CN111378711A - Industrialized production method of recombinant spider silk protein - Google Patents
Industrialized production method of recombinant spider silk protein Download PDFInfo
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- CN111378711A CN111378711A CN201911169166.5A CN201911169166A CN111378711A CN 111378711 A CN111378711 A CN 111378711A CN 201911169166 A CN201911169166 A CN 201911169166A CN 111378711 A CN111378711 A CN 111378711A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
- C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
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- Chemical & Material Sciences (AREA)
- Insects & Arthropods (AREA)
- Organic Chemistry (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention relates to a production process route suitable for the expression and amplification of a spider silk protonuclear inclusion body, which optimizes key quality parameters in the process and completes the industrial scale amplification according to optimization conditions. The fermentation medium adopted by the invention has simple components and low cost; the fermentation process adopts specific control conditions, so that the energy consumption is lower, the expression quantity is improved by more than 19 percent, and the purity is improved by more than 12 percent.
Description
Technical Field
The invention belongs to the field of bioengineering, and relates to an industrialized production method of recombinant spider silk protein.
Background
Spider silks are both hard, as steel, and elastic, as rubber. The outstanding performance is mainly shown in high strength, high elasticity and high breaking power, so that the material is the toughest material so far and is known as 'biological steel'. With the intensive research on spider silks, the spider silks are found to have the characteristics of biodegradability, super-contractility, high temperature resistance, low temperature resistance, compatibility with biological tissues and the like. Because of the unique physical and biological characteristics of spider silk, the spider silk has wide application prospects in the aspects of medicine, materials, military affairs, textile and the like. An artificial spider silk manufactured by Swedish scientist Jan Johansson by using non-irritant chemical substances has good biocompatibility, can be applied to regenerative medicine research such as spinal cord repair or repair of damaged heart tissue by helping stem cell growth, and can also be applied to textile industry application such as self-protection tools (NatureChemicalbiology, DOI:10.1038/nchembio.2269 (2017)).
In view of the great potential applicability of spider silk proteins, researchers at home and abroad have intensified the research on spider silks, and it is expected that spider silks can be put to practical use on a large scale like silk. Because spiders cannot be domesticated and the yield of natural spider silks is low, a large amount of spider silks can be obtained only by means of genetic engineering, and the potential application requirements of the spider silks are met. Researchers can carry out bioengineering preparation of spider silk protein by means of expression systems of escherichia coli, yeast, insect cells, mammalian cells and the like. The escherichia coli expression system has the advantages of fast growth, high yield, large production scale, low cost, simple culture conditions, clear genetic background and the like, is widely applied to recombinant expression research of spider silk proteins at present, but the expression level of related spider silk proteins is still limited in the current process, and cannot effectively meet the requirement of industrial preparation.
The applicant designs a protein sequence derived from natural spider silk according to the properties of the natural spider silk protein, and simultaneously introduces a gene encoding a recombinant spider silk protein sequence into escherichia coli for protein expression.
Disclosure of Invention
The method designs a process route suitable for the expression and amplification of the spider silk protein inclusion body, optimizes key quality parameters in the process, and completes the industrial scale amplification according to optimization conditions.
The invention provides a cobweb protein prokaryotic inclusion expression amplification process, which comprises fermentation and purification processes, wherein the fermentation process comprises recombinant escherichia coli activation, fermentation seed liquid culture, high-density fermentation, fermentation thallus crushing, inclusion body washing, inclusion body denaturation and salting-out purification.
Preferably, the temperature of the induction stage of the high-density fermentation is controlled to be 33-35 ℃, the pH value is 7.0-7.4, the DO 25-45%, the ventilation volume is 20L-100L, the oxygen proportion is 50-100%, and the addition amount of the inducer is 0.1-0.5M.
Preferably, the induction stage of the high-density fermentation is controlled by 35-45% of DO, 20L of ventilation, 100% of oxygen proportion and 0.1M of inducer addition.
The purification process of the spider silk protein protonuclear inclusion body comprises the steps of thallus crushing, inclusion body washing and salting-out of a denaturating solution, wherein preferably, the washing solution is urea containing 3M, the pH value is 6.0-7.0, and 1.0-2.0M sulfate is adopted for salting-out. .
Preferably, the washing solution also contains 1% triton X-100, and the pH value is 6.0.
The amplification process of the invention has the following advantages: the adopted fermentation medium has simple components and low cost; the fermentation process adopts specific control conditions, so that the energy consumption is lower, the expression quantity is improved by more than 19 percent, and the purity is improved by more than 12 percent.
