CN117737163A - Fermentation process for improving production level of recombinant humanized collagen - Google Patents
Fermentation process for improving production level of recombinant humanized collagen Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a fermentation process for improving the production level of recombinant humanized collagen, belonging to the technical field of biological fermentation. The invention adds malt extract powder to the fermentation basic culture medium containing yeast extract powder to replace part of the yeast extract powder to form the fermentation culture medium to ferment and produce recombinant humanized collagen, wherein the mass percentage of the malt extract powder to replace the yeast extract powder is 5-20%. According to the invention, three escherichia coli col 1, col 2 and col 3 with different genotypes are used as fermentation strains, and the unexpected finding that the escherichia coli col 1 has higher fermentation speed and higher fermentation yield under the fermentation process, shortens the fermentation period, reduces the total cost by more than 25%, improves the expression quantity of the recombinant humanized collagen by 15.38-115.38%, and further improves the production level of the recombinant humanized collagen.
Description
Technical Field
The invention relates to a fermentation process for improving the production level of recombinant humanized collagen, belonging to the technical field of biological fermentation.
Background
Collagen is a very important class of functional proteins in the human body, and is widely distributed in connective tissues of the human body, such as skin, cartilage and bone, tendons, ligaments, cornea, organ capsule, dura mater, etc. The human body contains type I, type II and type III collagen with relatively high content, and the content of the type I, type II and type III collagen accounts for 80-90% of the total collagen. Because of its unique biological structure, it has been widely used in medicine, health products and cosmetics industries.
Collagen undergoes two major stages of production, extraction and biosynthesis. Based on different gene coding technical modes, the recombinant collagen can be further divided into recombinant collagen, recombinant humanized collagen and recombinant human collagen. According to the naming guidance principle of the national drug administration for publishing recombinant collagen biomaterials, recombinant human collagen refers to the full-length amino acid sequence encoded by a specific type gene of human collagen prepared by a DNA recombination technology and has a triple helix structure; recombinant humanized collagen refers to full-length or partial amino acid sequence fragments encoded by specific genes of human collagen prepared by DNA recombination technology, or a combination containing functional fragments of human collagen; the recombinant collagen-like protein is an amino acid sequence or a fragment thereof which is prepared by a DNA recombination technology and is encoded by a specific designed and modified gene, or a combination of functional amino acid sequence fragments, and the gene encoding sequence or the amino acid sequence has low homology with the gene encoding sequence or the amino acid sequence of the human collagen protein.
Wherein the recombinant human collagen vector has optimal expression yield and bioactivity, and the recombinant humanized collagen is the next. The amino acid sequence repeating unit of the recombinant humanized collagen is the same as the specific functional region of the amino acid sequence of the human collagen, and the humanized collagen with 100 percent of similarity to the amino acid sequence of the human collagen can be actively absorbed and utilized by skin.
In the production of the recombinant humanized collagen, the fermentation time is further shortened, the protein expression quantity is improved, the production cost is reduced, and the industrial large-scale production of the recombinant humanized collagen is facilitated. The recombinant humanized collagen expression gene is embedded into an escherichia coli host, and high-purity recombinant humanized collagen is obtained through fermentation, so that practical application value is brought to industrial production.
Malt extract, malt extract powder. The malt extract powder is prepared by taking selected barley malt as a raw material, and extracting the barley malt without adding any other materials under the conditions of biotechnology means and equipment. Malt extract is rich in various carbohydrates, proteins, peptides, amino acids, purines, pyrimidines, vitamins, etc. Malt extract powder can be used for culturing microorganisms, but the influence of malt extract powder on the production of recombinant humanized collagen is still freshly reported at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a fermentation process for improving the production level of recombinant humanized collagen. The malt extract powder is used as one of the raw materials of the culture medium, provides the growth factors required by cell growth, and compared with the yeast extract powder, the rich carbohydrate substances in the malt extract powder bring high-quality carbon sources to cell metabolism besides providing nitrogen sources, and the price has advantages, and the yeast extract powder is replaced by a certain proportion, so that the production cost can be reduced, and the yield of the recombinant humanized collagen is improved.
