WO2025190352A1 - Method for preparing recombinant human albumin with high expression and low o-glycosylation level - Google Patents

Abstract

A method for preparing recombinant human albumin with high expression and low O-glycosylation level, comprising taking at least one measure during fermentation to improve the protein expression rate and reduce the O-glycosylation level of a protein; and specifically comprising: S1, seed solution preparation; S2, batch fermentation; S3, glycerol fed fermentation; S4, methanol fed fermentation; S5, fermentation product collection.

Description

Method for preparing recombinant human albumin with high expression and low O-glycosylation level Technical Field

The present invention relates to a method for preparing recombinant human albumin with high expression and low O-glycosylation level, belonging to the field of medicine.

Background Art

Human serum albumin is the most abundant protein in human plasma. Its non-glycosylated single-chain polypeptide consists of 585 amino acids with a molecular weight of approximately 66.5 kDa. It is widely used clinically to treat critical conditions such as shock, edema, or ascites caused by hemorrhagic trauma and burns, as well as hypoproteinemia.

Furthermore, while albumin itself lacks N-glycosylation sites, it may possess some degree of O-glycosylation at its S/T sites. Recombinant products expressed in yeast, in particular, often exhibit a certain degree of mannosylation due to the inherent characteristics of the expression system. This can lead to potential immunogenicity, easy clearance from the human body, and a short half-life. Therefore, there is an urgent need to identify methods for regulating the O-glycosylation modification of recombinant proteins expressed in yeast.

Currently, controlling the O-glycosylation level of yeast proteins is primarily achieved through genetic engineering. Chinese Patent CN200980105645.9 provides a yeast strain that can be used to produce low-level O-glycosylated proteins. This method involves replacing the gene encoding an endogenous chaperone protein with a mammalian homolog of the chaperone protein, disrupting or removing the O-mannosyltransferase gene, and/or overexpressing an endogenous or exogenous Ca2+ ATPase. Chinese Patent 201080030803.1 provides a method for controlling O-linked glycosylation in antibodies by modifying the nucleic acid sequence of the target protein to replace or delete at least one amino acid residue selected from S, T, or Y that is subject to O-linked glycosylation. However, reducing protein O-glycosylation levels through genetic engineering may negatively impact yeast cell viability and the functional properties of the target protein. For example, a defect in the mannosyltransferase gene may impair cell wall integrity, leading to low cell viability.

In summary, how to regulate the O-glycosylation level of yeast proteins is a major difficulty in producing recombinant human albumin using a yeast expression system.

Summary of the Invention

The present invention provides a method for preparing recombinant human albumin with high expression and low O-glycosylation level, which can increase protein expression level and reduce protein O-glycosylation, and the expressed recombinant protein contains extremely low level of glycosylation modification.

To achieve this object, the present invention provides the following technical solutions:

The present invention provides a method for preparing recombinant human albumin with high expression and low O-glycosylation level, the method comprising taking at least one measure to increase the protein expression rate and reduce the protein O-glycosylation level during the fermentation process; the measure comprising changing at least one of the following fermentation conditions:

1) Induction temperature;

2) induced pH;

3) dissolved oxygen content during the induction phase;

4) induction time point;

5) inducer concentration;

6) Method of adding inducer;

7) Flow rate of inducer addition.

In this embodiment, the measures can be taken by directly changing the induction conditions; or by indirectly changing the induction conditions by changing the fermentation conditions during the batch fermentation stage.

In the present invention, the induction time point is determined based on the culture time and/or culture parameters; the inducer addition method can be continuous flow addition or discontinuous flow addition, single flow addition or simultaneous flow addition with other carbon sources; the inducer addition flow rate can be a constant flow rate, an increasing flow rate, or a flow rate determined based on biomass or dissolved oxygen feedback.

Preferably, the method comprises the following steps:

S1. Seed solution preparation: Take the preserved bacterial strain and streak it on a plate. Pick a single colony and inoculate it into YPD liquid medium at 28-30℃. When the OD600 of the shake flask seeds reaches 10-12, use it as the seed solution for tank filling.

