A method for producing a recombinant elastin

Abstract

The application provides a recombinant elastin production method, and relates to the field of genetic recombination engineering bacteria fermentation and purification technology. The recombinant elastin production method comprises using specific fermentation medium, culture method, induction method, cell breaking method and purification method to obtain the recombinant elastin. The application can improve the fermentation expression amount and purification efficiency of the recombinant elastin, reduce the endotoxin level, thereby improving the production level of the recombinant elastin, and is suitable for stable industrial production. Meanwhile, the recombinant elastin produced by the application has excellent biocompatibility, no virus hidden danger and low rejection reaction compared with the elastin extracted from animal tissues.

Description

Technical Field

[0001] This invention relates to the field of recombinant engineered bacteria fermentation technology, and more specifically, to a method for producing recombinant elastin. Background Technology

[0002] Natural elastin is an extracellular matrix protein with low water solubility, amorphous structure, strong hydrophobicity, and extensive cross-linking. It is abundant in tissues such as skin, lungs, ligaments, and blood vessels, and plays an important role in maintaining tissue elasticity. Therefore, it has broad application prospects in the fields of medical aesthetics and skin care.

[0003] In existing technologies, the main source of elastin is extraction from animal tissues. However, with the development of biotechnology, recombinant elastin obtained through microbial fermentation using gene recombination technology has achieved great success. Compared with natural elastin, recombinant elastin produced using this technology solves the defects of viral risks in traditional extraction methods, while significantly improving the stability, hydrophilicity and biocompatibility of elastin.

[0004] For the application of recombinant elastin, it is crucial to produce it in large quantities and with high purity in a cost-effective manner. Especially as a biomaterial and tissue engineering scaffold, high yield and low cost are the two biggest obstacles to its large-scale use. While there are reports abroad of expression levels reaching 1.6 g / L in shake-flask stages, large-scale industrial production is rarely reported. This invention aims to provide a large-scale recombinant elastin production process.

[0005] Currently, engineered Escherichia coli is generally used for fermentation culture to produce recombinant elastin; however, the fermentation medium is mostly modified LB medium, while the traditional fermentation of genetically engineered Escherichia coli uses glucose as a carbon source. During the use of glucose, a large amount of organic acids are produced, which inhibits cell growth and protein expression. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for producing recombinant elastin, which can improve the fermentation expression level and purification efficiency of recombinant elastin and reduce the endotoxin level, thereby improving the fermentation production level of recombinant elastin and making it suitable for stable industrial production.

[0007] To solve the above-mentioned technical problems, the objective of this invention is achieved through the following technical solution: providing a method for producing recombinant elastin, characterized by comprising the following steps:

[0008] S1. Fermentation: Engineered strains are cultured using specific fermentation media, and engineered strains are induced in stages during the culture process;

[0009] S2, Cell disruption: Cell disruption is achieved using surfactants and salt ions;

[0010] S3. Purification: Adjust pH, control temperature, and add surfactants to purify the protein.

[0011] By adopting the above technical solutions, the fermentation expression level and purification efficiency of recombinant elastin can be improved, and the endotoxin level can be reduced, thereby improving the fermentation production level of recombinant elastin and making it suitable for stable industrial production.

[0012] Preferably, step S1 further includes the following step:

[0013] A1. Inoculate the engineered Escherichia coli seed culture into the specific fermentation medium in the fermenter and culture it. After the dissolved oxygen level rises significantly, feed medium is added to continue the culture.

[0014] A2. When the OD value of the engineered bacteria solution reaches 44-60, start slowly adding lactose solution for initial induction, while continuing to culture with fed culture medium.

[0015] A3. When the OD value of the engineered bacteria solution reaches 65-73, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time, feed culture medium is added to continue the culture.

[0016] A4. During the induction period, slowly add lactose solution to maintain the induction pressure, while simultaneously adding feed medium until fermentation is complete.

[0017] By adopting the above technical solution, the induction pressure can be increased in stages during the fermentation process, thereby increasing protein yield. Specifically, step A1 is the base material consumption stage; when the OD reaches 44-60, it is the initial induction stage, the purpose of which is to allow the strain to adapt to lactose; A3 is the formal induction stage; and A4 maintains the induction pressure to achieve continuous induction.

[0018] Preferably, in step A1, the specific fermentation medium comprises the following components at the following concentrations: diammonium hydrogen phosphate 0.5–3.5 g / L, potassium dihydrogen phosphate 3.0–13.5 g / L, citric acid monohydrate 0.2–1.6 g / L, magnesium sulfate heptahydrate 0.2–1.8 g / L, glucose monohydrate 2–15 g / L, glycerol 10–20 g / L, ferrous sulfate heptahydrate 30–70 mg / L, manganese sulfate pentahydrate 1–5 mg / L, zinc sulfate heptahydrate 8–15 mg / L, copper sulfate pentahydrate 3–10 mg / L, calcium chloride dihydrate 5–20 mg / L, borax 0.5–2 mg / L, and ammonium molybdate 0.1–2 mg / L.

[0019] By adopting the above technical solution, the combination of glycerol and glucose in the fermentation medium reduces the possibility that the presence of glucose will inhibit lactose induction, and also reduces the inhibition of bacterial growth and protein expression by organic acids produced during fermentation.

[0020] Further preferably, the following components are used: diammonium hydrogen phosphate 2.0–3.0 g / L; potassium dihydrogen phosphate 5.0–10.0 g / L; citric acid monohydrate 0.5–1.5 g / L; magnesium sulfate heptahydrate 0.2–1.0 g / L; glucose monohydrate 2–7 g / L; glycerol 10–18 g / L; ferrous sulfate heptahydrate 40–60 mg / L; manganese sulfate pentahydrate 2–4 mg / L; zinc sulfate heptahydrate 10–13 mg / L; copper sulfate pentahydrate 4–7 mg / L; calcium chloride dihydrate 8–13 mg / L; borax 0.7–1.5 mg / L; and ammonium molybdate 0.3–0.8 mg / L.

[0021] By adopting the above technical solution, the possibility of glucose inhibiting lactose induction is further reduced, and the inhibition of bacterial growth and protein expression by organic acids produced during fermentation is also reduced. In addition, the culture medium uses pure inorganic salt ions and food-grade raw materials, which do not involve the risks of pathogenicity, cultural taboos, or genetic modification associated with traditional animal and plant peptone / yeast powder.

[0022] Preferably, in step A1, the supplemental culture medium comprises the following components at the following concentrations: glucose monohydrate 150-300 g / L, glycerol 300-600 g / L, magnesium sulfate heptahydrate 0.1-4.5 g / L, complex amino acids 1-6.0 g / L, ferrous sulfate heptahydrate 10-50 mg / L, manganese sulfate pentahydrate 0.5-3 mg / L, zinc sulfate heptahydrate 2-10 mg / L, copper sulfate pentahydrate 1-5 mg / L, calcium chloride dihydrate 2-10 mg / L, borax 0.1-1 mg / L, and ammonium molybdate 0.1-1 mg / L.

[0023] By adopting the above technical solution, the combination of glycerol and glucose in the fed culture medium reduces the possibility that the presence of glucose will inhibit lactose induction, and also reduces the inhibition of bacterial growth and protein expression by organic acids produced during fermentation.

[0024] Further preferably, the following components are used: glucose monohydrate 175–250 g / L, glycerol 350–500 g / L, magnesium sulfate heptahydrate 1.0–3.0 g / L, proline 2.0–5.0 g / L, alanine 2.0–5.0 g / L, ferrous sulfate heptahydrate 20–40 mg / L, manganese sulfate pentahydrate 1.0–2.5 mg / L, zinc sulfate heptahydrate 4.0–8.0 mg / L, copper sulfate pentahydrate 3.0–6.0 mg / L, calcium chloride dihydrate 2.0–6.0 mg / L, borax 0.1–0.5 mg / L, and ammonium molybdate 0.2–0.7 mg / L.

[0025] By adopting the above technical solution, the possibility of glucose inhibiting lactose induction is further reduced, and the inhibition of bacterial growth and protein expression by organic acids produced during fermentation is also reduced. In addition, the culture medium uses pure inorganic salt ions and food-grade raw materials, which do not involve the risks of pathogenicity, cultural taboos, or genetic modification associated with traditional animal and plant peptone / yeast powder.