Drawings
FIG. 1 SDS-PAGE of the fermentation product and OD of the fermentation broth in example 1600And a trend chart of the change of the wet weight of the thalli.
Wherein, FIG. 1-a is SDS-PAGE electrophoresis, lane M is protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 3h after induction, lane 2 is the inclusion body denaturation solution fermented for 6h after induction, and lane 3 is the inclusion body denaturation solution fermented for 9h after induction. FIG. 1-b shows fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 2 SDS-PAGE electrophoresis of pre-process optimized fermentation batch and OD of fermentation broth in example 2600Heyu bacteriaAnd (5) a body wet weight change trend graph.
Wherein, FIG. 2-a is SDS-PAGE electrophoresis, lane M is protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 2h after induction, lane 2 is the inclusion body denaturation solution fermented for 4h after induction, lane 3 is the inclusion body denaturation solution fermented for 6h after induction, and lane 4 is the inclusion body denaturation solution fermented for 8h after induction. FIG. 2-b is a view showing fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 3 SDS-PAGE electrophoresis of first batch and OD of fermentation broth in example 2600And a trend chart of the change of the wet weight of the thalli.
Wherein, FIG. 3-a is a first SDS-PAGE electrophoresis, and lane M is a protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 2h after induction, lane 2 is the inclusion body denaturation solution fermented for 4h after induction, and lane 3 is the inclusion body denaturation solution fermented for 6h after induction. FIG. 3-b is a graph showing fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 4 SDS-PAGE electrophoretogram of second batch and OD of fermentation broth in example 2600And a trend chart of the change of the wet weight of the thalli.
Wherein, FIG. 4-a is a second SDS-PAGE electrophoresis, lane M is a protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 2h after induction, lane 2 is the inclusion body denaturation solution fermented for 4h after induction, and lane 3 is the inclusion body denaturation solution fermented for 6h after induction. FIG. 4-b is a graph showing fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 5 SDS-PAGE photograph of the third batch and OD of the fermentation broth in example 2600And a trend chart of the change of the wet weight of the thalli.
Wherein, FIG. 5-a is a third SDS-PAGE electrophoresis, lane M is a protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 2h after induction, lane 2 is the inclusion body denaturation solution fermented for 4h after induction, and lane 3 is the inclusion body denaturation solution fermented for 6h after induction. FIG. 5-b is a graph showing fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 6 SDS-PAGE electrophoretogram of first batch and OD of fermentation broth in example 3600And a trend chart of the change of the wet weight of the thalli.
Wherein FIG. 6-a is a first SDS-PAGE electrophoresisLane M is a protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 2h after induction, lane 2 is the inclusion body denaturation solution fermented for 4h after induction, and lane 3 is the inclusion body denaturation solution fermented for 6h after induction. FIG. 6-b is a graph showing fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 7 SDS-PAGE electrophoretogram of second batch and OD of fermentation broth in example 3600And a trend chart of the change of the wet weight of the thalli.
Wherein, FIG. 7-a is a second SDS-PAGE electrophoresis, lane M is a protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 2h after induction, lane 2 is the inclusion body denaturation solution fermented for 4h after induction, and lane 3 is the inclusion body denaturation solution fermented for 6h after induction. FIG. 7-b is a graph showing fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 8 SDS-PAGE photograph of the third batch and OD of fermentation broth in example 3600And a trend chart of the change of the wet weight of the thalli.
Wherein, FIG. 8-a is a third SDS-PAGE electrophoresis, lane M is a protein loading Marker; lane 1 is the inclusion body denaturation solution fermented for 2h after induction, lane 2 is the inclusion body denaturation solution fermented for 4h after induction, and lane 3 is the inclusion body denaturation solution fermented for 6h after induction. FIG. 8-b is a graph showing fermentation broth OD600And a trend chart of the change of the wet weight of the thalli.
FIG. 9 SDS-PAGE of target proteins obtained by different washing processes in example 4.
Lane M is protein loading Marker; lane 1 is the inclusion body denatured liquid after washing with the washing formula one, Lane 2 is the inclusion body denatured liquid after washing with the washing formula two, Lane 3 is the inclusion body denatured liquid after washing with the washing formula three, Lane 4 is the inclusion body denatured liquid after washing with the washing formula four, and Lane 5 is the inclusion body denatured liquid after washing with the washing formula five.