S2. Batch fermentation: BSM medium was added to the fermentor, the pH was adjusted to 5.0-5.8, and sterilized. After cooling, the dissolved oxygen was corrected, the temperature and ventilation were set to 28-29.5°C, sterilized PTM1 solution was added to the medium, and the pH of the medium was again adjusted to 5.0-5.8; the medium in the fermentor was partially discarded through the sampling port, and the shake flask seeds were connected to the fermentor. The dissolved oxygen was corrected and the rotation speed was set. During the fermentation, the dissolved oxygen was maintained above 20% by adjusting the rotation speed and ventilation volume;

S3, glycerol fed fermentation: when the dissolved oxygen suddenly increases, the glycerol feeding stage is entered, and glycerol is fed at a uniform rate. When the fermentation lasts for more than 30 hours, the addition of glycerol is stopped;

S4, methanol fed-batch fermentation: No carbon source was added to ensure complete depletion of glycerol; after starvation treatment, methanol was fed, the pH in the tank was maintained at 5.8-6.0, the temperature was lowered to 24-28°C, and various fermentation parameters were monitored in real time to maintain dissolved oxygen above 20%;

S5. After fermentation is complete, the tank is placed in a centrifuge and the fermentation product is centrifuged to collect the supernatant.

Preferably, the strain in step S1 is CBS7435-rHSA, and the CBS7435-rHSA strain is prepared by the following method:

R1. Design and synthesize the recombinant human albumin DNA sequence shown in SEQ ID NO.1;

R2. Ligate the sequence to the pPICZαA vector with restriction enzyme sites PmII and XbaI. Transform the ligation product into E. coli DH5α competent cells and plate on LB plates containing zeocin. Pick a single clone and transfer it into LB liquid medium containing zeocin. Re-extract the plasmid. After restriction enzyme digestion and sequencing verification, the expression plasmid was correctly constructed and named pPICZαA-rHSA.

R3. Linearize the plasmid vector with Sac I and electroporate it into CBS7435 competent cells. Spread the transformed strain on a YPD-resistant plate containing zeocin and pick the positive single clone to obtain the recombinant human albumin expression strain CBS7435-rHSA.

In the present invention, SEQ ID NO.1 is:

Preferably, between step S3 and step S4, mixed fed-batch fermentation is further included: glycerol feed and methanol feed are added simultaneously, the temperature is 24° C., and the pH is adjusted to 5.85.

Preferably, in steps S1 and S2, the temperature is 28°C; in step S2, the pH is adjusted to 5.0.

Preferably, in step S1 and steps S2-S4, the dissolved oxygen is above 30%.

Preferably, in step S4, the method of adding methanol feed is: adding methanol feed at a constant flow rate of 10-12 mL/h·L, or, first adding methanol feed at an initial rate of 3-5 mL/h·L, and gradually increasing the flow rate to 10-12 mL/h·L.

Preferably, in step S4, dissolved oxygen and methanol feeding are coupled, the coupling correlation is inversely correlated, and the critical value is 20-30%, that is, feeding is started when the dissolved oxygen is higher than the critical value, and is stopped when the dissolved oxygen is lower than the critical value. The working cycle of methanol feeding is 10-15s.

Preferably, each 20L of BSM medium includes the following components: 85% phosphoric acid 534-600mL, calcium sulfate dihydrate 18.6-20g, potassium sulfate 364-400g, magnesium sulfate dihydrate 298-320g, potassium hydroxide 82.6-100g, glycerol 800-850g; each 500mL of PTM1 solution includes the following components: copper sulfate pentahydrate 3-5g, potassium iodide 0.044-0.1g, sulfur monohydrate Manganese dioxide 1.5-2g, sodium molybdate dihydrate 0.1-0.2g, boric acid 0.01-0.03g, cobalt chloride 0.25-0.35g, zinc chloride 10-12g, ferrous sulfate heptahydrate 32.5-35g, biotin 0.1-0.2g, sulfuric acid 2.5-3.0mL; each 500mL YPD includes the following components: yeast extract 5-10g, peptone 10-20g, glucose 10-20g.

Preferably, the glycerol feed comprises: glycerol, purified water, defoaming agent and PTM1; the methanol feed comprises: methanol and defoaming agent.