[0026] Preferably, in step A2, the lactose solution comprises components with the following concentrations: lactose 40-80 g / L, more preferably 45-60 g / L.

[0027] Preferably, in step A1, the flow rate of the feeding medium is 10-15 mL / h / L; in step A2, the flow rate of the feeding medium is 15-20 mL / h / L, and the flow rate of the lactose solution is 3-5 mL / h / L; in step A3, the flow rate of the feeding medium is 15-20 mL / h / L, and the flow rate of the lactose solution is 5-12.5 mL / L; and in step A4, the flow rate of the feeding medium is 10-15 mL / h / L, and the flow rate of the lactose solution is 1-2 mL / h / L.

[0028] By adopting the above technical solution, the staged self-induction of lactose during the production process improved the protein expression level of the genetically engineered bacteria. Specifically, lactose was slowly added during the mid-logarithmic growth phase for self-induction, an appropriate amount of lactose was added once during the mid-to-late logarithmic growth phase to increase the induction pressure, and lactose was slowly added during the induction phase to maintain the induction pressure.

[0029] Preferably, the fermentation conditions are: fermentation temperature 34-37℃, pH adjusted to 6.5-7.0 with ammonia water, dissolved oxygen not less than 30%, and tank pressure 0.04-0.08MPa.

[0030] Preferably, step S2 further includes the following steps:

[0031] B1. After fermentation, centrifuge to separate the solid and liquid components;

[0032] B2. Redissolve the bacterial precipitate to 0.8-1.5 times the volume of the container, adjust the pH to 2-5, add sodium chloride, EDTA, and surfactant, and heat to 55-85℃;

[0033] B3. Adjust the tank pressure and stir continuously at 50-150 rpm for 2-5 hours.

[0034] By adopting the above technical solution, the acid and heat resistance of elastin is taken into account. In particular, by combining the use of surfactants and other salt ions and controlling the pressure, the cell disruption can be achieved efficiently without the need for an expensive high-pressure homogenizer. The operation is simple and saves production costs.

[0035] Preferably, step S3 further includes the following steps:

[0036] C1. When the temperature of the cell lysis solution drops to 30-40℃, add a small amount of salt ions to adjust the pH to 3.0-5.0, maintain the temperature and continue stirring, then centrifuge to collect the supernatant.

[0037] C2. Adjust the pH of the supernatant to 3.0-5.0, add EDTA and surfactant, heat to 30-40℃, adjust the tank pressure, maintain the temperature and continue stirring, centrifuge to collect the supernatant, and repeat this step.

[0038] C3. Add inorganic salts to the supernatant, heat to 38-50℃, stir, and centrifuge to collect the protein precipitate;

[0039] C4. The protein precipitate was reconstituted, desalted by ultrafiltration, and purified by weak anion exchange chromatography.

[0040] By adopting the above technical solution, the initial endotoxin can be effectively controlled based on the characteristics of recombinant elastin. By controlling the ionic environment, pH, and temperature of the purification solution and adding surfactants, the target protein is precipitated while other proteins are precipitated in very small amounts, thereby improving protein purity. Furthermore, the protein purity is further improved through chromatography purification, reaching over 95%. At the same time, endotoxin can be controlled below 10 EU / mL. The operation is simple, greatly saving purification costs, and the low endotoxin level expands the product's usability.

[0041] Preferably, in step C1, the added salt ion is 0.1M-0.2M sodium chloride, the temperature is maintained at 50-150 rpm and stirring is continued for 2-5 hours, and the supernatant is collected using a tubular centrifuge at 10000-12000 rpm; in step C2, 1-10 mM EDTA and 1.5%-3% surfactant are added, air is introduced to the tank pressure to 1.0-1.6 MPa, the temperature is maintained at 50-150 rpm and stirring is continued for 1-2 hours, and the supernatant is collected by centrifugation; in step C3, 10%-40% ammonium sulfate is added to the supernatant, the mixture is stirred at 50-150 rpm for 1-3 hours, and the protein precipitate is collected by centrifugation; in step S4, the protein precipitate is reconstituted with deionized water to 5-10 times its weight, and then subjected to DEAE weak anion chromatography.

[0042] The specific fermentation medium and fed-batch medium used in this invention employ a combination of glycerol and glucose, along with staged induction. This solves the problem of glucose inhibiting lactose induction in existing technologies, while also avoiding the inhibition of bacterial growth and protein expression caused by excessive organic acid production. Compared to traditional methods, this reduces the amount of lactose added and saves costs. Furthermore, this invention uses pure inorganic salt ions and food-grade raw materials, eliminating the risks associated with traditional animal and plant peptone / yeast powders, such as pathogenicity, cultural taboos, and genetic modification. In the production process, this invention uses lactose for staged self-induction. Lactose is slowly added during the mid-logarithmic growth phase for self-induction, and a suitable amount of lactose is added once during the mid-to-late logarithmic growth phase to increase the induction pressure. During the induction period, lactose is slowly added to maintain the induction pressure, thereby increasing the protein expression level of the genetically engineered bacteria. Furthermore, the cell-breaking method employed in this invention, taking advantage of the acid- and heat-resistant properties of elastin, combined with the use of surfactants and other salt ions and pressure control, achieves efficient cell breaking without the need for expensive high-pressure homogenizers. This method is simple to operate and saves production costs. In addition, the purification method designed based on the characteristics of recombinant elastin effectively controls the initial endotoxin. By controlling the ionic environment, pH, and temperature of the purification solution, and by adding surfactants, the target protein precipitates while other proteins precipitate in very small amounts, initially improving protein purity. Chromatographic purification further improves protein purity, achieving a purity of over 95%, while keeping endotoxin levels below 10 EU / mL. The simple operation significantly reduces purification costs, and the low endotoxin level expands the product's usability. Attached Figure Description

[0043] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1 SDS-PAGE gel electrophoresis images for comparison of cell disruption effects.

[0045] Figure 2 This is a diagram illustrating the purification effect;

[0046] Figure 3 This is a schematic diagram illustrating the comparison of cytotoxicity. Detailed Implementation

[0047] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] It should be understood that, when used in this specification and the appended claims, the terms "comprising" and "including" indicate the presence of the described features, integrals, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integrals, steps, operations, elements, components and / or collections thereof.

[0049] It should also be understood that the terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms unless the context clearly indicates otherwise.

[0050] It should also be further understood that the term "and / or" as used in this specification and the appended claims refers to any combination of one or more of the associated listed items and all possible combinations, and includes such combinations.

[0051] Furthermore, terms like "roughly" and "basically" are used to indicate that the content does not require absolute precision, but rather allows for a certain degree of deviation. For example, "roughly equal" does not simply mean absolute equality; in actual production and operation, achieving absolute "equality" is difficult, and a certain degree of deviation is generally present. Therefore, besides absolute equality, "roughly equal to" also includes the aforementioned situation where a certain degree of deviation exists. Using this as an example, in other cases, unless otherwise specified, terms like "roughly" and "basically" have similar meanings.

[0052] This invention provides a method for producing recombinant elastin, wherein the amino acid sequence of the recombinant elastin in this invention is as follows:

[0053] MASVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGSSVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPG SSVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGSSVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPGSR

[0054] Example 1

[0055] The method for producing the recombinant elastin described in this embodiment includes the following steps:

[0056] S1. Fermentation: Preparing specific fermentation medium for the production of recombinant elastin, culturing (fermenting) engineered strains, and inducing engineered strains;

[0057] S2, cell rupture;

[0058] S3, Purification.

[0059] Step S1 includes the following steps:

[0060] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 13mL / h / L.

[0061] A2. When the OD increases to 52, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 18 mL / h / L and the feeding rate of the lactose solution is 3 mL / h / L.

[0062] A3. When the OD increases to 72, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture with fed medium. Add 7 mL / L of lactose solution and the flow rate of fed medium is 18 mL / h / L.