The technical scheme of the invention is as follows:
a fermentation process for improving the production level of recombinant humanized collagen is to add malt extract powder to a fermentation basic culture medium containing yeast extract powder to replace part of the yeast extract powder to form a fermentation culture medium, inoculating a fermentation strain, and fermenting to produce the recombinant humanized collagen, wherein the proportion of the malt extract powder to the yeast extract powder is 5-20% by mass.
According to the invention, the ratio of malt extract powder to yeast extract powder is preferably 10-20%.
According to a preferred embodiment of the invention, the fermentation base medium has the following composition: 2-4% of yeast extract powder, 0.5-3% of glycerol, 0.1-0.4% of magnesium sulfate heptahydrate, 0.2-0.5% of ammonium sulfate, 0.06-0.15% of sodium sulfate, 0.2-1.5% of sodium chloride, 0.2-0.8% of monopotassium phosphate, 0.5-2.3% of disodium hydrogen phosphate dodecahydrate, 0.2-0.6% of microelement solution (film sterilization) and the balance of water in percentage by mass;
wherein the trace element solution comprises: boric acid 0.22-0.35%, sodium molybdate 0.01-0.02%, ferrous sulfate heptahydrate 0.14-0.27%, manganese sulfate monohydrate 0.055-0.12%, cupric chloride dihydrate 0.015-0.035%, zinc sulfate monohydrate 0.022-0.033%, potassium sodium tartrate tetrahydrate 0.15-0.32%, cobalt chloride 0.05-0.9%, concentrated sulfuric acid 0.05-0.1%, and the balance water.
According to the present invention, preferably, the fermentation strain is Escherichia coli 1 recombinant expressing a humanized type III collagen having an amino acid sequence shown in SEQ ID NO. 1.
Further preferably, the escherichia coli col 1 is constructed by using escherichia coli BL21 (DE 3) as a host bacterium.
In the invention, the fermentation method of the recombinant humanized collagen can be used for carrying out fermentation culture according to the prior art after the composition of the fermentation medium is regulated to produce the recombinant humanized collagen.
The invention relates to a fermentation process for improving the production level of recombinant humanized collagen, which specifically comprises the following steps:
a. seed liquid culture: inoculating Escherichia coli col 1 into seed culture medium, and culturing at 36-38deg.C and 170-200rpm to obtain seed solution, OD of the obtained seed solution 600 More than 3;
b. preparing a fermentation medium: adding malt extract powder to the fermentation basic culture medium to replace part of yeast extract powder to form a fermentation culture medium, wherein the malt extract powder is 5-20% of the yeast extract powder by mass percent, and sterilizing for later use;
c. fermentation: inoculating the seed liquid cultured in the step a into the sterilized fermentation medium in the step b to start fermentation, wherein the initial fermentation conditions are as follows: the ventilation is 1 to 1.3vvm, the stirring rotation speed is 130 to 150rpm, the fermentation temperature is 36 to 38 ℃, 50 percent ammonia water is used for adjusting the pH value to 6.8 to 7.5, and the dissolved oxygen is 98 to 102 percent;
in the fermentation process, dissolved oxygen is continuously reduced, and the dissolved oxygen is controlled to be 15-30% by associated stirring;
when the glycerol in the fermentation medium is exhausted, the dissolved oxygen can rise obviously, and the dissolved oxygen rises to 55-75%, and then the glycerol is supplemented;
when fermentation liquor OD 600 When the fermentation temperature is 60-80 ℃, adjusting the fermentation temperature to 25-30 ℃, adding IPTG after membrane sterilization to make the final concentration of the IPTG to be 0.3-0.6mM, and beginning to induce and express recombinant humanized collagen;
when fermentation liquor OD 600 When no more increases, the fermentation is ended.
Further preferably, the composition of the seed medium in step a is: yeast extract powder 0.5%, peptone 1.0%, sodium chloride 1.0% and the balance of water.
