A method for preparing recombinant human albumin with low O-glycosylation level

Abstract

The invention provides a method for preparing recombinant human albumin with low O-glycosylation level, comprising taking at least one measure to improve the growth rate of the strain and reduce the O-glycosylation level of the protein during the fermentation process, and specifically comprising S1, seed liquid preparation; S2, BSM culture medium is put into a fermentation tank, the pH is adjusted to 5.0-5.8, sterilization is carried out, dissolved oxygen is corrected after cooling, temperature and ventilation volume are set, sterilized PTM1 solution is added to the culture medium, and the pH of the culture medium is adjusted to 5.0-5.8 again; S3, shaking flask seeds are connected to the fermentation tank, dissolved oxygen is corrected, and rotation speed is set; S4, when the dissolved oxygen increases suddenly, a glycerol feeding stage is entered, and glycerol feeding is added at a uniform speed; S5, glycerol feeding and methanol feeding are added simultaneously, pH is adjusted to 6.0-6.5, and the temperature is lowered; S6, methanol feeding is added; S7, after the fermentation is completed, the tank is released, the fermentation product is centrifuged, and the supernatant is collected; the method of the invention can reduce protein O-glycosylation, and the expressed recombinant protein contains a very low level of glycosylation modification.

Description

Technical Field

[0001] The invention relates to a method for preparing recombinant human albumin with low O-glycosylation level, belonging to the field of medicine. Background Art

[0002] Human serum albumin is the most abundant protein in human plasma. Its non-glycosylated single-chain polypeptide contains 585 amino acids and has a molecular weight of about 66.5 kD. It is widely used in the clinic to treat critical illnesses such as shock, edema or ascites caused by blood loss, trauma and burns, as well as hypoproteinemia.

[0003] Unlike traditional genetically recombinant drugs, the total infusion dose of recombinant human albumin in clinical use generally needs to reach 10 g or more to have a therapeutic effect. Under high-dose protein background conditions, if recombinant human albumin contains a certain amount of glycosylated or glycated recombinant human albumin, the total infusion amount of these abnormal structural proteins will also be amplified during clinical use, which may cause clinical allergic reactions and other hazards.

[0004] Albumin itself does not have N-glycosylation sites, but it may have a certain degree of O-glycosylation at its S / T sites. In particular, recombinant products of yeast expression systems usually produce a certain degree of mannosylation based on the characteristics of the expression system itself, which has potential immunogenicity, is easily cleared in the human body, and has a short half-life. Therefore, it is urgent to find methods that can regulate the O-glycosylation modification of recombinant proteins in yeast expression systems.

[0005] The glycosylation process is catalyzed by glycosyltransferases, which connect the sugar on the sugar donor to the sugar acceptor through a glycosidic bond. Therefore, the study of protein glycosylation modification can start from three aspects: sugar donors, sugar acceptors and glycosyltransferases. The first mannose attached to the yeast protein is provided by polyterpenoid phosphomannose (Dol-P-Man). Studies have confirmed that knocking out the gene encoding Dol-P-Man synthase can completely lose the O-glycosylation of yeast proteins. In addition, the commonly used methods for removing O-glycosylation of yeast proteins include replacing or deleting amino acid residues that can be O-linked, destroying or removing genes encoding glycosyltransferases, etc. Although these methods are effective, they have obvious technical defects. Studies have shown that defects in mannosyltransferase genes may affect the integrity of the cell wall and lead to low cell viability; replacing or deleting amino acid residues that can be O-linked in the target protein may change the properties and functions of the target protein.

[0006] The present invention aims to provide a new method for reducing the level of O-glycosylation, which does not involve genetic modification technology and is simple to operate, and provides a reference for the glycosylation regulation of recombinant proteins in yeast expression systems. Summary of the invention

[0007] The invention provides a method for preparing recombinant human albumin with low O-glycosylation level, which can reduce protein O-glycosylation, and the expressed recombinant protein contains extremely low level of glycosylation modification.

[0008] To achieve this object, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a method for preparing recombinant human albumin with low O-glycosylation level, the method comprising taking at least one measure during the fermentation process to increase the growth rate of the strain and reduce the protein O-glycosylation level;

[0010] The measures include changing at least one of the following fermentation conditions:

[0011] 1) Inoculum size;

[0012] 2) Bacteria concentration in seed solution;

[0013] 3) Initial pH of the culture medium;

[0014] 4) Cultivation temperature;

[0015] 5) ventilation volume;

[0016] 6) Stirring speed.