[0063] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 13 mL / h / L, and the flow rate of the lactose solution was 1 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.5 with ammonia, the tank pressure was 0.08 MPa, the dissolved oxygen was not less than 30%, and the induction was carried out for 5 hours. When the OD of E. coli cells no longer increased and microscopic examination showed cell aging, the entire fermentation process was completed. After low-temperature ultrasonic cell destruction, the protein content was detected by SDS-PAGE and found to be 2.25 g / L.

[0064] The specific fermentation medium for engineered Escherichia coli used in the fermenter includes the following components at the following concentrations: diammonium hydrogen phosphate 2.5 g / L; potassium dihydrogen phosphate 8.0 g / L; citric acid monohydrate 1.0 g / L; magnesium sulfate heptahydrate 0.4 g / L; glucose monohydrate 5 g / L; glycerol 15 g / L; ferrous sulfate heptahydrate 50 mg / L; manganese sulfate pentahydrate 2.5 mg / L; zinc sulfate heptahydrate 11.25 mg / L; copper sulfate pentahydrate 5 mg / L; calcium chloride dihydrate 10 mg / L; borax 1.15 mg / L; and ammonium molybdate 0.5 mg / L.

[0065] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 200 g / L, glycerol 400 g / L, magnesium sulfate heptahydrate 1.5 g / L, proline 4.0 g / L, alanine 4.0 g / L, ferrous sulfate heptahydrate 28 mg / L, manganese sulfate pentahydrate 2.0 mg / L, zinc sulfate heptahydrate 6.0 mg / L, copper sulfate pentahydrate 5 mg / L, calcium chloride dihydrate 4.0 mg / L, borax 0.3 mg / L, and ammonium molybdate 0.5 mg / L.

[0066] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 50 g / L.

[0067] Step S2 includes the following steps:

[0068] B1. After fermentation, a disc centrifuge is used to separate the solid and liquid components.

[0069] B2. The bacterial precipitate is then reconstituted with purified water to 1.2 times the volume of the container, the pH is adjusted to 3.5, and 10 mM sodium chloride, 10 mM EDTA, and 1.8% Triton-114 are added. The mixture is then heated to 60°C.

[0070] B3. Introduce air until the tank pressure reaches 1.5 MPa, stir at 50-150 rpm for 4 hours, and the cell breakage rate can reach 98% after testing.

[0071] In addition, step S3 includes the following steps:

[0072] C1. When the temperature of the cell lysis solution drops to 30℃, add 0.1M sodium chloride to adjust the pH to 3.5, maintain the temperature at 50 rpm and continue stirring for 3 hours. Collect the supernatant using a tubular centrifuge at 12000 rpm.

[0073] C2. Adjust the pH of the supernatant to 3.5, add 3mM EDTA and 2.0% Triton-114, heat to 30°C, purge air to the pressure of the tank to 1.0Mpa, maintain the temperature at 60rpm and continue stirring for 1h, collect the supernatant using a tubular centrifuge at 12000rpm, and repeat this step once. At this point, the endotoxin level in the supernatant can be reduced to 100-300EU / ml.

[0074] C3. Add 25% ammonium sulfate to the supernatant, heat to 45°C, stir at 100 rpm for 1.5 h, and collect the protein precipitate using a tubular centrifuge at 12000 rpm.

[0075] C4. Protein precipitate is reconstituted with deionized water to 10 times its weight, desalted by ultrafiltration, and purified by DEAE weak anion exchange chromatography column to remove endotoxins. The protein purity can reach over 95%, and the endotoxin can be reduced to 5-10 EU / ml.

[0076] It should also be noted that the fermentation method of the present invention is applicable to Escherichia coli fermentation systems that can use this culture medium, while the cell disruption method is applicable to acid- and heat-resistant proteins, but the purification method is only applicable to elastin.

[0077] Example 2

[0078] Based on Example 1, the specific settings in this example are as follows:

[0079] In step S1:

[0080] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 10mL / h / L.

[0081] A2. When the OD increases to 45, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 15 mL / h / L and the feeding rate of the lactose solution is 3 mL / h / L.

[0082] A3. When the OD increases to 65, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture with fed medium. Add 7 mL / L of lactose solution and the flow rate of fed medium is 16 mL / h / L.

[0083] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 10 mL / h / L, and the flow rate of the lactose solution was 1 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.7 with ammonia, the tank pressure was 0.06 MPa, and the dissolved oxygen was not lower than 30%. After induction for 5 hours, the OD of E. coli cells stopped increasing, and microscopic examination showed cell aging. The entire fermentation process was then complete. After low-temperature ultrasonic cell disruption, the protein content was detected by SDS-PAGE to be 2.14 g / L.

[0084] The specific fermentation medium for engineered Escherichia coli used in the fermenter includes the following components at the following concentrations: diammonium hydrogen phosphate 2.1 g / L; potassium dihydrogen phosphate 5.0 g / L; citric acid monohydrate 0.6 g / L; magnesium sulfate heptahydrate 0.3 g / L; glucose monohydrate 4 g / L; glycerol 10 g / L; ferrous sulfate heptahydrate 35 mg / L; manganese sulfate pentahydrate 1.5 mg / L; zinc sulfate heptahydrate 9 mg / L; copper sulfate pentahydrate 7 mg / L; calcium chloride dihydrate 15 mg / L; borax 0.8 mg / L; and ammonium molybdate 0.2 mg / L.

[0085] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 175 g / L, glycerol 350 g / L, magnesium sulfate heptahydrate 0.9 g / L, proline 2.0 g / L, alanine 2.0 g / L, ferrous sulfate heptahydrate 15 mg / L, manganese sulfate pentahydrate 1.0 mg / L, zinc sulfate heptahydrate 4.0 mg / L, copper sulfate pentahydrate 1.5 mg / L, calcium chloride dihydrate 2.5 mg / L, borax 0.6 mg / L, and ammonium molybdate 0.8 mg / L.

[0086] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 45 g / L.

[0087] In step S2:

[0088] B1. After fermentation, a disc centrifuge is used to separate the solid and liquid components.

[0089] B2. The bacterial precipitate was then reconstituted with purified water to 1.2 times the volume of the container, the pH was adjusted to 4.0, and 10 mM sodium chloride, 10 mM EDTA, and 1.8% Triton-114 were added. The mixture was then heated to 55°C.

[0090] B3. Introduce air until the pressure in the tank reaches 1.5 MPa, stir at 100 rpm for 4 hours, and the cell breakage rate can reach 98% after testing.

[0091] Additionally, in step S3:

[0092] C1. When the temperature of the cell lysis solution drops to 30℃, add 0.15M sodium chloride to adjust the pH to 4.0, maintain the temperature at 50 rpm and continue stirring for 3 hours. Collect the supernatant using a tubular centrifuge at 12000 rpm.

[0093] C2. Adjust the pH of the supernatant to 4.0, add 4 mM EDTA and 2.0% Triton-114, heat to 30°C, purge air to the pressure of the tank to 1.4 MPa, maintain the temperature at 50-150 rpm and continue stirring for 1 hour, collect the supernatant using a tubular centrifuge at 12000 rpm, and repeat this step once. At this point, the endotoxin level in the supernatant can be reduced to 100-300 EU / ml.

[0094] C3. Add 15% ammonium sulfate to the supernatant, heat to 45°C, stir at 100 rpm for 1.5 h, and collect the protein precipitate using a tubular centrifuge at 12000 rpm.

[0095] C4. Protein precipitate is reconstituted with deionized water to 10 times its weight, desalted by ultrafiltration, and purified by DEAE weak anion exchange chromatography column to remove endotoxins. The protein purity can reach over 95%, and the endotoxin can be reduced to 5-10 EU / ml.

[0096] Example 3

[0097] Based on Example 1, the specific settings in this example are as follows:

[0098] In step S1:

[0099] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 10mL / h / L.

[0100] A2. When the OD increases to 45, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 18 mL / h / L and the feeding rate of the lactose solution is 4.5 mL / h / L.

[0101] A3. When the OD increases to 65, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture with fed medium. Add 7 mL / L of lactose solution and the flow rate of fed medium is 18 mL / h / L.

[0102] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 15 mL / h / L, and the flow rate of the lactose solution was 2 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.7 with ammonia, the tank pressure was 0.06 MPa, the dissolved oxygen was not lower than 30%, and the induction was carried out for 5 hours. When the OD of E. coli cells no longer increased and microscopic examination showed cell aging, the entire fermentation process was completed. After low-temperature ultrasonic cell destruction, the protein content was detected by SDS-PAGE and found to be 2.23 g / L.