Further preferably, the OD of the seed liquid in step a 600 3-5.
It is further preferred that the seed solution in step c is inoculated in an amount of 1 to 3 mass%.
Further preferably, the concentration of glycerol fed in step c is 50wt% and the feed rate is 0.4-0.7mL/min.
The application of malt extract powder in preparing recombinant humanized collagen by fermentation.
According to the invention, the fermented strain is escherichia coli 1 for recombinant expression of humanized type III collagen with an amino acid sequence shown as SEQ ID NO. 1.
Further preferably, the escherichia coli col 1 is constructed by using escherichia coli BL21 (DE 3) as a host bacterium.
According to the invention, the malt extract is preferably used to replace part of the yeast extract in the fermentation basal medium to participate in the fermentation process.
Further preferably, the mass percentage of the malt extract powder to replace yeast extract powder is 5-20%; further preferably 10 to 20%.
The beneficial effects are that:
the invention provides a fermentation process for improving the production level of recombinant humanized collagen, which is characterized in that malt extract powder is added into a fermentation basic culture medium containing yeast extract powder to replace part of the yeast extract powder to form a fermentation culture medium, so as to ferment and produce the recombinant humanized collagen, wherein the mass percentage of the malt extract powder to replace the yeast extract powder is 5-20%. According to the invention, three escherichia coli col 1, col 2 and col 3 with different genotypes are used as fermentation strains, and the unexpected finding that the escherichia coli col 1 has higher fermentation speed and higher fermentation yield under the fermentation process, shortens the fermentation period, reduces the total cost by more than 25%, improves the expression quantity of the recombinant humanized collagen by 15.38-115.38%, and further improves the production level of the recombinant humanized collagen.
Drawings
FIG. 1 is a SDS-PAGE diagram of samples of fermentation broth of Escherichia coli col 1 obtained under different fermentation processes.
FIG. 2 is a SDS-PAGE diagram of samples of fermentation broth of Escherichia coli col 2 obtained under different fermentation processes.
FIG. 3 is a SDS-PAGE diagram of samples of fermentation broth of Escherichia coli col 3 obtained under different fermentation processes.
FIG. 4 is a graph showing the yield of recombinant humanized type III collagen from Escherichia coli col 1, col 2, and col 3 under different fermentation processes.
Detailed Description
The technical scheme of the invention will be further described by combining specific embodiments. It should be understood that the embodiments described herein are exemplary only and should not be construed as limiting the scope of the invention in any way. The reagents and materials used in the examples were all commercially available products unless otherwise specified. The experimental procedures referred to in the examples, unless otherwise specified, are conventional in the art.
Microbial origin:
the escherichia coli col 1, col 2 and col 3 are genetically engineered bacteria for recombinant expression of the existing humanized III-type collagen. The method for constructing escherichia coli col 1, col 2 and col 3 is as follows:
the amino acid sequences of the recombinant III type humanized collagens col 1, col 2 and col 3 are respectively shown as SEQ ID NO.1-3, and codon optimization is carried out according to the codon preference of escherichia coli, so that the coding genes of the recombinant III type humanized collagens col 1, col 2 and col 3 are artificially synthesized; the coding genes and plasmid pET-3a are respectively cut by KpnI and NdeI restriction enzymes, and recombinant plasmids pET-3a-col 1, pET-3a-col 2 and pET-3a-col 3 are obtained after enzyme connection; the recombinant plasmids are respectively transformed into host bacterium escherichia coli BL21 (DE 3), and the recombinant genetic engineering bacteria escherichia coli col 1, escherichia coli col 2 and escherichia coli col 3 are obtained through verification and screening.
Composition of seed medium:
every 100mL of seed culture medium comprises the following components: yeast extract 0.5g, peptone 1.0g, sodium chloride 1.0g, water constant volume to 100mL.