[0017] Preferably, a method for preparing recombinant human albumin with low O-glycosylation level comprises the following steps:

[0018] S1. Seed solution preparation: Take the preserved strains and streak them on the plate, pick a single colony and inoculate it into YPD liquid medium for cultivation, and when the OD600 of the shake flask seeds reaches 10-12, use it as the seed solution for tank loading;

[0019] S2, put the BSM culture medium into the fermentation tank, adjust the pH to 5.0-5.8, sterilize, calibrate the dissolved oxygen after cooling, set the temperature and ventilation volume, add the sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0-5.8 again;

[0020] S3, discard part of the culture medium in the fermentation tank through the sampling port, connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen, set the speed, and maintain the dissolved oxygen above 20% by adjusting the speed and ventilation during the fermentation;

[0021] S4, when the dissolved oxygen increases suddenly, the glycerol feeding stage is entered, and glycerol feeding is added at a uniform rate;

[0022] S5, after 10-12 hours of glycerol feed addition, the mixed feed stage is entered, glycerol feed and methanol feed are added simultaneously, the pH is adjusted to 6.0-6.5, and the temperature is lowered;

[0023] S6, after 2-3 hours of feed addition, the methanol feed period begins, methanol feed is added, and various fermentation parameters are monitored in real time;

[0024] S7. After the fermentation is completed, the tank is placed, the fermentation product is centrifuged, and the supernatant is collected.

[0025] Preferably, ammonia water is used to adjust the pH value.

[0026] Preferably, the strain in step S1 is CBS7435-rHSA, and the CBS7435-rHSA strain is prepared by the following method:

[0027] R1, design and synthesize the recombinant human albumin DNA sequence, the sequence is shown in SEQ ID NO.1;

[0028] R2, the optimized sequence was connected to the pPICZα A vector, the restriction sites were PmII and XbaI, the ligation product was transferred into E. coli DH5α competent cells, spread on LB plates containing zeocin, single clones were picked and transferred into LB liquid culture medium containing zeocin, the plasmid was re-extracted, and the expression plasmid was correctly constructed after restriction digestion identification and sequencing verification, and was named pPICZα A-rHSA;

[0029] R3. The plasmid vector was linearized by Sac I and then electroporated into CBS7435 competent cells. The transformed strain was spread on a YPD resistance plate containing zeocin, and the positive single clone was picked to obtain the recombinant human albumin expression strain CBS7435-rHSA.

[0030] In the present invention, SEQ ID NO.1 is:

[0031]

[0032] Preferably, in step S1, each 1 L of YPD liquid culture medium comprises the following components: 5-10 g yeast extract, 10-20 g peptone, and 10-20 g glucose.

[0033] Preferably, in step S1, the culture temperature of the bacteria in the YPD liquid culture medium is 28-30°C.

[0034] Preferably, in step S1, the OD600 of the seeds in the shake flask is 10.

[0035] Preferably, in step S2, the pH value is adjusted to 5 twice.

[0036] Preferably, in step S2, each 20 L of BSM culture 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.

[0037] Further preferably, each 20 L of BSM culture medium includes the following components: 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, and the volume is fixed to 20 L.

[0038] Preferably, in step S2, each 500 mL of PTM1 solution includes the following components: 3-5 g of copper sulfate pentahydrate, 0.044-0.1 g of potassium iodide, 1.5-2 g of manganese sulfate monohydrate, 0.1-0.2 g of sodium molybdate dihydrate, 0.01-0.03 g of boric acid, 0.25-0.35 g of cobalt chloride, 10-12 g of zinc chloride, 32.5-35 g of ferrous sulfate heptahydrate, 0.1-0.2 g of biotin, and 2.5-3.0 mL of sulfuric acid.

[0039] Further preferably, in step S2, each 500 mL of PTM1 solution includes the following components: 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, fixed to 500 mL, and filtered for sterilization.

[0040] Preferably, the glycerol feed comprises: glycerol, purified water, a defoaming agent and PTM1.