More preferably,

YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high-pressure steam; if preparing solid medium, add 2% agar powder;

PTM1: 3 g copper sulfate pentahydrate, 0.044 g potassium iodide, 1.5 g manganese sulfate monohydrate, 0.1 g sodium molybdate dihydrate, 0.01 g boric acid, 0.25 g cobalt chloride, 10 g zinc chloride, 32.5 g ferrous sulfate heptahydrate, 0.1 g biotin, 2.5 mL sulfuric acid, dilute to 500 mL, and filter sterilize;

BSM: 85% phosphoric acid 534 mL, calcium sulfate dihydrate 18.6 g, potassium sulfate 364 g, magnesium sulfate dihydrate 298 g, potassium hydroxide 82.6 g, glycerol 800 g, dilute to 20 L;

Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL/L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

Methanol feed: 20 L of methanol, sterilized by filtration; 0.2 mL/L of defoaming agent, sterilized and dissolved in the sterilized methanol.

Preferably, aqueous ammonia is used to adjust the pH.

Preferably, a method for preparing recombinant human albumin with high expression and low O-glycosylation level comprises the following steps:

S1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

S2. Batch Fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank is about 19.5L before inoculation. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 30% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

S3. Glycerol fed-batch fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

S4. Methanol fed-batch fermentation:

After 36 hours of fermentation (wet weight reaches about 285g/L), stop adding glycerol, the dissolved oxygen will rise rapidly, and enter the methanol-fed fermentation stage. No carbon source is added to the tank within 1 hour to ensure that the glycerol is completely consumed. At this time, the temperature is reduced to 24°C and the pH is adjusted to 5.85. After starvation treatment, methanol is fed at an initial rate of 3-5mL/h·L, and the flow rate is gradually increased to 10-12mL/h·L. The fermentation parameters are monitored in real time, and the methanol flow rate is adjusted according to the dissolved oxygen content (if the DO value increases by more than 10% within 1 minute after stopping methanol feeding, it means that the carbon source is limited, otherwise it means that methanol is excessive). During this period, the dissolved oxygen is maintained above 20%. Fermentation ends at 132 hours and the tank is released. The fermentation product is centrifuged at 8000rpm for 20 minutes, the supernatant is collected, and stored at -20°C.

Preferably, a method for preparing recombinant human albumin with high expression and low O-glycosylation level comprises the following steps:

S1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

S2. Batch Fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank is about 19.5L before inoculation. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 30% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

S3. Glycerol fed-batch fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

S4. Mixed fed-batch fermentation

After 30 h of fermentation (wet weight reached about 242 g/L), the mixed feeding stage was entered, and glycerol feeding and methanol feeding were added simultaneously. At this time, the temperature was lowered to 24° C. and the pH was adjusted to 5.85.

S5. Methanol fed-batch fermentation

After 2 hours of mixed feed addition, the methanol feed phase began. The dissolved oxygen-speed coupling was decoupled, and the dissolved oxygen and methanol feed were coupled. The coupling correlation was inversely correlated, with a critical value of 30%. Feeding was initiated when the dissolved oxygen exceeded the critical value and stopped when it fell below it. The methanol feed cycle was 10 seconds. Fermentation was completed after 132 hours, and the tank was drained. The fermentation product was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected and stored at -20°C.

Compared with the prior art, the beneficial effects and significant improvements of the technical solution of the present invention are:

1. The method of the present invention for preparing recombinant human albumin with high expression and low O-glycosylation levels does not require the destruction or removal of any glycosyltransferase genes, thus avoiding the negative impact on cell integrity that may be caused by glycosyltransferase gene defects. Furthermore, there is no need to delete or replace amino acid residues containing glycosylation sites, and the expressed recombinant protein has the same amino acid sequence as the native protein.

2. The method of the present invention for preparing recombinant human albumin with high expression and low O-glycosylation level is highly operable, and the obtained recombinant human albumin has an extremely low glycosylation level and a high protein expression amount.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solution of the present invention, the following briefly introduces the drawings used in the embodiments of the present invention.

FIG1 shows the wet weight during the fermentation process of Examples 1-5 and Comparative Examples 1-2;

Figure 2 shows the protein expression levels of Examples 1-5 and Comparative Examples 1-2;

FIG3 shows the average protein expression rates of Examples 1-5 and Comparative Examples 1-2.

DETAILED DESCRIPTION

The present invention will be further described below in conjunction with specific examples. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the contents of the present invention, those skilled in the art may make various changes and modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

In order to more fully understand the present invention, the professional terms in the present invention are explained below.

"Recombinant proteins" in this context are proteins obtained using recombinant DNA or recombinant RNA technologies. In vitro recombinant protein production primarily encompasses four systems: prokaryotic protein expression, mammalian cell protein expression, yeast protein expression, and insect cell protein expression. The proteins produced vary in activity and application methods. Choosing the appropriate protein expression system based on your downstream application can improve your expression success rate.