The fermentation base medium composition in examples 1-4 and comparative example 1:
every 3000g of fermentation basic culture medium comprises the following components: 90g of yeast extract powder, 50g of glycerol, 25g of disodium hydrogen phosphate dodecahydrate, 14g of potassium dihydrogen phosphate, 8g of ammonium sulfate, 6g of magnesium sulfate heptahydrate, 2.5g of sodium sulfate, 15g of sodium chloride, 15g of microelement solution and water to a constant volume of 3000g;
wherein, the trace element solution comprises the following components in 15 g: boric acid 0.043g, manganese sulfate monohydrate 0.017g, ferrous sulfate heptahydrate 0.038g, cupric chloride dihydrate 0.005g, zinc sulfate monohydrate 0.004g, potassium sodium tartrate tetrahydrate 0.033g, cobalt chloride 0.13g, sodium molybdate 0.0022g, concentrated sulfuric acid 0.015g, water constant volume to 15g, and refrigerating at 4 ℃ after sterilizing by film.
The malt extract powder is a commercial product and can be purchased from Sichuan Baoshun biotechnology Co.
In the examples, the ratio of malt extract to yeast extract added is as follows:
TABLE 1 Yeast extract and malt extract in fermentation medium in mass percent
Example 1:
a fermentation process of recombinant humanized collagen comprises the following steps:
a. culturing seed liquid: respectively collecting 100 μl of preserved Escherichia coli col 1, col 2 and col 3, inoculating into 100mL seed culture medium, culturing at 37deg.C and 170rpm for 14 hr to obtain seed solution, and adjusting OD of the seed solution 600 4.5;
b. preparing a fermentation medium: based on 3000g of fermentation basic culture medium, replacing part of yeast extract powder in the fermentation basic culture medium with malt extract powder, preparing a fermentation culture medium, wherein the mass of the yeast extract powder and the malt extract powder in the fermentation culture medium are respectively 85.5g and 4.5g, the proportion of other components is unchanged, filling the prepared fermentation culture medium into a 5L fermentation tank, and sterilizing at 121 ℃ for 25min for later use;
c. fermentation culture: inoculating the seed solution obtained in the step a into the fermentation culture medium of the step b according to the mass percent of inoculation of 3% for fermentation culture, wherein the initial fermentation conditions are as follows: stirring at 150rpm, introducing air at 1.3vvm, fermenting at 37deg.C, regulating pH to 7.0 with 50% ammonia water, and dissolving oxygen at 100%; along with the fermentation, the dissolved oxygen is continuously reduced, and when the dissolved oxygen is reduced to 20%, the rotating speed is increased to control the dissolved oxygen to 15-30%; when the glycerol in the fermentation medium is exhausted, the dissolved oxygen can be obviously increased, the dissolved oxygen can be increased to 55-65% from 15-30% before, and the glycerol is added;
the glycerol concentration of the supplement was 50wt% and the rate of supplement was carried out with reference to the following table:
TABLE 2 rate of glycerol supplementation
When fermentation liquor OD 600 At 80, the fermentation temperature is regulated to 28 ℃, IPTG (sterilizing by passing through a membrane) is added to a final concentration of 0.6mM, and the induction expression of recombinant human type III collagen is started;
in the process of inducing and expressing recombinant humanized III type collagen, 10mL of fermentation liquor is taken every 2h to detect OD of the fermentation liquor 600 When fermentation liquor OD 600 And when the fermentation is not increased any more, ending the fermentation, and collecting fermentation liquor.
Example 2:
the fermentation process of the recombinant humanized collagen is different from that of the embodiment 1 in that the weight of yeast extract powder and malt extract powder in a fermentation medium is 81g and 9g respectively, and the rest is the same as the embodiment 1.
Example 3:
the fermentation process of the recombinant humanized collagen is different from that of the example 1 in that the weight of the yeast extract and the malt extract in the fermentation medium are 76.5g and 13.5g respectively, and the rest is the same as that of the example 1.