[0041] Further preferably, the glycerol phase feed: 4200 g glycerol, 2800 g purified water, 0.2 mL / L defoamer, 180 mL PTM, are mixed and placed in a 10 L storage bottle for sterilization.

[0042] Preferably, the methanol feed comprises: methanol and a defoaming agent.

[0043] Further preferably, methanol feed: 20 L of methanol, filtered and sterilized; 0.2 mL / L of defoaming agent, sterilized and dissolved in the sterilized methanol.

[0044] Compared with the prior art, the beneficial effects and significant improvements of the technical solution of the present invention are:

[0045] 1. The method of the present invention is to reduce the protein O-glycosylation level by increasing the growth rate of the strain during the fermentation process, and the operation is simple and easy to control.

[0046] 2. The method of the present invention does not require the destruction or removal of any glycosyltransferase gene, thus avoiding the negative impact on cell integrity that may be caused by glycosyltransferase gene defects.

[0047] 3. The method of the present invention does not require deletion or replacement of amino acid residues containing glycosylation sites, and the expressed recombinant protein has the same amino acid sequence as the natural protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments of the present invention are briefly introduced below.

[0049] Figure 1 OD600 values ​​during the fermentation process of Examples 1-3 and Comparative Examples 1-3;

[0050] Figure 2 The wet weight results during the fermentation process of Examples 1-3 and Comparative Examples 1-3 are shown. DETAILED DESCRIPTION

[0051] The present invention is further described below in conjunction with specific examples. The examples are only used 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 content of the present invention, those skilled in the art can make various changes and modifications to the present invention, and these equivalent forms also fall within the scope limited by the appended claims of the application.

[0052] In order to more fully understand the present invention, the professional terms in the present invention are explained below.

[0053] The "recombinant protein" of the present invention is a protein obtained by applying the technology of recombinant DNA or recombinant RNA. The production of in vitro recombinant proteins mainly includes four major systems: prokaryotic protein expression, mammalian cell protein expression, yeast protein expression and insect cell protein expression. The produced proteins are different in terms of activity and application methods. Choose a suitable protein expression system according to your downstream application to improve the success rate of expression.

[0054] The "culture medium" of the present invention is an artificially prepared nutrient for the growth and maintenance of microorganisms, plant tissues and animal tissues, generally containing carbohydrates, nitrogen-containing substances, inorganic salts (including trace elements), vitamins and water, etc. Different culture media can add some compounds that cannot be synthesized by themselves, i.e., growth factors, according to actual needs.

[0055] The "fermentation" of the present invention refers to the process in which people use the life activities of microorganisms under aerobic or anaerobic conditions to prepare microbial cells themselves, or direct metabolites or secondary metabolites. Fermentation is sometimes also written as fermentation, and its definition varies depending on the occasion of use. Fermentation usually refers to a certain decomposition process of organic matter by organisms. Fermentation is a biochemical reaction that humans have been exposed to for a long time, and it is now widely used in the food industry, biological and chemical industries. It is also a basic process in bioengineering, namely fermentation engineering. Research on its mechanism and process control is still ongoing.

[0056] The "strains" of the present invention are microorganisms used as living cell catalysts in the fermentation process, including four categories: bacteria, actinomycetes, yeasts and molds. They come from a large number of microorganisms in nature, from which useful strains are separated and screened, then improved and stored for use in production.

[0057] The recombinant human albumin expression strain CBS7435-rHSA used in the present invention is self-constructed. All embodiments of the present invention use the above strain for fermentation culture. The construction method thereof comprises the following steps:

[0058] S1. Design and synthesize a recombinant human albumin DNA sequence, the sequence of which is shown in SEQ ID NO.1;

[0059] S2, the optimized sequence was connected to the pPICZα A vector, the restriction sites were PmII and XbaI, the ligation product was transferred into E. coli DH5α competent cells, spread on LB plates containing zeocin, single clones were picked and transferred into LB liquid medium containing zeocin, the plasmid was re-extracted, and the expression plasmid was correctly constructed after restriction digestion identification and sequencing verification, and was named pPICZα A-rHSA;

[0060] S3. The plasmid vector was linearized by Sac I and then electroporated into CBS7435 competent cells. The transformed strain was spread on a YPD resistance plate containing zeocin, and the positive single clone was picked to obtain the recombinant human albumin expression strain CBS7435-rHSA.