[0103] The specific fermentation medium for engineered Escherichia coli used in the fermenter includes the following components at the following concentrations: diammonium hydrogen phosphate 3.0 g / L; potassium dihydrogen phosphate 12.0 g / L; citric acid monohydrate 1.5 g / L; magnesium sulfate heptahydrate 1.5 g / L; glucose monohydrate 7 g / L; glycerol 18 g / L; ferrous sulfate heptahydrate 65 mg / L; manganese sulfate pentahydrate 4.5 mg / L; zinc sulfate heptahydrate 13 mg / L; copper sulfate pentahydrate 9 mg / L; calcium chloride dihydrate 5 mg / L; borax 0.5 mg / L; and ammonium molybdate 1.0 mg / L.

[0104] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 250 g / L, glycerol 350 g / L, magnesium sulfate heptahydrate 4.0 g / L, proline 3.0 g / L, alanine 3.0 g / L, ferrous sulfate heptahydrate 45 mg / L, manganese sulfate pentahydrate 2.8 mg / L, zinc sulfate heptahydrate 8 mg / L, copper sulfate pentahydrate 3 mg / L, calcium chloride dihydrate 8 mg / L, borax 0.8 mg / L, and ammonium molybdate 0.8 mg / L.

[0105] The engineered E. coli lactose culture medium used in the fermenter contains the following components at a concentration of 60 g / L lactose.

[0106] In step S2

[0107] B1. After fermentation, a disc centrifuge is used to separate the solid and liquid components.

[0108] B2. Then, the bacterial precipitate is redissolved in purified water to 1.5 times the volume of the tank, the pH is adjusted to 4.5, 10mM sodium chloride, 10mM EDTA, and 2.2% Triton-114 are added, and the mixture is heated to 70℃.

[0109] B3. Introduce air until the pressure in the tank reaches 1.5 MPa, stir at 150 rpm for 2 hours, and the cell breakage rate can reach 98%.

[0110] Additionally, in step S3:

[0111] C1. When the temperature of the cell lysis solution drops to 30℃, add 0.15M sodium chloride to adjust the pH to 4.0, maintain the temperature at 50 rpm and continue stirring for 3 hours. Collect the supernatant using a tubular centrifuge at 12000 rpm.

[0112] C2. Adjust the pH of the supernatant to 4.0, add 4 mM EDTA and 2.0% Triton-114, heat to 30°C, purge air to the pressure of the tank to 1.4 MPa, maintain the temperature at 150 rpm and continue stirring for 1 hour, collect the supernatant using a tubular centrifuge at 12000 rpm, and repeat this step once. At this point, the endotoxin level in the supernatant can be reduced to 100-300 EU / ml.

[0113] C3. Add 25% ammonium sulfate to the supernatant, heat to 45°C, stir at 100 rpm for 1.5 h, and collect the protein precipitate using a tubular centrifuge at 12000 rpm.

[0114] C4. Protein precipitate is reconstituted with deionized water to 10 times its weight, desalted by ultrafiltration, and purified by DEAE weak anion exchange chromatography column to remove endotoxins. The protein purity can reach over 95%, and the endotoxin can be reduced to 5-10 EU / ml.

[0115] Example 4

[0116] Based on Example 1, the specific settings in this example are as follows:

[0117] In step S1:

[0118] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 13mL / h / L.

[0119] A2. When the OD increases to 45, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 16 mL / h / L and the feeding rate of the lactose solution is 4 mL / h / L.

[0120] A3. When the OD increases to 65, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture by feeding culture medium. Add 5 mL / L of lactose solution and the flow rate of the feeding culture medium is 18 mL / h / L.

[0121] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 15 mL / h / L, and the flow rate of the lactose solution was 2 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.7 with ammonia, the tank pressure was 0.06 MPa, and the dissolved oxygen was not less than 30%. After induction for 5 hours, the OD of E. coli cells stopped increasing, and microscopic examination showed cell aging. The entire fermentation process was then complete. After low-temperature ultrasonic cell disruption, the protein content was detected by SDS-PAGE to be 2.03 g / L.

[0122] The specific fermentation medium for engineered Escherichia coli used in the fermenter includes the following components at the following concentrations: diammonium hydrogen phosphate 2.0 g / L; potassium dihydrogen phosphate 5.0 g / L; citric acid monohydrate 0.2 g / L; magnesium sulfate heptahydrate 1.8 g / L; glucose monohydrate 15 g / L; glycerol 20 g / L; ferrous sulfate heptahydrate 40 mg / L; manganese sulfate pentahydrate 1 mg / L; zinc sulfate heptahydrate 15 mg / L; copper sulfate pentahydrate 3 mg / L; calcium chloride dihydrate 18 mg / L; borax 1.5 mg / L; and ammonium molybdate 1.0 mg / L.

[0123] The fed culture medium consists of the following components at the following concentrations: glucose monohydrate 150 g / L, glycerol 600 g / L, magnesium sulfate heptahydrate 0.2 g / L, proline 1.0 g / L, alanine 6.0 g / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate pentahydrate 0.5 mg / L, zinc sulfate heptahydrate 2 mg / L, copper sulfate pentahydrate 8 mg / L, calcium chloride dihydrate 10 mg / L, borax 0.1 mg / L, and ammonium molybdate 0.1 mg / L.

[0124] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 80 g / L.

[0125] In step S2

[0126] B1. After fermentation, a disc centrifuge is used to separate the solid and liquid components.

[0127] B2. Then, the bacterial precipitate is redissolved in purified water to 1.5 times the volume of the tank, the pH is adjusted to 4.5, 10mM sodium chloride, 10mM EDTA, and 2.2% Triton-114 are added, and the mixture is heated to 70℃.

[0128] B3. Introduce air until the pressure in the tank reaches 1.5 MPa, stir at 150 rpm for 2 hours, and the cell breakage rate can reach 98%.

[0129] Additionally, in step S3:

[0130] C1. When the temperature of the cell lysis solution drops to 30℃, add 0.15M sodium chloride to adjust the pH to 4.0, maintain the temperature at 50 rpm and continue stirring for 3 hours. Collect the supernatant using a tubular centrifuge at 12000 rpm.

[0131] C2. Adjust the pH of the supernatant to 4.0, add 4 mM EDTA and 2.0% Triton-114, heat to 30°C, purge air to the pressure of the tank to 1.4 MPa, maintain the temperature at 150 rpm and continue stirring for 1 hour, collect the supernatant using a tubular centrifuge at 12000 rpm, and repeat this step once. At this point, the endotoxin level in the supernatant can be reduced to 100-300 EU / ml.

[0132] C3. Add 35% ammonium sulfate to the supernatant, heat to 45°C, stir at 80 rpm for 1.5 h, and collect the protein precipitate using a tube centrifuge at 11000 rpm.

[0133] C4. Protein precipitate is reconstituted with deionized water to 10 times its weight, desalted by ultrafiltration, and purified by DEAE weak anion exchange chromatography column to remove endotoxins. The protein purity can reach over 95%, and the endotoxin can be reduced to 5-10 EU / ml.

[0134] Example 5

[0135] Based on Example 1, the specific settings in this example are as follows:

[0136] In step S1:

[0137] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 12mL / h / L.

[0138] A2. When the OD increases to 45, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The flow rate of the feeding medium is 18 mL / h / L and the flow rate of the lactose solution is 3 mL / h / L.

[0139] A3. When the OD increases to 65, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture with fed medium. Add 12.5 mL / L of lactose solution and the flow rate of fed medium is 20 mL / h / L.

[0140] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 12 mL / h / L, and the flow rate of the lactose solution was 2 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.7 with ammonia, the tank pressure was 0.06 MPa, and the dissolved oxygen was not lower than 30%. After induction for 5 hours, the OD of E. coli cells stopped increasing, and microscopic examination showed cell aging. The entire fermentation process was then complete. After low-temperature ultrasonic cell disruption, the protein content was detected by SDS-PAGE to be 2.09 g / L.