Example 4:
the fermentation process of the recombinant humanized collagen is different from that of the embodiment 1 in that the weight of yeast extract powder and malt extract powder in a fermentation medium is 72g and 18g respectively, and the rest is the same as the embodiment 1.
Comparative example 1:
a fermentation process of recombinant humanized collagen was different from example 1 in that the weight of yeast extract powder in the fermentation medium was 90g, malt extract powder was not added, and the rest was the same as in example 1.
The fermentation times for the induction of recombinant humanized type III collagen expression in examples 1-4 and comparative example 1 are shown in the following table:
TABLE 3 fermentation time Table for inducible expression of recombinant humanized III collagen
As shown in Table 3, the fermentation time of the recombinant humanized III-type collagen can be properly reduced and the fermentation speed can be improved by adding malt extract powder to the fermentation medium instead of part of yeast extract powder.
Example 5:
unlike example 1, the fermentation process of recombinant humanized collagen was characterized in that the components of the fermentation basal medium were as follows:
every 3000g of fermentation basic culture medium comprises the following components: 120g of yeast extract powder, 24g of glycerol, 30g of disodium hydrogen phosphate dodecahydrate, 6g of potassium dihydrogen phosphate, 12g of ammonium sulfate, 3g of magnesium sulfate heptahydrate, 3g of sodium sulfate, 30g of sodium chloride, 6g of microelement solution and water to a constant volume of 3000g;
wherein, each 15g of microelement solution comprises the following components: boric acid 0.033g, manganese sulfate monohydrate 0.009g, ferrous sulfate heptahydrate 0.021g, cupric chloride dihydrate 0.003g, zinc sulfate monohydrate 0.004g, potassium sodium tartrate tetrahydrate 0.023g, cobalt chloride 0.015g, sodium molybdate 0.0015g, concentrated sulfuric acid 0.008g, water constant volume to 15g, and refrigerating at 4 ℃ after sterilization by using a film.
Based on 3000g of fermentation basic culture medium, replacing part of yeast extract powder in the fermentation basic culture medium with malt extract powder to prepare a fermentation culture medium, wherein the mass of the yeast extract powder and the malt extract powder in the fermentation culture medium are respectively 108g and 12g;
the remainder was the same as in example 1.
Example 6:
unlike example 1, the fermentation process of recombinant humanized collagen was characterized in that the components of the fermentation basal medium were as follows:
every 3000g of fermentation basic culture medium comprises the following components: 60g of yeast extract powder, 90g of glycerol, 60g of disodium hydrogen phosphate dodecahydrate, 24g of potassium dihydrogen phosphate, 15g of ammonium sulfate, 12g of magnesium sulfate heptahydrate, 4.5g of sodium sulfate, 45g of sodium chloride, 18g of microelement solution and water to a constant volume of 3000g;
wherein, each 15g of microelement solution comprises the following components: boric acid 0.052g, manganese sulfate monohydrate 0.013g, ferrous sulfate heptahydrate 0.03g, cupric chloride dihydrate 0.004g, zinc sulfate monohydrate 0.004g, potassium sodium tartrate tetrahydrate 0.048g, cobalt chloride 0.075g, sodium molybdate 0.003g, concentrated sulfuric acid 0.011g, water constant volume 15g, and refrigerating at 4 ℃ after film sterilization.
Based on 3000g of fermentation basic culture medium, replacing part of yeast extract powder in the fermentation basic culture medium with malt extract powder to prepare a fermentation culture medium, wherein the mass of the yeast extract powder and the malt extract powder in the fermentation culture medium are respectively 48g and 12g;
the remainder was the same as in example 1.
Experimental example 1:
analysis of recombinant humanized type III collagen production, the procedure was as follows:
(1) Sample treatment: taking appropriate amount of fermentation broth (according to fermentation broth OD) 600 Calculating the sampling amount: sample size (μL) =5/OD 600 X 1000, in order to keep the quantity of thalli in each processed sample consistent), putting the samples into a centrifuge 12000rpm for centrifugation for 2min, collecting thalli, adding 100mL of ultrapure water and 25 mu L of 5 Xprotein loading buffer solution, blowing and mixing uniformly by a pipetting gun, resuspending thalli, and boiling for 10min at 100 ℃ in a metal bath to obtain a sample solution.
