[0062] Since the amount of sodium chloride added in step 1 of comparative example 1 was reduced, the target protein was not completely precipitated in phase change at the same stirring time, resulting in a significant reduction in the yield of the final product.

[0063] Comparative Example 2

[0064] 1) To 60 L of fermentation broth (29.1°C), 130 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, followed by centrifugation at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.6 kg.

[0065] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring at room temperature for 1 hour, the mixture was centrifuged at 4000 rpm / min for 15 minutes. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.3 kg.

[0066] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 54 L.

[0067] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 57 L of the clarified filtrate was collected.

[0068] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain humanized elastin with a purity of 86.6% (see Figure 3 ) 49.2 g of freeze-dried powder, that is, the yield per liter of fermentation liquid is 0.848 g / L, and the yield is 70%.

[0069] Since too much sodium chloride was added in step 1 of comparative example 2, the impurity protein was salted out, which significantly reduced the purity of the final product.

[0070] With reference to comparative experiments such as Comparative Example 1 and Comparative Example 2 and Example 1, the results show that when 90 g / L to 117 g / L of sodium chloride is added to the 60 L fermentation broth in step 1, the target protein purification rate is better (specifically, the target protein purity and yield can reach the highest at the same time). This shows that the concentration of sodium chloride in step 1 is relatively important. Too high a concentration leads to salting out of impurity proteins, while too low a concentration leads to insufficient phase-changing precipitation of the target protein, resulting in a reduced yield. That is, when the sodium chloride concentration is not appropriate, the purity and yield of the final product cannot be balanced, and ultimately the target protein purification rate is reduced.

[0071] Comparative Example 3

[0072] 1) To 60 L of fermentation broth (28.9°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 19 kg.

[0073] 2) To the precipitate collected in step 1, 0.01 mol / L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer containing 11.7 g / L sodium chloride, pH 6.8, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring at room temperature for 1 hour, the mixture was centrifuged at 4000 r / min for 15 minutes. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.6 kg.

[0074] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 54.5 L.

[0075] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 57.8 L of the clarified filtrate was collected.

[0076] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain humanized elastin with a purity of 85.8% (see Figure 3 ) 53 g of freeze-dried powder, that is, the yield per liter of fermentation liquid is 0.883 g / L, and the yield is 74.6%.

[0077] Because a relatively high pH buffer was added in step 2 of Comparative Example 3, impurities were not fully dissolved, significantly reducing the purity of the final product. A pH below 4 is detrimental to equipment and personnel operation and is therefore undesirable. Comparative experiments conducted with Example 1, for example, show that in step 2 of dissolving and cleaning impurities (removing impurities), a pH range of 4 to 6 ensures that impurities are well dissolved, thereby achieving a higher purification rate of the target protein.

[0078] Comparative Example 4

[0079] 1) To 60 L of fermentation broth (29.3°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, followed by centrifugation at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.6 kg.

[0080] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 15 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring at room temperature for 1 hour, the mixture was centrifuged at 4000 r / min for 15 minutes. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.3 kg.

[0081] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 54 L.

[0082] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 57 L of the clarified filtrate was collected.

[0083] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD roll-type membrane ultrafiltration device, and the retentate was freeze-dried to obtain humanized elastin with a purity of 84.7% (see Figure 3 ) 51.8 g of freeze-dried powder, that is, the yield per liter of fermentation liquid is 0.879 g / L, and the yield is 73%.

[0084] Because excessive sodium chloride was added to the buffer in step 2 of Comparative Example 4, i.e., the sodium chloride content in the buffer salt system was too high, impurities were not fully dissolved, significantly reducing the purity of the final product. Comparative experiments conducted with Example 1, for example, demonstrated that excessive sodium chloride concentration in step 2, which involves cleaning impurities by dissolution, hindered impurity dissolution, significantly reduced the purity of the final product, and ultimately failed to achieve a high purification rate for the target protein.

[0085] Comparative Example 5

[0086] 1) To 60 L of fermentation broth (29.0°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 19 kg.

[0087] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring for 2 h at room temperature, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.5 kg.

[0088] 3) Add 250 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, then centrifuge at 4000 r / min for 15 min, discard the precipitate, and collect the supernatant, the volume of the supernatant is 54.9 L.

[0089] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 250 g / L urea solution to recover the residual target protein, and 60 L of the clarified filtrate was collected.

[0090] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 43.6 g of humanized elastin freeze-dried powder with a purity of 97.2%, i.e., a yield of 0.726 g / L per liter of fermentation broth, and a yield of 61.4%.