[0141] The specific fermentation medium for engineered Escherichia coli used in the fermenter includes the following components at the following concentrations: diammonium hydrogen phosphate 3.5 g / L; potassium dihydrogen phosphate 10.0 g / L; citric acid monohydrate 0.5 g / L; magnesium sulfate heptahydrate 0.2 g / L; glucose monohydrate 2 g / L; glycerol 20 g / L; ferrous sulfate heptahydrate 30 mg / L; manganese sulfate pentahydrate 5 mg / L; zinc sulfate heptahydrate 10 mg / L; copper sulfate pentahydrate 4 mg / L; calcium chloride dihydrate 8 mg / L; borax 0.6 mg / L; and ammonium molybdate 0.6 mg / L.

[0142] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 300 g / L, glycerol 300 g / L, magnesium sulfate heptahydrate 3.0 g / L, proline 2.0 g / L, alanine 5.0 g / L, ferrous sulfate heptahydrate 20 mg / L, manganese sulfate pentahydrate 2.5 mg / L, zinc sulfate heptahydrate 10 mg / L, copper sulfate pentahydrate 6 mg / L, calcium chloride dihydrate 6 mg / L, borax 0.5 mg / L, and ammonium molybdate 0.2 mg / L.

[0143] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 40 g / L.

[0144] In step S2

[0145] B1. After fermentation, a disc centrifuge is used to separate the solid and liquid components.

[0146] B2. Then, the bacterial precipitate is redissolved in purified water to 1.5 times the volume of the tank, the pH is adjusted to 4.5, 10mM sodium chloride, 10mM EDTA, and 2.2% Triton-114 are added, and the mixture is heated to 70℃.

[0147] B3. Introduce air until the pressure in the tank reaches 1.5 MPa, stir at 150 rpm for 2 hours, and the cell breakage rate can reach 98%.

[0148] Additionally, in step S3:

[0149] C1. When the temperature of the cell lysis solution drops to 30℃, add 0.18M sodium chloride to adjust the pH to 4.0, maintain the temperature at 50 rpm and continue stirring for 3 hours. Collect the supernatant using a tubular centrifuge at 12000 rpm.

[0150] C2. Adjust the pH of the supernatant to 4.0, add 6mM EDTA and 2.0% Triton-114, heat to 30°C, purge air to the pressure of the tank to 1.4Mpa, maintain the temperature at 150rpm and continue stirring for 1h, collect the supernatant using a tubular centrifuge at 12000rpm, and repeat this step once. At this point, the endotoxin level in the supernatant can be reduced to 100-300EU / ml.

[0151] C3. Add 10% ammonium sulfate to the supernatant, heat to 45°C, stir at 150 rpm for 2 hours, and collect the protein precipitate using a tube centrifuge at 12000 rpm.

[0152] C4. Protein precipitate is reconstituted with deionized water to 10 times its weight, desalted by ultrafiltration, and purified by DEAE weak anion exchange chromatography column to remove endotoxins. The protein purity can reach over 95%, and the endotoxin can be reduced to 5-10 EU / ml.

[0153] Example 6

[0154] The method for producing the recombinant elastin described in this embodiment includes the following steps:

[0155] S1. Fermentation: Preparing specific fermentation medium for the production of recombinant elastin, culturing (fermenting) engineered strains, and inducing engineered strains;

[0156] S2, cell rupture;

[0157] S3, Purification.

[0158] Step S1 includes the following steps:

[0159] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 34℃. Adjust the pH to 6.5 with ammonia water, maintain a tank pressure of 0.04MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 10mL / h / L.

[0160] A2. When the OD increases to 44, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 15 mL / h / L and the feeding rate of the lactose solution is 3 mL / h / L.

[0161] A3. When the OD increases to 65, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture by feeding culture medium. Add 5 mL / L of lactose solution and the flow rate of the feeding culture medium is 15 mL / h / L.

[0162] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 10 mL / h / L, and the flow rate of the lactose solution was 1 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.5 with ammonia, the tank pressure was 0.04 MPa, the dissolved oxygen was not less than 30%, and the induction was carried out for 5 hours. When the OD of E. coli cells no longer increased and microscopic examination showed cell aging, the entire fermentation process was completed. After low-temperature ultrasonic cell destruction, the protein content was detected by SDS-PAGE and found to be 2.11 g / L.

[0163] The specific fermentation medium for engineered E. coli used in the fermenter includes the following components at the following concentrations: diammonium hydrogen phosphate 0.5 g / L; potassium dihydrogen phosphate 3.0 g / L; citric acid monohydrate 0.2 g / L; magnesium sulfate heptahydrate 0.2 g / L; glucose monohydrate 2 g / L; glycerol 10 g / L; ferrous sulfate heptahydrate 30 mg / L; manganese sulfate pentahydrate 1 mg / L; zinc sulfate heptahydrate 8 mg / L; copper sulfate pentahydrate 3 mg / L; calcium chloride dihydrate 5 mg / L; borax 0.5 mg / L; and ammonium molybdate 0.1 mg / L.

[0164] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 150 g / L, glycerol 300 g / L, magnesium sulfate heptahydrate 0.1 g / L, proline 1 g / L, alanine 1 g / L, ferrous sulfate heptahydrate 10 mg / L, manganese sulfate pentahydrate 0.5 mg / L, zinc sulfate heptahydrate 2 mg / L, copper sulfate pentahydrate 1 mg / L, calcium chloride dihydrate 2 mg / L, borax 0.1 mg / L, and ammonium molybdate 0.1 mg / L.

[0165] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 40 g / L.

[0166] Step S2 includes the following steps:

[0167] B1. After fermentation, a disc centrifuge is used to separate the solid and liquid components.

[0168] B2. The bacterial precipitate is then reconstituted with purified water to 0.8 times the volume of the container, the pH is adjusted to 2.0, and 5 mM sodium chloride, 5 mM EDTA, and 1.5% Triton-114 are added. The mixture is then heated to 55°C.

[0169] B3. Introduce air until the tank pressure reaches 1.2 MPa, stir at 50-150 rpm for 2 hours, and the cell breakage rate can reach 95% after testing.

[0170] In addition, step S3 includes the following steps:

[0171] C1. When the temperature of the cell lysis solution drops to 30℃, add 0.1M sodium chloride to adjust the pH to 3.0. Maintain the temperature at 50 rpm and continue stirring for 2 hours. Collect the supernatant using a tubular centrifuge at 10,000 rpm.

[0172] C2. Adjust the pH of the supernatant to 3.0, add 1 mM EDTA and 1.5% Triton-114, heat to 30°C, purge air to the pressure of the tank to 1.0 MPa, maintain the temperature at 50 rpm and continue stirring for 1 hour, collect the supernatant using a tubular centrifuge at 12000 rpm, and repeat this step once. At this point, the endotoxin level in the supernatant can be reduced to 100-300 EU / ml.

[0173] C3. Add 10% ammonium sulfate to the supernatant, heat to 38°C, stir at 50 rpm for 1 hour, and collect the protein precipitate using a tubular centrifuge at 12000 rpm.

[0174] C4. Protein precipitate is reconstituted with deionized water to 5 times its weight, desalted by ultrafiltration, and purified by DEAE weak anion exchange chromatography column to remove endotoxins. The protein purity can reach over 95%, and the endotoxin can be reduced to 5-10 EU / ml.

[0175] Example 7

[0176] The method for producing the recombinant elastin described in this embodiment includes the following steps:

[0177] S1. Fermentation: Preparing specific fermentation medium for the production of recombinant elastin, culturing (fermenting) engineered strains, and inducing engineered strains;

[0178] S2, cell rupture;

[0179] S3, Purification.

[0180] Step S1 includes the following steps:

[0181] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 7.0 with ammonia water, maintain a tank pressure of 0.08MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 15mL / h / L.

[0182] A2. When the OD increases to 60, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 20 mL / h / L and the feeding rate of the lactose solution is 5 mL / h / L.

[0183] A3. When the OD increases to 73, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture with fed medium. Add 12.5 mL / L of lactose solution and the flow rate of fed medium is 20 mL / h / L.