The "culture medium" of the present invention is a synthetic nutrient medium used for the growth and maintenance of microorganisms, plant tissues, and animal tissues. It generally contains carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins, and water. Depending on the specific needs of each culture medium, compounds that the culture medium cannot synthesize itself, namely growth factors, may be added.

"Fermentation" in the present invention refers to the process by which microorganisms, under aerobic or anaerobic conditions, are used to produce microbial cells themselves, or direct metabolites or secondary metabolites. Fermentation is sometimes also written as "酦酿" (酦酿), and its definition varies depending on the context of use. Fermentation, as it is commonly referred to, generally refers to the decomposition process of organic matter by an organism. Fermentation is a biochemical reaction that humans have been exposed to for a long time and is now widely used in the food, biological, and chemical industries. It is also a fundamental process in bioengineering, namely fermentation engineering. Research into its mechanisms and process control is ongoing.

The "strains" of the present invention are microorganisms used as living cell catalysts in the fermentation process, including four major categories: bacteria, actinomycetes, yeasts, and molds. They are derived from a large number of microorganisms in nature, from which useful strains are isolated and screened, then modified and stored for use in production.

The term "protein expression rate" as used herein refers to the amount of protein secreted per unit time. This term is represented by the "average protein expression rate," which is the average protein secretion rate during the induced fermentation process. This rate can be determined by calculating the ratio of maximum protein expression to expression time, expressed in g/L·h. The protein expression time starts at the time of inducer addition and ends at the time when protein expression reaches its peak.

The "inducer" of the present invention refers to a substance that can directly or indirectly interact with an inducible promoter and thus promote the initiation of expression of the promoter.

The "culture" of the present invention is a general term for liquid or solid culture medium in which a microbial community grows after artificial inoculation and cultivation.

"Batch fermentation" in this context refers to a culture method in which a sterilized culture medium is inoculated with a live production bacterium, without adding or removing any other substances to the fermentation broth (for aerobic microorganisms, a continuous supply of oxygen is required). In other words, aside from continuous aeration (aerobic fermentation) and the addition of acid or alkali solutions to adjust the pH of the fermentation broth, no other materials are exchanged with the environment, and the culture medium is added all at once before fermentation begins.

"Fed-batch fermentation" in the present invention refers to fermentation culture that is fed with additional culture medium. In the present invention, glycerol is fed during the "glycerol fed-batch fermentation" phase, methanol and glycerol are fed simultaneously during the "mixed fed-batch fermentation" phase, and methanol is fed during the "methanol fed-batch fermentation" phase.

The recombinant human albumin expression strain CBS7435-rHSA used in the present invention was self-constructed. All examples in the present invention used the above strain for fermentation culture. The construction method thereof includes the following steps:

S1. Design and synthesize the recombinant human albumin DNA sequence, the sequence of which is shown in SEQ ID NO.1;

S2. The optimized sequence was ligated into the pPICZαA vector with restriction enzyme sites PmII and XbaI. The ligation product was transformed into E. coli DH5α competent cells and plated on LB plates containing zeocin. A single clone was picked and transferred into LB liquid medium containing zeocin. The plasmid was re-extracted and confirmed by restriction enzyme digestion and sequencing. The expression plasmid was correctly constructed and named pPICZαA-rHSA.

S3. Linearize the plasmid vector by Sac I digestion and then electroporate into CBS7435 competent cells. Spread the transformed strain on YPD resistance plates containing zeocin and pick the positive single clone to obtain the recombinant human albumin expression strain CBS7435-rHSA.

Wherein, SEQ ID NO.1 is:

The culture medium used in the following examples was prepared as follows:

YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high-pressure steam; if preparing solid culture medium, add 2% agar powder.

PTM1: 3 g copper sulfate pentahydrate, 0.044 g potassium iodide, 1.5 g manganese sulfate monohydrate, 0.1 g sodium molybdate dihydrate, 0.01 g boric acid, 0.25 g cobalt chloride, 10 g zinc chloride, 32.5 g ferrous sulfate heptahydrate, 0.1 g biotin, 2.5 mL sulfuric acid, dilute to 500 mL, and filter sterilize.