(2) BSA control: taking 20 mu L of 1.0mg/mL BSA (bovine serum albumin) control solution frozen at-20 ℃, adding 5 mu L of 5 Xprotein loading buffer solution, and boiling for 5min at 100 ℃ in a metal bath to obtain the control solution.
(3) Preparation of separation gel and concentrated gel: the gel was prepared by the preparation method of the purchased kit, and after mixing, the gel solution was separated by a pipette and carefully poured along a slide into a slit between a long glass plate and a short glass plate at about 7.5 mL/block, and polymerized at room temperature (different room temperatures, different polymerization times). After polymerization of the separated gum solution, the upper aqueous layer was blotted with filter paper, and then the concentrated gum solution was poured in, and a sample comb was inserted, taking care to avoid air bubbles.
(4) And (3) sample running: taking 5 mu L of the sample solution treated in the step (1) and 3 mu L of the control solution treated in the step (2) by a liquid-transferring gun respectively, carefully adding the sample solution into each gel concave sample groove sequentially, adding a marker into one groove, and adding the marker into different hole grooves for distinguishing two plates, wherein the loading amount of the marker is 5 mu L. The electrophoresis tank is placed on the electrophoresis apparatus, and the power supply is switched on, so that the anode and the cathode are good. And (3) regulating the voltage to 160V, starting electrophoresis, stopping electrophoresis when the bromophenol blue mark migrates to the bottom of the gel, and turning off the power supply.
(5) Film processing: taking out the electrophoresis tank, taking down the two plates, tilting the electrophoresis tank from the middle of the long glass slide by using a scraping blade, scraping the concentrated glue, and taking down. Put in a glass plate, rinsed 1 time with ultrapure water. Then adding coomassie brilliant blue staining solution, allowing the staining solution to overflow the gel, placing on a shaking table at a rotating speed of about 50r/min, staining overnight, recovering the staining solution after completion, and rinsing with ultrapure water for 2 times. Transferring the gel to a clean glass plate, adding a decoloring solution, and spreading the gel on a shaking table at a rotating speed of about 50r/min for about 1h, and changing a new decoloring solution at a rotating speed of about 50r/min for about 30min until the background color is completely removed, wherein the stripes are clearly visible. Placing the decolored glue on a sample stage of a gel imager for imaging.
(6) Data analysis: and (3) carrying out gray level analysis on the electrophoresis image by using BLT Bio analysis software, and calculating the content of the recombinant humanized collagen in the sample solution and the concentration of the recombinant humanized collagen in the fermentation broth by taking the gray level value of the 3 mu L BSA reference solution as a reference. Wherein:
concentration of recombinant humanized collagen in fermentation broth = content of recombinant humanized collagen in sample solution (μg)/sample volume of fermentation broth (μl)
The SDS-PAGE diagram of the fermentation liquid samples obtained by the escherichia coli 1 under different fermentation processes is shown in fig. 1, the SDS-PAGE diagram of the fermentation liquid samples obtained by the escherichia coli 2 under different fermentation processes is shown in fig. 2, and the SDS-PAGE diagram of the fermentation liquid samples obtained by the escherichia coli 3 under different fermentation processes is shown in fig. 3, wherein the size of target collagen is 70kDa.
The results of the calculated yields of recombinant humanized type III collagen in examples 1-4 and comparative example 1 are shown in Table 4 and FIG. 4:
TABLE 4 recombinant humanized III collagen fermentation yield
As can be seen from Table 4 and FIG. 4, the recombinant humanized type III collagen expressed by Escherichia coli col 1 was higher, up to 5.32g/L, using the fermentation medium containing malt extract. From the yield data of recombinant humanized type III collagen of Escherichia coli col 2 and col 3, it is known that malt extract can be used for collagen production without significantly affecting the yield of collagen by replacing part of yeast extract, but the fermentation yield of recombinant humanized type III collagen is significantly improved by adding malt extract to the fermentation medium for Escherichia coli col 1, especially at a malt extract content of 10-20%.