FIG. 10 SDS-PAGE of the denatured liquid of example 4.
Lane M is protein loading Marker; lane 1 is the direct denatured liquid of fermented inclusion body, lane 2 is the centrifugal supernatant of the direct denatured liquid of fermented inclusion body, and lane 3 is the protein denatured liquid after salting out of the centrifugal supernatant of the direct denatured liquid of fermented inclusion body.
FIG. 11 is an SDS-PAGE electrophoresis of the denatured liquid of example 5.
Lane M is protein loading Marker; lane 1 is the direct denatured liquid of fermented inclusion body, and lane 2 is the protein denatured liquid of the supernatant of the fermented inclusion body denatured liquid after salting out.
Detailed Description
The present invention is further illustrated below by reference to specific examples, which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Example 1 recombinant spider silk protein Small Scale (3L) high Density fermentation Process
Step 1: recombinant strain activation
Glycerol strain seeds (shown in Chinese patent No. CN201810093672.x, the same is shown below) frozen in a recombinant spidroin working seed bank at-80 ℃ are streaked on an LB solid culture medium (yeast extract 5g/L, peptone 10g/L, sodium chloride 10g/L and agarose 15g/L), and are cultured in a constant temperature and humidity box at 37 ℃ for 12 hours.
Step 2: seed liquid culture
The monoclonal colonies on the plates were picked up and cultured in LB liquid medium (yeast extract 5g/L, peptone 10g/L, sodium chloride 10g/L) at 37 ℃ and 220rpm to OD600And (4) keeping the concentration approximately equal to 2, and observing the bacteria under a microscope to obtain the seed liquid for fermentation.
And step 3: fermentation process
Washing Sideris BIOSTAT B bioreactor, respectively correcting pH meter probe of fermentation tank with standard solution with pH7.0 and pH 4.0, preparing inorganic salt culture medium (citric acid monohydrate 1.7g/L, potassium dihydrogen phosphate 12g/L, diammonium hydrogen phosphate 4g/L, glucose 20g/L, magnesium sulfate heptahydrate 1.2g/L) as fermentation culture medium, pouring into tank, autoclaving at 121 deg.C for 20min, cooling to 50 deg.C, and adjusting pH to 7.2 + -0.2 with concentrated ammonia water.
Inoculating the seed solution obtained in step 2 into a bioreactor according to the proportion of 1:15(V/V, seed solution/fermentation culture medium), and adding trace elements (trace element formula: FeSO) according to the proportion of 1ml/L4·7H2O 10g/L、ZnSO4·7H2O 2.25g/L、CuSO4·5H2O 15g/L、MnSO4·5H2O 5g/L、CaCl2·7H2O 1g/L、CoCl·6H2O 1g/L、Na2MoO4·2H2O 1.125g/L、H3BO30.0625g/L, HCl 41.75.75 ml, Biotin 0.5 g/L). OD measurement by periodic sampling after fermentation600And wet weight of the cells. The fermentation temperature in the thallus proliferation stage is 37 deg.C, pH7.2 + -0.2, the rotation speed is 300rpm, the DO value is 30-40%, the DO continuously decreases in the period of about 6h, and the DO is maintained at 45% by increasing the stirring rotation speed, the ventilation amount and the oxygen. When the carbon source is completely consumed, the dissolved oxygen value rapidly rises, and the OD of the thallus is60030, at which point the feed phase is entered, which is at 12ml/h-1L-1The rate of addition of glycerol was 1024g/L and magnesium sulfate heptahydrate was 20 g/L. When the thallus grows to 0D600Approximately equal to 40-55 percent, reducing the fermentation temperature to 30 ℃, adding IPTG with the final concentration of 0.5M into the fermentation tank for induction expression after the temperature is stable, maintaining DO not lower than 50 percent, setting the induction time for 8h, setting the induction temperature to 35-37 ℃, and inducing the pH to 7.0 +/-2. The relevant fermentation results are shown in FIG. 1.
Example 2 recombinant spider silk protein bench scale-up (30L) high-Density fermentation validation
Step 1: recombinant strain activation
Glycerol strain seeds frozen in a working seed bank at the temperature of-80 ℃ are streaked in an LB solid culture medium (5 g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 15g/L of agarose), and are cultured in a constant temperature and humidity box at the temperature of 37 ℃ for 12 hours.