BSM: 534 mL of 85% phosphoric acid, 18.6 g of calcium sulfate dihydrate, 364 g of potassium sulfate, 298 g of magnesium sulfate dihydrate, 82.6 g of potassium hydroxide, and 800 g of glycerol. Make up to 20 L.

Glycerol feed: 4200 g of glycerol, 2800 g of purified water, 0.2 mL/L of defoamer, and 80 mL of PTM1 were mixed and placed in a 10 L storage bottle for sterilization.

Methanol feed: 20 L of methanol, sterilized by filtration; 0.2 mL/L of defoaming agent, sterilized and dissolved in the sterilized methanol.

The present invention will be further described in detail below with reference to specific embodiments.

Example 1

1.1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

1.2 Batch fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank is about 19.5L before inoculation. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 30% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

1.3 Glycerol fed-batch fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

1.4 Methanol fed-batch fermentation

After 30 hours of fermentation (wet weight reaches approximately 235 g/L), the addition of glycerol is stopped, the dissolved oxygen quickly recovers, and the fermentation enters the methanol-fed fermentation stage. No carbon source is added to the tank within 1 hour to ensure that the glycerol is completely consumed. After starvation treatment, methanol is fed at a flow rate of 10 mL/h·L. At this time, the pH is adjusted to 6.0, and other parameters remain unchanged. The fermentation parameters are monitored in real time to maintain the dissolved oxygen above 30%. Fermentation is completed after 132 hours, and the tank is released. The fermentation product is centrifuged at 8000 rpm for 20 minutes, the supernatant is collected, and stored at -20°C.

Example 2

2.1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

2.2 Batch fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank is about 19.5L before inoculation. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 30% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

2.3 Glycerol fed-batch fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

2.4 Methanol fed-batch fermentation

After 30h of fermentation (wet weight reaches about 238g/L), stop adding glycerol, the dissolved oxygen will recover rapidly, and enter the methanol-fed fermentation stage. No carbon source is added to the tank within 1h to ensure that the glycerol is completely consumed. At this time, the temperature is reduced to 25°C and the pH is adjusted to 6.0. After starvation treatment, methanol is fed at an initial rate of 3-5mL/h·L, and the flow rate is gradually increased to 10-12mL/h·L. The fermentation parameters are monitored in real time, and the methanol flow rate is adjusted according to the dissolved oxygen content (if the DO value increases by more than 10% within 1min after stopping methanol feeding, it means that the carbon source is limited, otherwise it means that methanol is excessive). During this period, the dissolved oxygen is maintained above 20%. Fermentation ends at 132h and the tank is released. The fermentation product is centrifuged at 8000rpm for 20 minutes, the supernatant is collected, and stored at -20°C.

Example 3

3.1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

3.2 Batch fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank is about 19.5L before inoculation. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 20% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

3.3 Glycerol fed-batch fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

3.4 Methanol Fed-Batch Fermentation

After 30 hours of fermentation (wet weight reaches about 228g/L), the addition of glycerol is stopped, the dissolved oxygen rises rapidly, and the fermentation enters the methanol-fed fermentation stage. No carbon source is added to the tank within 1 hour to ensure that the glycerol is completely consumed. After starvation treatment, methanol is fed at a constant flow rate of 10mL/h·L. At this time, the temperature is lowered to 25°C, the pH is adjusted to 5.85, and the fermentation parameters are monitored in real time to maintain the dissolved oxygen above 20%. Fermentation is completed after 132 hours and the tank is released. The fermentation product is centrifuged at 8000rpm for 20 minutes, the supernatant is collected, and stored at -20°C.

Example 4

4.1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

4.2 Batch Fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank is about 19.5L before inoculation. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 30% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

4.3 Glycerol fed-batch fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

4.4 Methanol Fed-Batch Fermentation

After 36 hours of fermentation (wet weight reaches about 285g/L), stop adding glycerol, the dissolved oxygen will rise rapidly, and enter the methanol-fed fermentation stage. No carbon source is added to the tank within 1 hour to ensure that the glycerol is completely consumed. At this time, the temperature is reduced to 24°C and the pH is adjusted to 5.85. After starvation treatment, methanol is fed at an initial rate of 3-5mL/h·L, and the flow rate is gradually increased to 10-12mL/h·L. The fermentation parameters are monitored in real time, and the methanol flow rate is adjusted according to the dissolved oxygen content (if the DO value increases by more than 10% within 1 minute after stopping methanol feeding, it means that the carbon source is limited, otherwise it means that methanol is excessive). During this period, the dissolved oxygen is maintained above 20%. Fermentation ends at 132 hours and the tank is released. The fermentation product is centrifuged at 8000rpm for 20 minutes, the supernatant is collected, and stored at -20°C.