Experimental example 2:
purifying recombinant humanized III type collagen obtained by escherichia coli col 1 fermentation, and the steps are as follows:
(1) And (3) thallus crushing: taking fermentation liquor of escherichia coli col 1 in examples 1-6 and comparative example 1, centrifuging the fermentation liquor at 7500rpm and at 4 ℃ for 15min, collecting thalli, weighing 100g of wet thalli, putting into a beaker, adding 1500g of affinity A liquid, stirring uniformly, and breaking thalli for 30min under the pressure of 700-800 bar;
(2) And (3) centrifuging: centrifuging the damaged thalli for 20min at 3000g and 4 ℃ and collecting a supernatant;
(3) Affinity chromatography: loading the collected supernatant by Ni affinity chromatography, and collecting elution peak by using mixed solution of affinity A solution and affinity B solution with the volume ratio of 4:1, wherein the volume of the collected solution is 500mL;
(4) Replacement: adopting an ultrafiltration membrane bag of 20kDa, adding the ion A solution into the solution obtained in the step (3) for isovolumetric displacement to obtain 500mL of displaced solution;
(5) Ion exchange chromatography: loading the solution subjected to the replacement in the step (4) by adopting ion chromatography, and finally collecting an elution peak by adopting mixed solution of ion A solution and ion B solution in a volume ratio of 9:1, and collecting 300mL of liquid volume;
(6) Desalting: removing inorganic salt in the collection liquid in the step (5) by adopting a 20kDa ultrafiltration membrane bag and adding water, and ending desalting when the conductivity of the permeate end is less than 100 mu s/cm, and collecting 300mL of sample solution;
(7) And (3) freeze-drying: and freeze-drying the desalted sample solution by adopting freeze-drying equipment under the freeze-drying conditions shown in table 5, wherein the final sample is white spongy solid and has no visible foreign matters, so that the purified recombinant humanized III-type collagen is obtained.
Wherein the affinity A liquid comprises the following components: 20mM Tris, 0.5M NaCl, 20mM imidazole;
the affinity B liquid comprises the following components: 20mM Tris, 0.5M NaCl, 500mM imidazole;
the ion A liquid comprises the following components: 20mM Tris;
the ion B liquid comprises the following components: 20mM Tris, 1M NaCl.
TABLE 5 recombinant humanized collagen lyophilization conditions
| Sequence number | Target temperature/. Degree.C | Vacuum degree/Pa | Constant temperature time |
| 1 | -50 | 0 | 5h 20min |
| 2 | -15 | 10 | 15h |
| 3 | 30 | 10 | 20h |
The physicochemical properties of the recombinant humanized type III collagen sample obtained by the purification were analyzed, and the results are shown in the following table:
TABLE 6 comparison of physicochemical Properties of recombinant humanized type III collagen
As is clear from Table 6, the recombinant humanized type III collagen obtained by fermentation of Escherichia coli col 1 in examples 1 to 6 and comparative example 1 had little difference in physicochemical properties, and all met the quality standard of YY/T1888-2023.
Claims (10)
1. A fermentation process for improving the production level of recombinant humanized collagen is to add malt extract powder to a fermentation basic culture medium containing yeast extract powder to replace part of the yeast extract powder to form a fermentation culture medium, inoculating a fermentation strain, and fermenting to produce the recombinant humanized collagen, wherein the proportion of the malt extract powder to the yeast extract powder is 5-20% by mass.
2. The fermentation process of claim 1, wherein the malt extract is present in an amount of 10 to 20% relative to the yeast extract.