[0061] Wherein, SEQ ID NO.1 is:

[0062]

[0063] The present invention is further described in detail below with reference to specific embodiments.

[0064] Example 1

[0065] 1.1. Culture medium preparation

[0066] YPD: 10 g yeast extract, 20 g peptone, 20 g glucose, dilute to 1 L, and sterilize with high pressure steam.

[0067] 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 to sterilize.

[0068] 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, 800 g of glycerol, make up to 20 L.

[0069] Glycerol feed: 4200 g glycerol, 2800 g purified water, 0.2 mL / L defoamer, 80 mL PTM1, mixed and placed in a 10 L storage bottle for sterilization.

[0070] Methanol feed: 20 L methanol, filtered and sterilized; 0.2 mL / L defoaming agent, sterilized and dissolved in the sterilized methanol.

[0071] 1.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA for plate streaking, pick a single colony and inoculate it into 1 LYPD liquid culture medium, culture it at 30°C and 220rpm, and when the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank loading.

[0072] 1.3. Add 20L BSM medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30 min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 30℃, the ventilation volume to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87mL of sterilized PTM1 solution to the medium and adjust the pH of the medium to 5.0 again.

[0073] 1.4. Discard about 1 L of the culture medium in the fermentation tank through the sampling port, so that the volume in the tank before inoculation is about 19 L. Connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen to 100%, set the speed to 300 rpm, and maintain the dissolved oxygen above 20% by adjusting the speed (300 rpm-1000 rpm) and ventilation during fermentation.

[0074] 1.5. When the dissolved oxygen increases suddenly, the glycerol feeding stage begins and glycerol is added at a uniform rate. The parameter requirements remain unchanged.

[0075] 1.6. After 10 hours of glycerol feed addition, the mixed feed stage is entered. Glycerol feed and methanol feed are added simultaneously. The pH is adjusted to 6.0 and the temperature is reduced to 25°C. Other parameter requirements remain unchanged.

[0076] 1.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and various fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0077] 1.8. After 132 h of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0078] Example 2

[0079] 2.1. Culture medium preparation

[0080] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high pressure steam.

[0081] 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 to sterilize.

[0082] 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, 800 g of glycerol, make up to 20 L.

[0083] Glycerol feed: 4200 g glycerol, 2800 g purified water, 0.2 mL / L defoamer, 80 mL PTM1, mixed and placed in a 10 L storage bottle for sterilization.

[0084] Methanol feed: 20 L methanol, filtered and sterilized; 0.2 mL / L defoaming agent, sterilized and dissolved in the sterilized methanol.

[0085] 2.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA for plate streaking, pick a single colony and inoculate it into 500 mL YPD liquid medium, culture it at 30°C and 220 rpm, and when the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank loading.

[0086] 2.3. Add 20L BSM medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30 min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 30℃, the ventilation volume to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87mL of sterilized PTM1 solution to the medium and adjust the pH of the medium to 5.0 again.

[0087] 2.4. Discard about 500 mL of the culture medium in the fermentation tank through the sampling port to make the volume in the tank about 19.5 L before inoculation. Connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen to 100%, set the speed to 300 rpm, and maintain the dissolved oxygen above 20% by adjusting the speed (300 rpm-1000 rpm) and ventilation during fermentation.

[0088] 2.5. When the dissolved oxygen increases suddenly, the glycerol feeding stage begins and glycerol is added at a uniform rate. The parameter requirements remain unchanged.

[0089] 2.6. After 10 hours of glycerol feed addition, the mixed feed stage was entered, and glycerol feed and methanol feed were added simultaneously. The pH was adjusted to 6.0 and the temperature dropped to 25°C.

[0090] 2.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and various fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0091] 2.8. After 132 h of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0092] Example 3

[0093] 3.1. Culture medium preparation

[0094] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 1 L, and sterilize with high pressure steam.

[0095] 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 to sterilize.

[0096] 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, 800 g of glycerol, make up to 20 L.

[0097] Glycerol feed: 4200 g glycerol, 2800 g purified water, 0.2 mL / L defoamer, 80 mL PTM1, mixed and placed in a 10 L storage bottle for sterilization.

[0098] Methanol feed: 20 L methanol, filtered and sterilized; 0.2 mL / L defoaming agent, sterilized and dissolved in the sterilized methanol.