Example 5

5.1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

5.2 Batch Fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank is about 19.5L before inoculation. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 30% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

5.3 Glycerol fed-batch fermentation:

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

5.4, mixed fed-batch fermentation:

After 30 h of fermentation (wet weight reached about 242 g/L), the mixed feeding stage was entered, and glycerol feeding and methanol feeding were added simultaneously. At this time, the temperature was lowered to 24° C. and the pH was adjusted to 5.85.

5.5 Methanol fed-batch fermentation:

After 2 hours of mixed feed addition, the methanol feed phase began. The dissolved oxygen-speed coupling was decoupled, and the dissolved oxygen and methanol feed were coupled. The coupling correlation was inversely correlated, with a critical value of 30%. Feeding was initiated when the dissolved oxygen exceeded the critical value and stopped when it fell below it. The methanol feed cycle was 10 seconds. Fermentation was completed after 132 hours, and the tank was drained. The fermentation product was centrifuged at 8000 rpm for 20 minutes, and the supernatant was collected and stored at -20°C.

Comparative Example 1

6.1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 30°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 30°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 30°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

6.2 Batch Fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 6.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 30℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 6.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank before inoculation is about 19.5L. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 20% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

6.3 Glycerol Fed-Batch Fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

6.4 Methanol Fed-Batch Fermentation

After 30 hours of fermentation (wet weight reaches approximately 207 g/L), glycerol addition is stopped, dissolved oxygen rapidly recovers, and the fermentation enters the methanol-fed fermentation stage. No carbon source is added to the tank within 1 hour to ensure complete depletion of glycerol. After starvation treatment, methanol is fed at a constant flow rate of 10 mL/h·L. The parameters in the tank remain unchanged, and the fermentation parameters are monitored in real time to maintain dissolved oxygen above 20%. Fermentation is completed after 132 hours, and the tank is released. The fermentation product is centrifuged at 8000 rpm for 20 minutes, and the supernatant is collected and stored at -20°C.

Comparative Example 2

7.1. Preparation of seed solution

Take the preserved strain CBS7435-rHSA, streak it on a plate, and incubate it at 28°C for 2-3 days. Pick a single colony with good growth and inoculate it into 10 mL of YPD liquid medium. Incubate it at 28°C and 220 rpm for approximately 24 hours. Take 5 mL of this culture and continue to expand it. Inoculate it into 500 mL of YPD liquid medium and incubate it at 28°C and 220 rpm. When the OD600 of the seed in the shake flask reaches 10, use it as the seed liquid for tank filling.

7.2 Batch Fermentation

Put 20L of BSM culture medium into the fermenter, adjust the pH to 6.0 with ammonia water, sterilize at 121℃ for 30min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20L/min, and the tank pressure to 0.05Mpa. Add 87mL of sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 6.0 again. Discard about 500mL of the culture medium in the fermenter through the sampling port, so that the volume in the tank before inoculation is about 19.5L. Pump the shake flask seeds into the fermenter, correct the dissolved oxygen to 100%, and set the speed to 300rpm. Select "speed linkage" for the dissolved oxygen control mode, and maintain the dissolved oxygen above 20% by adjusting the speed (300rpm-1000rpm) and ventilation during fermentation.

7.3 Glycerol Fed-Batch Fermentation

When the dissolved oxygen suddenly increases and the rotation speed suddenly drops, the fermentation enters the glycerol feeding stage, and the glycerol feed is fed at a flow rate of 15 mL/h·L. The parameters in the tank remain unchanged.

7.4 Methanol Fed-Batch Fermentation

After 30 hours of fermentation (wet weight reaches approximately 219 g/L), glycerol addition is stopped, dissolved oxygen rapidly recovers, and the fermentation enters the methanol-fed fermentation stage. No carbon source is added to the tank for 1 hour to ensure complete depletion of glycerol. After starvation treatment, methanol is fed at a constant flow rate of 10 mL/h·L. The parameters in the tank remain unchanged, and all fermentation parameters are monitored in real time to maintain dissolved oxygen above 20%. Fermentation is completed after 132 hours, and the tank is released. The fermentation product is centrifuged at 8000 rpm for 20 minutes, and the supernatant is collected and stored at -20°C.