3. The fermentation process of claim 1, wherein the fermentation base medium has a composition of: 2-4% of yeast extract powder, 0.5-3% of glycerol, 0.1-0.4% of magnesium sulfate heptahydrate, 0.2-0.5% of ammonium sulfate, 0.06-0.15% of sodium sulfate, 0.2-1.5% of sodium chloride, 0.2-0.8% of monopotassium phosphate, 0.5-2.3% of disodium hydrogen phosphate dodecahydrate, 0.2-0.6% of microelement solution (film sterilization) and the balance of water in percentage by mass;
wherein the trace element solution comprises: boric acid 0.22-0.35%, sodium molybdate 0.01-0.02%, ferrous sulfate heptahydrate 0.14-0.27%, manganese sulfate monohydrate 0.055-0.12%, cupric chloride dihydrate 0.015-0.035%, zinc sulfate monohydrate 0.022-0.033%, potassium sodium tartrate tetrahydrate 0.15-0.32%, cobalt chloride 0.05-0.9%, concentrated sulfuric acid 0.05-0.1%, and the balance water.
4. The fermentation process of claim 1, wherein the fermentation strain is escherichia coli col 1 for recombinant expression of humanized type III collagen having an amino acid sequence as shown in SEQ ID No. 1.
5. The fermentation process of claim 4, wherein the escherichia coli col 1 is constructed by using escherichia coli BL21 (DE 3) as a host.
6. A fermentation process for improving the production level of recombinant humanized collagen is characterized by comprising the following steps of:
a. seed liquid culture: inoculating Escherichia coli col 1 into seed culture medium, and culturing at 36-38deg.C and 170-200rpm to obtain seed solution, OD of the obtained seed solution 600 More than 3;
b. preparing a fermentation medium: adding malt extract powder to the fermentation basic culture medium to replace part of yeast extract powder to form a fermentation culture medium, wherein the malt extract powder is 5-20% of the yeast extract powder by mass percent, and sterilizing for later use;
c. fermentation: inoculating the seed liquid cultured in the step a into the sterilized fermentation medium in the step b to start fermentation, wherein the initial fermentation conditions are as follows: the ventilation is 1 to 1.3vvm, the stirring rotation speed is 130 to 150rpm, the fermentation temperature is 36 to 38 ℃, 50 percent ammonia water is used for adjusting the pH value to 6.8 to 7.5, and the dissolved oxygen is 98 to 102 percent;
in the fermentation process, dissolved oxygen is continuously reduced, and the dissolved oxygen is controlled to be 15-30% by associated stirring;
when the glycerol in the fermentation medium is exhausted, the dissolved oxygen can rise obviously, and the dissolved oxygen rises to 55-75%, and then the glycerol is supplemented;
when fermentation liquor OD 600 When the fermentation temperature is 60-80 ℃, adjusting the fermentation temperature to 25-30 ℃, adding IPTG after membrane sterilization to make the final concentration of the IPTG to be 0.3-0.6mM, and beginning to induce and express recombinant humanized collagen;
when fermentation liquor OD 600 When no more increases, the fermentation is ended.
7. The fermentation process of claim 6, wherein one or more of the following conditions are met:
i. the composition of the seed culture medium in step a is: yeast extract powder 0.5%, peptone 1.0%, sodium chloride 1.0%, and the balance of water in mass percent;
OD of the seed liquid in step a 600 3-5;
c, inoculating 1-3% of the seed liquid in the step;
and iv, the concentration of the glycerol added in the step c is 50wt%, and the feeding speed is 0.4-0.7mL/min.
8. The application of malt extract powder in preparing recombinant humanized collagen by fermentation.
9. The use according to claim 8, wherein the fermented strain is escherichia coli col 1 recombinantly expressing a humanized type III collagen with the amino acid sequence shown in SEQ ID No. 1;
further preferably, the escherichia coli col 1 is constructed by using escherichia coli BL21 (DE 3) as a host bacterium.
10. The use according to claim 8, wherein the malt extract is used in the fermentation process in place of part of the yeast extract in the fermentation base medium;
further preferably, the mass percentage of the malt extract powder to replace yeast extract powder is 5-20%; further preferably 10 to 20%.
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