[0099] 3.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA for plate streaking, pick a single colony and inoculate it into 1 LYPD liquid culture medium, culture it at 28°C and 220rpm, and when the OD600 of the shake flask seeds reaches 10, use it as the seed solution for tank loading.

[0100] 3.3. Add 20L BSM medium into the fermenter, adjust the pH to 5.0 with ammonia water, sterilize at 121℃ for 30 min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87mL of sterilized PTM1 solution to the medium and adjust the pH of the medium to 5.0 again.

[0101] 3.4. Discard about 1 L of the culture medium in the fermentation tank through the sampling port, so that the volume in the tank before inoculation is about 19 L. Connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen to 100%, set the speed to 300 rpm, and maintain the dissolved oxygen above 20% by adjusting the speed (300 rpm-1000 rpm) and ventilation during fermentation.

[0102] 3.5. When the dissolved oxygen increases suddenly, the glycerol feeding stage begins and glycerol is added at a uniform rate. The parameter requirements remain unchanged.

[0103] 3.6. After 10 hours of glycerol feed addition, the mixed feed stage was entered, and glycerol feed and methanol feed were added simultaneously. The pH was adjusted to 6.0 and the temperature dropped to 25°C.

[0104] 3.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and various fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0105] 3.8. After 132 h of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0106] Comparative Example 1

[0107] 4.1. Culture medium preparation:

[0108] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high pressure steam.

[0109] 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 to sterilize.

[0110] 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, 800 g of glycerol, make up to 20 L.

[0111] Glycerol feed: 4200 g glycerol, 2800 g purified water, 0.2 mL / L defoamer, 80 mL PTM1, mixed and placed in a 10 L storage bottle for sterilization.

[0112] Methanol feed: 20 L methanol, filtered and sterilized; 0.2 mL / L defoaming agent, sterilized and dissolved in the sterilized methanol.

[0113] 4.2. Preparation of seed solution: Take the preserved strain CBS7435-rHSA for plate streaking, pick a single colony and inoculate it into 500 mL YPD liquid medium, culture it at 30°C and 220 rpm, and when the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank loading.

[0114] 4.3. Add 20L BSM medium into the fermenter, adjust the pH to 6.0 with ammonia water, sterilize at 121℃ for 30 min, and correct the dissolved oxygen to 0% after cooling. Set the temperature to 30℃, the ventilation volume to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87mL of sterilized PTM1 solution to the medium and adjust the pH of the medium to 6.0 again.

[0115] 4.4. Discard about 500 mL of the culture medium in the fermentation tank through the sampling port to make the volume in the tank about 19.5 L before inoculation. Connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen to 100%, set the speed to 300 rpm, and maintain the dissolved oxygen above 20% by adjusting the speed (300 rpm-1000 rpm) and ventilation during fermentation.

[0116] 4.5. When the dissolved oxygen increases suddenly, the glycerol feeding stage begins and glycerol is added at a uniform rate. The parameter requirements remain unchanged.

[0117] 4.6. After 10 hours of glycerol feed addition, the mixed feed stage is entered. Glycerol feed and methanol feed are added simultaneously. The temperature is reduced to 25°C. Other parameter requirements remain unchanged.

[0118] 4.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and various fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0119] 4.8. After 132 h of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0120] Comparative Example 2

[0121] 5.1. Culture medium preparation

[0122] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high pressure steam.

[0123] 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 to sterilize.

[0124] 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, 800 g of glycerol, make up to 20 L.

[0125] Glycerol feed: 4200 g glycerol, 2800 g purified water, 0.2 mL / L defoamer, 80 mL PTM1, mixed and placed in a 10 L storage bottle for sterilization.

[0126] Methanol feed: 20 L methanol, filtered and sterilized; 0.2 mL / L defoaming agent, sterilized and dissolved in the sterilized methanol.

[0127] 5.2. Preparation of seed solution: Take the preserved strain CBS7435-rHSA for streaking, pick a single colony and inoculate it into 500 mL YPD liquid medium, culture it at 30°C and 220 rpm, and when the OD600 of the shake flask seeds reaches 10, use it as the seed solution for tank loading.