Example 6

During the fermentation process of Examples 1-5 and Comparative Examples 1-2, samples were taken every 6 hours to measure the wet weight. The results are shown in FIG1 .

After 36 hours of fermentation, the protein expression level of Examples 1-5 and Comparative Examples 1-2 was measured, and samples were taken every 6 hours for albumin quantification. The results are shown in FIG2 .

Since the fermentation time when the protein expression reached the maximum value was different in Examples 1-5 and Comparative Examples 1-2, the average protein expression rate was characterized by the ratio of the maximum expression amount to the induced expression time, and the results are shown in FIG3 .

The above results show that by changing the fermentation conditions, the average protein expression rate of each experimental group (Examples 1-5) is improved compared with the control group (Comparative Examples 1-2), among which the average protein expression rate of Example 4 and Example 5 is the highest, which is 3.6 times and 3.7 times that of Comparative Example 1, respectively.

Example 7

The fermentation broths obtained in Examples 1-5 and Comparative Examples 1-2 were purified and then quantified for glycoprotein using the sulfuric acid-phenol method. Mannose, dried to a constant weight under reduced pressure, was used as a reference. Control solutions of varying concentrations were prepared and developed with appropriately diluted test samples using the sulfuric acid-phenol method, and the absorbance was measured. A standard curve was fitted using the absorbance and reference concentration to determine the mannose content of the samples. The total protein content of the samples was determined using the Kjeldahl method. Finally, the ratio of mannose content to total protein in the samples was calculated [W/W, mannose mg·g(Pro) ^-1 ]. The results are shown in Table 1.

Table 1 Determination of mannose content in recombinant human albumin by sulfuric acid phenol method

The results in Table 1 indicate that changes in induction conditions affect the mannosylation level of recombinant human albumin. The correlation coefficient between the average protein expression rate and the mannose content in recombinant human albumin was calculated using the CORREL function. The resulting correlation coefficient was -0.9311, indicating a strong negative correlation. This indicates that the method of the present invention can reduce the O-glycosylation level of recombinant human albumin by increasing the protein expression rate.

The applicant declares that, in the description of the above specification:

The descriptions of terms such as "this embodiment", "an embodiment of the present invention", "as shown in...", "further", "a further improved technical sub-scheme", etc., mean that the specific features, structures, materials or characteristics described in the embodiment or example are included in at least one embodiment or example of the present invention; in this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in an appropriate manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine different embodiments or examples and features of different embodiments or examples described in this specification without causing any contradiction.

Finally, it should be noted that:

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same.

Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some or all of the technical features therein, and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. Non-essential improvements, adjustments or replacements made by those skilled in the art based on the contents of this specification are all within the scope of protection required by the present invention.

Claims (10)