Step 2: seed liquid culture
The monoclonal colonies on the plates were picked up and cultured in LB liquid medium (yeast extract 5g/L, peptone 10g/L, sodium chloride 10g/L) at 37 ℃ and 220rpm to OD600And (4) keeping the concentration approximately equal to 2, and observing the bacteria under a microscope to obtain the seed liquid for fermentation.
And step 3: fermentation process
Cleaning Sidoris Cplus bioreactor, calibrating pH meter probe of fermentation tank with standard solution of pH7.0 and pH 4.0, preparing inorganic salt culture medium 20L, pouring into fermentation tank, sterilizing at 121 deg.C for 20min, cooling to 50 deg.C, and adjusting pH to 7.2 + -0.2 with concentrated ammonia water.
Inoculating the seed solution obtained in the step 2 into a fermentation tank according to the proportion of 1:15(V/V, seed solution/fermentation culture medium), and adding trace elements (trace element formula FeSO) according to the proportion of 1ml/L4·7H2O 10g/L、ZnSO4·7H2O 2.25g/L、CuSO4·5H2O 15g/L、MnSO4·5H2O 5g/L、CaCl2·7H2O 1g/L、CoCl·6H2O 1g/L、Na2MoO4·2H2O 1.125g/L、H3BO30.0625g/L, HCl 41.75.75 ml, Biotin 0.5 g/L). OD measurement by periodic sampling after fermentation600And wet weight of the cells. The fermentation temperature in the thallus proliferation stage is 37 deg.C, pH7.2 + -0.2, the rotation speed is 300rpm, the DO value is 30-40%, the DO continuously decreases in the period of about 6h, and the DO is maintained at 45% by increasing the stirring rotation speed, the ventilation amount and the oxygen. When the carbon source is completely consumed, the dissolved oxygen value rapidly rises, and the OD of the thallus is60030, at which point the feed phase is entered, which is at 12ml/h-1L-1The rate of addition of glycerol was 1024g/L and magnesium sulfate heptahydrate was 20 g/L. When the thallus grows to 0D600Approximately equals to 40-55%, the fermentation temperature is reduced to 30 ℃, IPTG with the final concentration of 0.5M is added into the fermentation tank for induction expression after the temperature is stable, DO is maintained to be not less than 50%, and the induction conditions of different fermentation batches are shown in Table 1.
TABLE 1 optimization of recombinant spider silk protein pilot scale high-density fermentation process conditions
The results in table 1 show that the yield of the target protein is increased by more than 19% and the purity is increased by more than 12% after the process optimization compared with the yield before the process optimization. The applicant confirms that the fermentation process of the invention completes the verification and optimization of small scale on the basis of small scale conditions, obviously improves the fermentation yield, and can completely carry out industrialized scale-up production.
FIGS. 2 to 5 are SDS-PAGE electrophoresis of four batches of recombinant spidroin proteins in Table 1FIG. and fermentation liquid OD600And a trend chart of the change of the wet weight of the thallus, wherein the results of the attached chart show the fermentation product and the OD of the fermentation liquid of the embodiment600The absorption value and the wet weight change trend of the thalli are consistent with those of 3L small-scale high-density fermentation.
Example 3 Pilot-Scale (300L) high Density fermentation validation of recombinant spider silk proteins
Step 1: recombinant strain activation
Freezing at-80 deg.CRecombinant spider silk proteinsThe glycerol strain seeds in the working seed bank are streaked on an LB solid culture medium (5 g/L of yeast extract, 10g/L of peptone, 10g/L of sodium chloride and 15g/L of agarose), and are cultured in a constant temperature and humidity box at 37 ℃ for 12 h.
Step 2: seed liquid culture in shake flask
The monoclonal colonies on the plates were picked up and cultured in LB liquid medium (yeast extract 5g/L, peptone 10g/L, sodium chloride 10g/L) at 37 ℃ and 220rpm to OD600And (4) keeping the concentration at approximately equal to 2, and observing the mixed bacteria-free state under a microscope to obtain the shake flask seed liquid.
And step 3: seeding tank culture
Inoculating the shake flask seed liquid obtained in the step 2 into a 30L divoxin BVT-3000 type seed tank, culturing by adopting an inorganic salt culture medium at the temperature of 37 ℃, the pH value of 7.2 +/-0.2 and the rotation speed of 300rpm, keeping the dissolved oxygen at 45 percent by ventilation and rotation speed conditions, and finally obtaining the OD600Approximately equals to 40, the wet weight reaches about 80g/L, and the seeding tank fermentation liquor can be obtained by observing the bacteria under a microscope.