[0184] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 15 mL / h / L, and the flow rate of the lactose solution was 2 mL / h / L. The temperature was 37℃, the pH was adjusted to 7.0 with ammonia, the tank pressure was 0.08 MPa, the dissolved oxygen was not less than 30%, and the induction was carried out for 5 hours. When the OD of E. coli cells no longer increased and microscopic examination showed cell aging, the entire fermentation process was completed. After low-temperature ultrasonic cell destruction, the protein content was detected by SDS-PAGE and found to be 2.0 g / L.

[0185] The specific fermentation medium for engineered Escherichia coli used in the fermenter includes the following components at the following concentrations: diammonium hydrogen phosphate 3.5 g / L; potassium dihydrogen phosphate 13.5 g / L; citric acid monohydrate 1.6 g / L; magnesium sulfate heptahydrate 1.8 g / L; glucose monohydrate 15 g / L; glycerol 20 g / L; ferrous sulfate heptahydrate 70 mg / L; manganese sulfate pentahydrate 5 mg / L; zinc sulfate heptahydrate 15 mg / L; copper sulfate pentahydrate 10 mg / L; calcium chloride dihydrate 20 mg / L; borax 2 mg / L; and ammonium molybdate 2 mg / L.

[0186] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 300 g / L, glycerol 600 g / L, magnesium sulfate heptahydrate 4.5 g / L, proline 6 g / L, alanine 6 g / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 3 mg / L, zinc sulfate heptahydrate 10 mg / L, copper sulfate pentahydrate 8 mg / L, calcium chloride dihydrate 10 mg / L, borax 1 mg / L, and ammonium molybdate 1 mg / L.

[0187] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 80 g / L.

[0188] Step S2 includes the following steps:

[0189] B1. After fermentation, a disc centrifuge is used to separate the solid and liquid components.

[0190] B2. Next, reconstitute the bacterial precipitate with purified water to 1.5 times the volume of the container, adjust the pH to 5.0, add 15 mM sodium chloride, 15 mM EDTA, and 2.0% Triton-114, and heat to 85°C.

[0191] B3. Introduce air to the tank pressure to 1.5 MPa, stir at 50-150 rpm for 5 hours, and the cell breakage rate can reach 95% after testing.

[0192] In addition, step S3 includes the following steps:

[0193] C1. When the temperature of the cell lysis solution drops to 40℃, add 0.2M sodium chloride to adjust the pH to 5.0, maintain the temperature at 50 rpm and continue stirring for 3 hours. Collect the supernatant using a tubular centrifuge at 12000 rpm.

[0194] C2. Adjust the pH of the supernatant to 5.0, add 10mM EDTA and 3% Triton-114, heat to 40℃, purge air to the pressure of the tank to 1.6Mpa, maintain the temperature at 50-150rpm and continue stirring for 2h, collect the supernatant using a tubular centrifuge at 12000rpm, and repeat this step once. At this point, the endotoxin level in the supernatant can be reduced to 100-300EU / ml.

[0195] C3. Add 40% ammonium sulfate to the supernatant, heat to 50℃, stir at 50-150 rpm for 3 hours, and collect the protein precipitate using a tubular centrifuge at 12000 rpm.

[0196] C4. Protein precipitate is reconstituted with deionized water to 10 times its weight, desalted by ultrafiltration, and purified by DEAE weak anion exchange chromatography column to remove endotoxins. The protein purity can reach over 95%, and the endotoxin can be reduced to 5-10 EU / ml.

[0197] Comparative Example 1

[0198] Based on Example 1, the following parameters are set:

[0199] Fermentation conditions were as follows: culture temperature 37℃, pH adjusted to 6.8 with ammonia, dissolved oxygen not less than 30%, tank pressure 0.05MPa, and inoculum size 5%.

[0200] Feed-through strategy: control glucose concentration at 1.0%, dissolved oxygen at 30%, and pH at 6.8.

[0201] Induction method: When the OD value of the bacterial culture reached 72.0, IPTG was added to a final concentration of 0.5 mM to induce expression for 5 h. After centrifugation, the bacterial cells were harvested, and after low-temperature sonication to break the cells, the protein content was detected by SDS-PAGE and found to be 1.75 g / L.

[0202] The fermentation medium for engineered E. coli used in the fermenter comprises the following components at the following concentrations: tryptone 1.0 g / L, yeast extract 20.0 g / L, glucose 30.0 g / L, sodium chloride 2.0 g / L, dipotassium hydrogen phosphate 8.7 g / L, sodium dihydrogen phosphate 4.2 g / L, ammonium sulfate 5.6 g / L, gelatin (gelatin hydrolysate) 10.0 g / L, magnesium sulfate heptahydrate 2.5 g / L, EDTA 1.0 g / L, ferric chloride hexahydrate 100.0 mg / L, manganese sulfate tetrahydrate 20.0 mg / L, zinc sulfate 8.0 mg / L, boric acid 2.5 mg / L, sodium molybdate 2.5 mg / L, cobalt chloride 2.5 mg / L, and copper chloride 2.5 mg / L.

[0203] Comparative Example 2

[0204] Based on Example 1, the following parameters are set:

[0205] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 13mL / h / L.

[0206] A2. When the OD increases to 52, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 18 mL / h / L and the feeding rate of the lactose solution is 3 mL / h / L.

[0207] A3. When the OD increases to 72, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture by feeding culture medium. Add 1750 mL of lactose solution and the flow rate of the feeding culture medium is 18 mL / h / L.

[0208] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 13 mL / h / L, and the flow rate of the lactose solution was 1 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.5 with ammonia, the tank pressure was 0.08 MPa, the dissolved oxygen was not lower than 30%, and the induction was carried out for 5 hours. When the OD of E. coli cells no longer increased and microscopic examination showed cell aging, the entire fermentation process was completed. After low-temperature ultrasonic cell destruction, the protein content was detected by SDS-PAGE to be 1.81 g / L.

[0209] The fermentation medium for engineered Escherichia coli used in the fermenter contains the following components at the following concentrations: diammonium hydrogen phosphate 2.5 g / L, potassium dihydrogen phosphate 8.0 g / L, citric acid monohydrate 1.0 g / L, magnesium sulfate heptahydrate 0.4 g / L, glucose monohydrate 20 g / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 2.5 mg / L, sulfuric acid heptahydrate 11.25 mg / L, copper sulfate pentahydrate 5 mg / L, calcium chloride dihydrate 10 mg / L, borax 1.15 mg / L, and ammonium molybdate 0.5 mg / L.

[0210] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 600 g / L, magnesium sulfate heptahydrate 1.5 g / L, proline 4.0 g / L, alanine 4.0 g / L, ferrous sulfate heptahydrate 28 mg / L, manganese sulfate pentahydrate 2.0 mg / L, zinc sulfate heptahydrate 6.0 mg / L, copper sulfate pentahydrate 5 mg / L, calcium chloride dihydrate 4.0 mg / L, borax 0.3 mg / L, and ammonium molybdate 0.5 mg / L.

[0211] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 50 g / L.

[0212] Comparative Example 3

[0213] Based on Example 1, the following parameters are set:

[0214] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 13mL / h / L.

[0215] A2. When the OD increases to 52, increase the feeding rate of the feeding medium and start adding lactose solution at the same time. The feeding rate of the feeding medium is 18 mL / h / L and the feeding rate of the lactose solution is 3 mL / h / L.

[0216] A3. When the OD increases to 72, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time continue to culture by feeding culture medium. Add 1750 mL of lactose solution and the flow rate of the feeding culture medium is 18 mL / h / L.

[0217] A4. During the induction period, lactose solution was slowly added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation. The flow rate of the fed culture medium was 13 mL / h / L, and the flow rate of the lactose solution was 1 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.5 with ammonia, the tank pressure was 0.08 MPa, the dissolved oxygen was not less than 30%, and the induction was carried out for 5 hours. When the OD of E. coli cells no longer increased and microscopic examination showed cell aging, the entire fermentation process was completed. After low-temperature ultrasonic cell destruction, the protein content was detected by SDS-PAGE to be 1.90 g / L.