A method for preparing recombinant human albumin with high expression and low O-glycosylation level, characterized in that the method comprises taking at least one measure to increase the protein expression rate and reduce the protein O-glycosylation level during the fermentation process; The measures include changing at least one of the following fermentation conditions: 1) Induction temperature; 2) induced pH; 3) dissolved oxygen content during the induction phase; 4) induction time point; 5) inducer concentration; 6) Method of adding inducer; 7) Flow rate of inducer addition. The method for preparing recombinant human albumin with high expression and low O-glycosylation level according to claim 1, characterized in that it comprises the following steps: S1. Seed solution preparation: Take the preserved bacterial strain and streak it on a plate. Pick a single colony and inoculate it into YPD liquid medium at 28-29.5℃. When the OD600 of the seed in the shake flask reaches 10-12, it is used as the seed solution for tank filling. S2. Batch fermentation: BSM medium was added to the fermentor, the pH was adjusted to 5.0-5.8, and sterilized. After cooling, the dissolved oxygen was corrected, the temperature and ventilation were set to 28-29.5°C, sterilized PTM1 solution was added to the medium, and the pH of the medium was again adjusted to 5.0-5.8; the medium in the fermentor was partially discarded through the sampling port, and the shake flask seeds were connected to the fermentor. The dissolved oxygen was corrected and the rotation speed was set. During the fermentation, the dissolved oxygen was maintained above 20% by adjusting the rotation speed and ventilation volume; S3, glycerol fed fermentation: when the dissolved oxygen suddenly increases, the glycerol feeding stage is entered, and glycerol is fed at a uniform rate. When the fermentation lasts for more than 30 hours, the addition of glycerol is stopped; S4, methanol fed-batch fermentation: No carbon source was added to ensure complete depletion of glycerol; after starvation treatment, methanol was fed, the pH in the tank was maintained at 5.8-6.0, the temperature was lowered to 24-28°C, and various fermentation parameters were monitored in real time to maintain dissolved oxygen above 20%; S5. After fermentation is completed, the tank is placed, the fermentation product is centrifuged, and the supernatant is collected. The method for preparing a recombinant human albumin with high expression and low O-glycosylation level according to claim 2, characterized in that the strain in step S1 is CBS7435-rHSA, and the CBS7435-rHSA strain is prepared by the following method: R1. Design and synthesize the recombinant human albumin DNA sequence, the sequence of which is shown in SEQ ID NO.1; R2. Ligate the sequence to the pPICZαA vector with restriction enzyme sites PmII and XbaI. Transform the ligation product into E. coli DH5α competent cells and plate on LB plates containing zeocin. Pick a single clone and transfer it into LB liquid medium containing zeocin. Re-extract the plasmid. After restriction enzyme digestion and sequencing verification, the expression plasmid was correctly constructed and named pPICZαA-rHSA. R3. Linearize the plasmid vector with Sac I and electroporate it into CBS7435 competent cells. Spread the transformed strain on a YPD-resistant plate containing zeocin and pick the positive single clone to obtain the recombinant human albumin expression strain CBS7435-rHSA. The method for preparing a recombinant human albumin with high expression and low O-glycosylation level as claimed in claim 2, characterized in that, between step S3 and step S4, it also includes mixed fed-batch fermentation: glycerol feed and methanol feed are added simultaneously, the temperature is 24°C, and the pH is adjusted to 5.85. The method for preparing a recombinant human albumin with high expression and low O-glycosylation level according to claim 2 or 4, characterized in that in steps S1 and S2, the temperature is 28° C.; in step S2, the pH is adjusted to 5.0. The method for preparing a recombinant human albumin with high expression and low O-glycosylation level according to claim 5, characterized in that in step S1 and steps S2-S4, the dissolved oxygen is greater than 30%. A method for preparing recombinant human albumin with high expression and low O-glycosylation level according to claim 2, characterized in that in step S4, the method of adding methanol feed is: adding methanol feed at a constant flow rate of 10-12 mL/h·L, or first adding methanol feed at an initial rate of 3-5 mL/h·L, and gradually increasing the flow rate to 10-12 mL/h·L. The method for preparing a recombinant human albumin with high expression and low O-glycosylation level according to claim 2 or 4, characterized in that in step S4, the dissolved oxygen and methanol feeding are coupled, the coupling correlation is inversely correlated, the critical value is 20-30%, that is, the feeding is started when the dissolved oxygen is higher than the critical value, and is stopped when the dissolved oxygen is lower than the critical value, and the working cycle of the methanol feeding is 10-15 s. The method for preparing recombinant human albumin with high expression and low O-glycosylation level according to claim 2, characterized in that: Each 20 L of BSM medium includes the following components: 534-600 mL of 85% phosphoric acid, 18.6-20 g of calcium sulfate dihydrate, 364-400 g of potassium sulfate, 298-320 g of magnesium sulfate dihydrate, 82.6-100 g of potassium hydroxide, and 800-850 g of glycerol; Each 500 mL of PTM1 solution contains the following components: 3-5 g copper sulfate pentahydrate, 0.044-0.1 g potassium iodide, 1.5-2 g manganese sulfate monohydrate, 0.1-0.2 g sodium molybdate dihydrate, 0.01-0.03 g boric acid, 0.25-0.35 g cobalt chloride, 10-12 g zinc chloride, 32.5-35 g ferrous sulfate heptahydrate, 0.1-0.2 g biotin, and 2.5-3.0 mL sulfuric acid. Each 500 mL of YPD contains the following components: 5-10 g of yeast extract, 10-20 g of peptone, and 10-20 g of glucose. The method for preparing recombinant human albumin with high expression and low O-glycosylation level according to claim 9, wherein the glycerol feed comprises: glycerol, purified water, a defoaming agent and PTM1; and the methanol feed comprises: methanol and a defoaming agent.