And 4, step 4: fermentation process
Cleaning a 300L fermentation tank of DIVOXIN BVT-3000 type, respectively correcting a pH meter probe of the fermentation tank by using standard solutions with pH being 7.0 and pH being 4.0, preparing 200L of inorganic salt fermentation medium, pouring the inorganic salt fermentation medium into the fermentation tank, sterilizing the inorganic salt fermentation medium on line at 121 ℃ for 20min, and adjusting the pH to be 7.2 +/-0.2 by using concentrated ammonia water after the temperature is reduced to 50 ℃.
Inoculating the seed solution obtained in step 3 into a fermentation tank according to the proportion of 1:15(V/V, seed solution/fermentation culture medium), and adding trace elements (trace element formula: FeSO) according to 1ml/L4·7H2O 10g/L、ZnSO4·7H2O 2.25g/L、CuSO4·5H2O 15g/L、MnSO4·5H2O 5g/L、CaCl2·7H2O 1g/L、CoCl·6H2O 1g/L、Na2MoO4·2H2O 1.125g/L、H3BO30.0625g/L, HCl 41.75.75 ml, Biotin 0.5 g/L). OD measurement by periodic sampling after fermentation600And wet weight of the cells. The fermentation temperature in the thallus proliferation stage is 37 deg.C, pH7.2 + -0.2, the rotation speed is 300rpm, the DO value is 30-40%, the DO continuously decreases in the period of about 6h, and the DO is maintained at 45% by increasing the stirring rotation speed, the ventilation amount and the oxygen. When the carbon source is completely consumed, the dissolved oxygen value rapidly rises, and the OD of the thallus is60030, at which point the feed phase is entered, which is at 12ml/h-1L-1The rate of addition of glycerol was 1024g/L and magnesium sulfate heptahydrate was 20 g/L. After the thallus grows to OD600Approximately ranging from 40 to 55 ℃, reducing the fermentation temperature to 30 ℃, adding IPTG into the fermentation tank for induction expression after the temperature is stable, wherein the induction time is 6 hours, the temperature is 33 to 35 ℃, the pH value is 7.2 +/-0.2, the DO is maintained to be not less than 50 percent, and the induction conditions of different fermentation batches are shown in Table 2. The results in table 2 show that the yield and purity of the target protein in the pilot plant test are greatly improved compared with those in the pilot plant test, the repeatability is better, the ventilation and the use amount of induction are reduced, and the energy consumption is reduced.
TABLE 2 stability study of Pilot-plant high-Density fermentation Process conditions for recombinant spider silk proteins
FIGS. 6-8 are SDS-PAGE electrophoresis and OD600 and wet weight change trend of fermentation broth of three batches in Table 2, respectively, and the results show that the OD600 absorption value and wet weight change trend of fermentation broth of this example are better than those of 30L small-scale large-scale high-density fermentation.
Example 4 purification of recombinant spider silk proteins
The spider silk protein has extremely low solubility in water, and is designed by a purification method according to its characteristics, and is divided into the following three steps.
Collecting the fermented thalli by a centrifuge, using 3M urea to resuspend until the thalli concentration is 200g/L, then carrying out homogenization and crushing, using the high-pressure homogenization pressure to be 800bar in the process, repeating for 3 times, after fully crushing, centrifugally collecting precipitates, and obtaining the precipitates as the crude inclusion bodies.
The crude inclusion bodies are re-suspended to 3g/L by using a washing solution for washing, and then the centrifugation is carried out for a plurality of times until the centrifugation supernatant has no color and turbidity and the centrifugation sediment (namely the crude inclusion bodies) is fair and compact. The inclusion bodies harvested after the formulas 1-4 are washed can obtain higher recovery rate, and the purity of the target protein harvested after the formulas 1 and 4 are washed is obviously higher than that of other formulas.
TABLE 3 optimization of formulation conditions for inclusion body washes
The washed inclusion bodies were thoroughly denatured with a denaturing solution of urea 6M at pH 6.0, and the supernatant was collected by centrifugation. Removing insoluble impurities, salting out the target protein with ammonium sulfate of 2.0M, 1.5M and 1.0M concentrations, centrifuging, and collecting precipitate to obtain the target protein. FIG. 10 shows that the salting-out results of the target protein denaturant are greatly influenced by using ammonium sulfate with different concentrations, and the purity of the target protein is higher by using ammonium sulfate with a concentration of 1.0M.