[0218] The fermentation medium for engineered Escherichia coli used in the fermenter contains the following components at the following concentrations: diammonium hydrogen phosphate 2.5 g / L, potassium dihydrogen phosphate 8.0 g / L, citric acid monohydrate 1.0 g / L, magnesium sulfate heptahydrate 0.4 g / L, glycerol 20 g / L, ferrous sulfate heptahydrate 50 mg / L, manganese sulfate pentahydrate 2.5 mg / L, sulfuric acid heptahydrate 11.25 mg / L, copper sulfate pentahydrate 5 mg / L, calcium chloride dihydrate 10 mg / L, borax 1.15 mg / L, and ammonium molybdate 0.5 mg / L.

[0219] The fed culture medium contains the following components at the following concentrations: glycerol 600 g / L, magnesium sulfate heptahydrate 1.5 g / L, proline 4.0 g / L, alanine 4.0 g / L, ferrous sulfate heptahydrate 28 mg / L, manganese sulfate pentahydrate 2.0 mg / L, zinc sulfate heptahydrate 6.0 mg / L, copper sulfate pentahydrate 5 mg / L, calcium chloride dihydrate 4.0 mg / L, borax 0.3 mg / L, and ammonium molybdate 0.5 mg / L.

[0220] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 50 g / L.

[0221] Comparative Example 4

[0222] Based on Example 1, the following parameters are set:

[0223] A1. Inoculate the engineered Escherichia coli at a rate of 5% into a 500L fermenter containing 250L of fermentation medium and culture at a temperature of 37℃. Adjust the pH to 6.8 with ammonia water, maintain a tank pressure of 0.05MPa, and ensure dissolved oxygen is not lower than 30%. After the dissolved oxygen has significantly recovered, add feed medium at a flow rate of 13mL / h / L.

[0224] A2. When the OD increases to 52, increase the feeding rate of the feeding medium to 18 mL / h / L.

[0225] A3. When the OD increases to 72, add an appropriate amount of lactose solution to start induction, and at the same time continue to culture by feeding culture medium. Add 1750 mL of lactose solution and the feed culture medium flow rate is 18 mL / h / L.

[0226] A4. During the induction period, fed culture medium was added until the end of fermentation. The feed rate of the culture medium was 13 mL / h / L. The temperature was 34℃, the pH was adjusted to 6.5 with ammonia, the tank pressure was 0.08 MPa, the dissolved oxygen was not less than 30%, and the induction period was 5 hours. When the OD of E. coli cells stopped increasing, microscopic examination showed that the cells were aging and the entire fermentation process was over. After low-temperature ultrasonic cell destruction, the protein content was detected by SDS-PAGE and found to be 1.93 g / L.

[0227] The fermentation medium for engineered Escherichia coli used in the fermenter contains the following components at the following concentrations: diammonium hydrogen phosphate 2.5 g / L; potassium dihydrogen phosphate 8.0 g / L; citric acid monohydrate 1.0 g / L; magnesium sulfate heptahydrate 0.4 g / L; glucose monohydrate 5 g / L; glycerol 15 g / L; ferrous sulfate heptahydrate 50 mg / L; manganese sulfate pentahydrate 2.5 mg / L; sulfuric acid heptahydrate 11.25 mg / L; copper sulfate pentahydrate 5 mg / L; calcium chloride dihydrate 10 mg / L; borax 1.15 mg / L; and ammonium molybdate 0.5 mg / L.

[0228] The fed culture medium contains the following components at the following concentrations: glucose monohydrate 200 g / L; glycerol 400 g / L; magnesium sulfate heptahydrate 1.5 g / L; proline 4.0 g / L; alanine 4.0 g / L; ferrous sulfate heptahydrate 28 mg / L; manganese sulfate pentahydrate 2.0 mg / L; zinc sulfate heptahydrate 6.0 mg / L; copper sulfate pentahydrate 5 mg / L; calcium chloride dihydrate 4.0 mg / L; borax 0.3 mg / L; and ammonium molybdate 0.5 mg / L.

[0229] The engineered E. coli lactose culture medium used in the fermenter contains the following components at the following concentrations: lactose 50 g / L.

[0230] Experimental Example 1

[0231] Determination of recombinant elastin cytotoxicity

[0232] L929 mouse fibroblasts were diluted to 1×10⁻⁶ cells using DMEM / F12 medium containing 10% fetal bovine serum. 3Single-cell suspension of 100 μL / mL was inoculated into each well of a 96-well culture plate and incubated at 37°C for 24 h in a 5% CO2 incubator. The original culture medium was discarded, and 100 μL of recombinant elastin and hydrolyzed elastin (Sigma) were added, with concentrations of 100 mg / L, 500 mg / L, 1000 mg / L, and 5000 mg / L respectively. A negative control group (cell culture medium only) and a positive control group (4% DMSO) were also set up, with 8 wells in each group. The culture plate was transferred to a 37°C, 5% CO2 incubator. After 96 hours, the plate was removed, and 50 μL of MTT (thiazolyl blue) was added to each well. The plate was incubated at 37°C for another 2 h. The liquid was aspirated, and immediately 150 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was then incubated at room temperature with gentle shaking for 10–15 min. The absorbance of each well was measured at 490 nm using a microplate reader, and the relative cell proliferation rate was calculated.

[0233] RGR (%) = (Experimental group absorbance / Negative control group absorbance) × 100%

[0234] The results are as follows Figure 3 As shown.

[0235] Depend on Figure 3 It can be seen that the relative cell proliferation rate of the recombinant elastin system at different concentrations is greater than 100%, and is significantly higher than that of hydrolyzed elastin. The cytotoxicity is grade 0, and the biocompatibility is good.

[0236] Experimental Example 2

[0237] Following standard methods, solutions obtained from Examples 1, 2, 3, Comparative Examples 1, 2, 3, and 4 were subjected to protein gel electrophoresis to detect the fermentation production level of recombinant elastin (fermentation production level (g / L·h) = concentration of recombinant elastin (g / L) / fermentation period (h)). The results are shown in Table 1. Cell disruption effect is as follows... Figure 1 As shown, the purification effect of recombinant elastin is as follows: Figure 2 As shown:

[0238] Table 1. Recombinant Elastin Production Level

[0239]

[0240] As shown in Table 1, compared with Comparative Examples 1-4, Examples 1-5 used a combination of glycerol and glucose for culture and induction, a compound amino acid and inorganic salt ion formulation, and staged lactose induction, which promoted cell growth and target protein expression. The fermentation production level of Examples 1-5 reached 0.117 g / L / h, which was more than 20% higher than that of the comparative examples. Comparative Example 1 used a traditional peptone / yeast powder formulation and IPTG induction, Comparative Example 2 used glucose alone as a carbon source, and Comparative Example 3 used glycerol alone as a carbon source. When glucose is used as a carbon source, it easily produces organic acids during the growth of E. coli, which inhibits cell growth and protein expression. Glycerol is viscous and easily causes oxygen transfer to be blocked, resulting in low utilization and slow growth of E. coli, which affects protein expression. This invention uses a suitable ratio of glucose and glycerol as a carbon source, avoiding excessive organic acid generation during E. coli growth without affecting the cell growth rate. Inorganic salt ions promote carbon source utilization, while complex amino acids and other complex ions promote both cell growth and protein expression. Comparative Example 4 uses a single lactose induction. Production level comparison shows that staged lactose induction is significantly superior to single lactose induction. In summary, the fermentation method provided by this invention, compared to Comparative Examples 1-4, yields a higher concentration of recombinant elastin, a shorter fermentation cycle, and a higher fermentation production level of recombinant elastin. Furthermore, during fermentation, excessively low flow rates of lactose and fed-batch medium result in slow growth and impaired expression, while excessively high flow rates lead to organic acid accumulation, inhibiting cell growth and protein expression. The cell lysis process utilizes the thermal phase change property of the target protein, employing salt ions and heating to precipitate the target protein while precipitating only a small amount of other proteins, facilitating purification and improving protein purity.