[0091] Because the amount of urea added in step 3 of Comparative Example 5 was less than the process requirement of 300 g / L, the target protein was not fully dissolved, resulting in a significant reduction in yield and yield. Experiments conducted in Comparative Example 4 and Example 1, for example, demonstrated that in step 3 using a urea solution to dissolve the target protein, a urea content in the solvent of 300 g / L to 360 g / L ensured optimal process conditions and reduced costs.

[0092] Comparative Example 6

[0093] 1) To 60 L of fermentation broth (29.0°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, and then centrifuged at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 19 kg.

[0094] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring at room temperature for 2 h, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18 kg.

[0095] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:2 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 36 L.

[0096] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 38 L of the clarified filtrate was collected.

[0097] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 34 g of humanized elastin freeze-dried powder with a purity of 95.5%, i.e., a yield of 0.567 g / L per liter of fermentation broth, and a yield of 48%.

[0098] Because the proportion of solvent used for dissolving the precipitate in step 3 of Comparative Example 6 was reduced, the target protein was not fully dissolved in step 3, resulting in a significant reduction in yield and yield. Comparative experiments conducted in Example 3 and Example 1, for example, demonstrated that controlling the solvent proportion in step 3 to be no less than three times that of the precipitate ensured that the process was completed under optimal conditions.

[0099] Comparative Example 7

[0100] 1) To 60 L of fermentation broth (29.3°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, followed by centrifugation at 4000 rpm for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 18.95 kg.

[0101] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:2.6 (Kg / L). The stirring speed was set to 200 rpm. After stirring for 2 h at room temperature, the mixture was centrifuged at 4000 r / min for 15 min, the supernatant was discarded, and the precipitate was collected. The precipitate weighed 18.58 kg.

[0102] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 55.2 L.

[0103] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 59.3 L of the clarified filtrate was collected.

[0104] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 52.3 g of humanized elastin freeze-dried powder with a purity of 90.2%, i.e., a yield of 0.872 g / L per liter of fermentation broth, and a yield of 74.2%.

[0105] In Comparative Example 7, the solvent ratio used for dissolving the precipitate in step 2 was reduced, resulting in insufficient dissolution of the impurity protein in step 2, significantly reducing the purity of the target protein. Comparative experiments conducted with reference to Examples 3, 1, and Comparative Example 6 demonstrated that controlling the solvent ratio in steps 2 and 3 to be no less than three times that of the precipitate ensured that the process was completed under optimal conditions.

[0106] Comparative Example 8

[0107] 1) To 60 L of fermentation broth (heated to 40.0°C), 117 g / L sodium chloride was added. The mixture was stirred at 200 rpm for 1 h at room temperature, followed by centrifugation at 4000 rpm for 15 min. The supernatant was discarded and the precipitate weighed 18.8 kg.

[0108] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring for 2 h at room temperature, the mixture was centrifuged at 4000 r / min for 15 min, the supernatant was discarded, and the precipitate was collected. The precipitate weighed 17.9 kg.

[0109] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 54 L.

[0110] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 57 L of the clarified filtrate was collected.

[0111] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 53 g of humanized elastin freeze-dried powder with a purity of 96.5%, i.e., a yield of 0.883 g / L per liter of fermentation broth, and a yield of 74.6%.

[0112] In step 1 of comparative example 8, the fermentation liquid was heated, and the temperature after heating was higher than 35°C. Although this had little effect on the process effect, the higher process temperature increased energy consumption, also increased the requirements for the thermal insulation performance of the equipment, and increased production costs.

[0113] Comparative Example 9

[0114] 1) To 60 L of fermentation broth (cooled to 23.5°C) was added 117 g / L of sodium chloride. The mixture was stirred at 200 rpm for 1 hour at room temperature, followed by centrifugation at 4000 rpm for 15 minutes. The supernatant was discarded and the precipitate weighed 17.2 kg.

[0115] 2) To the precipitate collected in step 1, 0.01 mol / L citric acid-trisodium citrate buffer containing 11.7 g / L sodium chloride, pH 4.7, was added at a mass volume ratio of 1:3 (Kg / L). The stirring speed was set to 200 rpm. After stirring for 2 h at room temperature, the mixture was centrifuged at 4000 r / min for 15 min. The supernatant was discarded and the precipitate was collected. The precipitate weighed 16.2 kg.

[0116] 3) Add 360 g / L urea solution to the precipitate collected in step 2 at a mass volume ratio of 1:3 (Kg / L), set the stirring speed to 200 rpm, stir at room temperature for 1.5 h, and then centrifuge at 4000 r / min for 15 min. Discard the precipitate and collect the supernatant. The volume of the supernatant is 49 L.

[0117] 4) The supernatant collected in step 3 was filtered and clarified through a 0.45 μm hollow fiber filter. The retentate was diluted with 5 L of 360 g / L urea solution to recover the residual target protein, and 52.7 L of the clarified filtrate was collected.