Example 5Purification treatment of recombinant spider silk protein pilot expression protein
Collecting the pilot-scale fermentation thalli through a tubular centrifuge, using 3M urea to resuspend until the thalli is 200g/L, then carrying out homogenization and crushing, using high-pressure homogenization pressure of 800bar in the process, repeating for 2 times, after fully crushing, centrifugally collecting precipitates, and obtaining the precipitates as the crude inclusion bodies.
The crude inclusion bodies are resuspended to 3g/L by using a washing solution formula I for washing, and then the centrifugation is repeated for a plurality of times until the centrifugation supernatant has no color and turbidity and the centrifugation sediment (namely the crude inclusion bodies) is fair and compact.
The washed inclusion bodies were thoroughly denatured with a denaturing solution of urea 6M at pH 6.0, and the supernatant was collected by centrifugation. After insoluble impurities are removed, salting out is carried out on the target protein by using ammonium sulfate with the concentration of 1.0M, and the inclusion body finished product is obtained by centrifugally collecting precipitates. FIG. 11 shows that the process is amplified to pilot-plant expression of the target protein after verification, and better repeatability can be obtained.
Claims (5)
1. The spider silk protein prokaryotic inclusion expression amplification process comprises fermentation and purification processes, wherein the fermentation process comprises recombinant escherichia coli activation, fermentation seed liquid culture, high-density fermentation, fermentation thallus crushing, inclusion body washing, inclusion body denaturation and salting-out purification.
2. The spider silk protein proto-nuclear inclusion expression amplification process of claim 1, characterized in that the temperature is controlled at 33-35 ℃, the pH value is 7.0-7.4, the DO is 25-45%, the ventilation volume is 20L-100L, the oxygen proportion is 50-100%, and the addition of an inducer is 0.1-0.5M in the induction stage of high-density fermentation.
3. The amplification process for expression of the proto-entrapment of spider silk protein according to claim 2, wherein the DO is controlled to be 35% -45% in the induction stage of the high-density fermentation, the ventilation is controlled to be 20L, the oxygen proportion is 100%, and the addition amount of the inducer is 0.1M.
4. The process for amplifying expression of protonuclear inclusion body of spider silk protein as claimed in claim 1, wherein the process for purifying inclusion body of spider silk protein comprises breaking thallus, washing inclusion body, denaturing inclusion body, and salting out of denatured liquid, wherein the washing liquid of inclusion body is urea containing 3M, pH value is 6.0-7.0, and 1.0-2.0M sulfate is used for salting out.
5. The amplification process of claim 4, wherein the washing solution contains 3M urea, 1% Triton X-100, and pH 6.0.
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| CN114292322A (en) * | 2021-12-30 | 2022-04-08 | 中国科学院青岛生物能源与过程研究所 | Preparation method and application of water-soluble recombinant spider silk protein |
| CN114672531A (en) * | 2020-12-24 | 2022-06-28 | 江苏万邦医药科技有限公司 | Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control |
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| CN102532295A (en) * | 2004-07-22 | 2012-07-04 | Am丝绸有限责任公司 | Recombinant spider silk proteins |
| CN105755025A (en) * | 2016-04-14 | 2016-07-13 | 东华大学 | Recombinant spider silk protein preparation method |
| CN108456246A (en) * | 2017-02-22 | 2018-08-28 | 常州京森生物医药研究所有限公司 | Recombinant spider silk proteins and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102532295A (en) * | 2004-07-22 | 2012-07-04 | Am丝绸有限责任公司 | Recombinant spider silk proteins |
| CN105755025A (en) * | 2016-04-14 | 2016-07-13 | 东华大学 | Recombinant spider silk protein preparation method |
| CN108456246A (en) * | 2017-02-22 | 2018-08-28 | 常州京森生物医药研究所有限公司 | Recombinant spider silk proteins and its preparation method and application |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114672531A (en) * | 2020-12-24 | 2022-06-28 | 江苏万邦医药科技有限公司 | Method for improving escherichia coli protein expression quantity through stage dissolved oxygen control |
| CN114292322A (en) * | 2021-12-30 | 2022-04-08 | 中国科学院青岛生物能源与过程研究所 | Preparation method and application of water-soluble recombinant spider silk protein |
| CN114292322B (en) * | 2021-12-30 | 2023-09-19 | 中国科学院青岛生物能源与过程研究所 | Preparation method and application of water-soluble recombinant spider silk protein |
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