[0241] Depend on Figure 1 As shown in Figure 1 (lane 1 is the marker, lanes 2, 3, 4, 5, and 6 are recombinant elastin pseudo-standard solutions with concentrations of 0.1 mg / mL, 0.15 mg / mL, 0.20 mg / mL, 0.25 mg / mL, and 0.30 mg / mL, respectively; lane 7 is the 20-fold diluted solution of the 500L fermenter sample subjected to ultrasonic cell disruption in Example 1; lane 8 is the 20-fold diluted solution of the 500L fermenter sample subjected to cell disruption in Example 1; the sample loading volume for all samples is 10 μL), with the recombinant elastin concentration as the x-axis and its grayscale value as the y-axis (see Table 2), the linear regression (y = 25265x + 1815.5, R0) was calculated by software. 2 =0.9993), the fermentation yield of recombinant elastin in a 500L fermenter is 2.25g / L, and after cell rupture it is 2.21g / L, with a cell rupture complete rate of 98%, which can fully meet the conditions for industrial production.

[0242] Table 2. Cell disruption effect of recombinant elastin

[0243] 2 0.1 4296.648 3 0.15 5681.355 4 0.2 6873.305 5 0.25 8075.134 6 0.3 9415.962 7 2.25 4656.305 8 2.21 4606.234

[0244] according to Figure 2 In the figure, lane 1 is the marker, lane 2 is the recombinant elastin purified B1 sample, lane 3 is the recombinant elastin purified B2 sample, and lane 4 is the recombinant elastin purified B3 sample. The loading volume for each sample is 10 μL. Figure 3 It is evident that the purification method provided by this invention can achieve simple and efficient purification of recombinant elastin, effectively removing impurities and endotoxins. The final product purity, as determined by grayscale analysis, can reach over 95%, and the endotoxin level can be controlled at 5–10 EU / mL. The operation is simple, greatly saving purification costs, and the low endotoxin level also expands the product's usability range.

[0245] In summary, the recombinant elastin production process provided in this application uses a fermentation medium that is completely different from the traditional modified LB medium. During the fermentation process, a mixture of glycerol and glucose as carbon sources, along with amino acids and trace elements, is used for feeding. Lactose is slowly added during the mid-logarithmic growth phase for self-induction, and an appropriate amount of lactose is added once during the mid-to-late logarithmic growth phase to increase the induction pressure. Lactose is slowly added during the induction phase to maintain the induction pressure, which effectively improves the fermentation expression level of recombinant elastin. Furthermore, by combining the thermal phase change characteristics of recombinant elastin, a cell disruption process that does not require a high-pressure homogenizer has been developed, eliminating the need for a complex and lengthy purification and endotoxin removal post-treatment process.

[0246] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present invention, and these modifications or substitutions should all be covered within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for producing recombinant elastin, characterized in that, Includes the following steps: S1. Fermentation: Engineered strains are cultured using specific fermentation media, and engineered strains are induced in stages during the culture process; The specific fermentation medium comprises the following components at the following concentrations: diammonium hydrogen phosphate 0.5~3.5 g / L, potassium dihydrogen phosphate 3.0~13.5 g / L, citric acid monohydrate 0.2~1.6 g / L, magnesium sulfate heptahydrate 0.2~1.8 g / L, glucose monohydrate 2~15 g / L, glycerol 10~20 g / L, ferrous sulfate heptahydrate 30~70 mg / L, manganese sulfate pentahydrate 1~5 mg / L, zinc sulfate heptahydrate 8~15 mg / L, copper sulfate pentahydrate 3~10 mg / L, calcium chloride dihydrate 5~20 mg / L, borax 0.5~2 mg / L, and ammonium molybdate 0.1~2 mg / L. The phased induction engineered microbial strains include: When the OD value of the engineered bacteria solution reaches 44-60, lactose solution is added for initial induction, and fed culture medium is added simultaneously for continued culture. When the OD value of the engineered bacteria solution reaches 65-73, lactose solution is added once to increase the induction pressure, and fed culture medium is added at the same time to continue culturing. During the induction period, lactose solution was added to maintain the induction pressure, while fed culture medium was added continuously until the end of fermentation; S2, Cell disruption: Cell disruption is achieved using surfactants and salt ions; S3. Purification: Adjust pH, control temperature, and add surfactants to purify the protein; specifically including: C1. When the temperature of the cell lysis solution drops to 30-40℃, add a small amount of salt ions to adjust the pH to 3.0-5.0, maintain the temperature and continue stirring, then centrifuge to collect the supernatant. C2. Adjust the pH of the supernatant to 3.0-5.0, add EDTA and surfactant, heat to 30-40℃, adjust the tank pressure, maintain the temperature and continue stirring, centrifuge to collect the supernatant, and repeat this step. C3. Add inorganic salts to the supernatant, heat to 38-50℃, stir, and centrifuge to collect the protein precipitate; C4. The protein precipitate was reconstituted, desalted by ultrafiltration, and purified by weak anion exchange chromatography.

2. The method for producing recombinant elastin according to claim 1, characterized in that, Step S1 specifically includes the following steps: A1. Inoculate the engineered Escherichia coli seed culture into the specific fermentation medium in the fermenter and culture it. After the dissolved oxygen level rises significantly, feed medium is added to continue the culture. A2. When the OD value of the engineered bacteria solution reaches 44-60, start adding lactose solution for initial induction, and at the same time add fed culture medium to continue culturing. A3. When the OD value of the engineered bacteria solution reaches 65-73, add an appropriate amount of lactose solution to increase the induction pressure, and at the same time, feed culture medium is added to continue the culture. A4. During the induction period, add lactose solution to maintain the induction pressure, and simultaneously add fed culture medium until the end of fermentation.

3. The method for producing recombinant elastin according to claim 2, characterized in that, In step A1, the supplemental culture medium comprises the following components at the following concentrations: glucose monohydrate 150-300 g / L, glycerol 300-600 g / L, magnesium sulfate heptahydrate 0.1-4.5 g / L, complex amino acids 1-6.0 g / L, ferrous sulfate heptahydrate 10-50 mg / L, manganese sulfate pentahydrate 0.5-3 mg / L, zinc sulfate heptahydrate 2-10 mg / L, copper sulfate pentahydrate 1-8 mg / L, calcium chloride dihydrate 2-10 mg / L, borax 0.1-1 mg / L, and ammonium molybdate 0.1-1 mg / L.

4. The method for producing recombinant elastin according to claim 2, characterized in that: In step A2, the lactose solution comprises the following components at the following concentrations: lactose 40-80 g / L.

5. The method for producing recombinant elastin according to claim 2, characterized in that: In step A1, the flow rate of the feeding medium is 10-15 mL / h / L; in step A2, the flow rate of the feeding medium is 15-20 mL / h / L, and the flow rate of the lactose solution is 3-5 mL / h / L; in step A3, the flow rate of the feeding medium is 15-20 mL / h / L, and the flow rate of the lactose solution is 5-12.5 mL / L; in step A4, the flow rate of the feeding medium is 10-15 mL / h / L, and the flow rate of the lactose solution is 1-2 mL / h / L.

6. The method for producing recombinant elastin according to claim 2, characterized in that: The fermentation conditions are as follows: fermentation temperature 34~37℃, pH adjusted to 6.5~7.0 with ammonia water, dissolved oxygen not less than 30%, and tank pressure 0.04~0.08MPa.

7. The method for producing recombinant elastin according to claim 1, characterized in that, Step S2 specifically includes the following steps: B1. After fermentation, centrifuge to separate the solid and liquid components; B2. Redissolve the bacterial precipitate to 0.8-1.5 times the volume of the container, adjust the pH to 2-5, add sodium chloride, EDTA, and surfactant, and heat to 55-85℃; B3. Adjust the tank pressure and stir continuously at 50-150 rpm for 2-5 hours.

8. The method for producing recombinant elastin according to claim 1, characterized in that: In step C1, the added salt ion is 0.1M~0.2M sodium chloride; in step C2, 1-10 mM EDTA and 1.5%-3% surfactant are added, air is introduced to the pressure of the container to 1.0-1.6 MPa, the temperature is maintained at 50-150 rpm and stirring is continued for 1-2 hours, and the supernatant is collected by centrifugation; in step C3, 10%-40% ammonium sulfate is added to the supernatant, stirring is carried out at 50-150 rpm for 1-3 hours, and the protein precipitate is collected by centrifugation; in step C4, the protein precipitate is reconstituted with deionized water to 5-10 times its weight, and then subjected to DEAE weak anion exchange chromatography.

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