[0118] 5) The filtrate collected in step 4 was ultrafiltered and concentrated using a 3KD spiral membrane ultrafiltration device, and the retentate was freeze-dried to obtain 36.7 g of humanized elastin freeze-dried powder with a purity of 97.6%, i.e., a yield of 0.612 g / L per liter of fermentation broth, and a yield of 52.6%.

[0119] Since the fermentation broth was cooled in step 1 of Comparative Example 9, and the temperature after cooling was lower than 28° C., the target protein in step 1 was not fully precipitated, resulting in a decrease in the yield and yield of the target protein. Referring to comparative experiments such as those conducted in Comparative Example 8 and Example 1, the results showed that controlling the fermentation broth temperature in step 1 within the range of 28 to 35° C. can ensure that the process is completed under optimal conditions.

[0120] Table 1. Summary of purification effects of various process schemes in the examples

[0121]

[0122]

[0123] In summary, the present invention purifies humanized elastin produced by induced expression in recombinant Pichia pastoris fermentation by adding a sodium salt (e.g., sodium chloride) to the crude product solution to induce phase change of the humanized elastin at room temperature. Impurities are then dissolved in a buffer solution containing a low concentration of sodium salt (e.g., sodium chloride) (most soluble impurities can be removed during the buffer solution dissolution and impurity removal process), and the humanized elastin is dissolved in a urea solution at room temperature. The humanized elastin produced during fermentation can then be purified by filtration, clarification, ultrafiltration concentration, and freeze-drying. This purification process eliminates the need for complex thermal phase change temperature control and can be performed entirely at room temperature. This simplifies the purification process, reduces the requirements for production equipment, and shortens the purification time. Furthermore, this purification process achieves a yield of over 0.8 g / L of pure product (purity greater than 95%) with a yield greater than 70%. This provides an ideal downstream separation and purification process for large-scale production of humanized elastin by fermentation, and is of great significance for the industrial production of humanized elastin.

Claims

1. A method for purifying humanized elastin, characterized by: The following steps are involved: 1) Mixing a fermentation broth of an engineered Pichia pastoris with sodium salt and stirring at 20-30° C. to cause the target protein to undergo phase change and precipitate, followed by solid-liquid separation to obtain a solid phase A, wherein the target protein is humanized elastin expressed by the engineered Pichia pastoris in the fermentation broth; 2) Mixing solid phase A with a solvent containing a buffer salt system at a pH of 4 to 6 and stirring at 20 to 30°C to dissolve impurities in solid phase A, followed by solid-liquid separation to obtain solid phase B; 3) Mixing solid phase B with a solvent containing urea to redissolve the target protein, followed by solid-liquid separation to obtain a target protein extract; 4) The target protein extract is filtered and clarified through a microfiltration system, and the filtrate is collected; 5) The filtrate collected in step 4) is concentrated by an ultrafiltration system to collect the retentate, and the retentate is freeze-dried to obtain a pure humanized elastin product; In step 1, the temperature of the fermentation broth of the Pichia pastoris engineered bacteria is 28-35° C.; In step 1, the sodium salt is sodium chloride, and the amount of sodium chloride is 90 g / L to 117 g / L; the stirring time is 1 to 3 hours, and the stirring speed is 180 to 200 rpm; In step 3, the redissolution specifically includes the following steps: using ≥3L of 300g / L~360g / L urea aqueous solution to 1Kg of solid phase B, mixing the solid phase B with the urea aqueous solution, and then stirring at 20~30°C for 1.5~2h at a stirring speed of 180~200rpm.

2. The method for purifying humanized elastin according to claim 1, characterized in that: In the step 2, the solvent consists of a buffer solution and a sodium salt for improving the solubility of impurities in the buffer solution.

3. The method for purifying humanized elastin according to claim 2, characterized in that: In the step 2, the sodium salt is sodium chloride.

4. The method for purifying humanized elastin according to claim 1, characterized in that: In step 2, 1 kg of solid phase A is mixed with ≥ 3 L of 0.01-0.03 mol / L citric acid-trisodium citrate buffer containing 9.8 g / L-11.7 g / L sodium chloride and pH 4-6; the stirring time is 1-3 h, and the stirring speed is 180-200 rpm.

5. The method for purifying humanized elastin according to claim 1, characterized in that: In steps 1, 2, and 3, the conditions for solid-liquid separation are: centrifugation at 20-30° C. and a speed of not less than 4000 rpm for 10-15 minutes.

6. The method for purifying humanized elastin according to claim 1, characterized in that: In step 4, the conditions for filtration and clarification include: a microfiltration pore size of 0.22-1 μm; and in step 5, the conditions for concentration include: a molecular weight cut-off of 1-6 KD.

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