[0128] 5.3. Add 20L BSM medium into the fermenter, adjust the pH to 6.0 with ammonia water, sterilize at 121℃ for 30 min, and adjust the dissolved oxygen to 0% after cooling. Set the temperature to 30℃, the ventilation volume to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87mL of sterilized PTM1 solution to the medium and adjust the pH of the medium to 6.0 again.

[0129] 5.4. Discard about 500 mL of the culture medium in the fermentation tank through the sampling port to make the volume in the tank about 19.5 L before inoculation. Connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen to 100%, set the speed to 300 rpm, and maintain the dissolved oxygen above 20% by adjusting the speed (300 rpm-1000 rpm) and ventilation during fermentation.

[0130] 5.5. When the dissolved oxygen increases suddenly, the glycerol feeding stage begins and glycerol is added at a uniform rate. The parameter requirements remain unchanged.

[0131] 5.6. After 10 hours of glycerol feed addition, the mixed feed stage begins. Glycerol feed and methanol feed are added simultaneously. The temperature drops to 25°C. Other parameter requirements remain unchanged.

[0132] 5.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and various fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0133] 5.8. After 132 h of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0134] Comparative Example 3

[0135] 6.1. Culture medium preparation:

[0136] YPD: 5 g yeast extract, 10 g peptone, 10 g glucose, dilute to 500 mL, and sterilize by high pressure steam.

[0137] 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 to sterilize.

[0138] 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, 800 g of glycerol, make up to 20 L.

[0139] Glycerol feed: 4200 g glycerol, 2800 g purified water, 0.2 mL / L defoamer, 80 mL PTM1, mixed and placed in a 10 L storage bottle for sterilization.

[0140] Methanol feed: 20 L methanol, filtered and sterilized; 0.2 mL / L defoaming agent, sterilized and dissolved in the sterilized methanol.

[0141] 6.2. Seed solution preparation: Take the preserved strain CBS7435-rHSA for plate streaking, pick a single colony and inoculate it into 500 mL YPD liquid medium, culture it at 28°C and 220 rpm, and when the OD600 of the shake flask seeds reaches 12, use it as the seed solution for tank loading.

[0142] 6.3. Add 20L BSM medium into the fermenter, adjust the pH to 6.0 with ammonia water, sterilize at 121℃ for 30 min, and adjust the dissolved oxygen to 0% after cooling. Set the temperature to 28℃, the ventilation volume to 20 L / min, and the tank pressure to 0.05 Mpa. Add 87mL of sterilized PTM1 solution to the medium and adjust the pH of the medium to 6.0 again.

[0143] 6.4. Discard about 500 mL of the culture medium in the fermentation tank through the sampling port to make the volume in the tank about 19.5 L before inoculation. Connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen to 100%, set the speed to 300 rpm, and maintain the dissolved oxygen above 20% by adjusting the speed (300 rpm-1000 rpm) and ventilation during fermentation.

[0144] 6.5. When the dissolved oxygen increases suddenly, the glycerol feeding stage begins and glycerol is added at a uniform rate. The parameter requirements remain unchanged.

[0145] 6.6. After 10 hours of glycerol feed addition, the mixed feed stage is entered. Glycerol feed and methanol feed are added simultaneously. The temperature is reduced to 25°C. Other parameter requirements remain unchanged.

[0146] 6.7. After 2 hours of mixed feed addition, the methanol feed period begins. Methanol feed is added in a flow manner, and various fermentation parameters are monitored in real time, and specific adjustments are made according to the situation.

[0147] 6.8. After 132 h of fermentation, the fermentation product was centrifuged at 8000 rpm for 20 minutes and the supernatant was collected.

[0148] Example 4

[0149] During the fermentation of Examples 1-3 and Comparative Examples 1-3, samples were taken every 12 h to measure OD600 values ​​and wet weights.

[0150] The results are as follows Figure 1 and Figure 2 The experimental results show that Example 1 and Example 3 have the fastest growth rates, and the order of growth rate from large to small is: Example 3> Example 1> Example 2> Comparative Example 2> Comparative Example 3> Comparative Example 1.

[0151] In summary, the growth rate of bacteria in the early stage of fermentation can be promoted by changing the inoculation amount of the strain, the concentration of the seed liquid, the temperature, and the pH fermentation conditions.

[0152] Example 5

[0153] After the fermentation broth obtained in Example 1-3 and Comparative Example 1-3 was purified, the glycoprotein was quantified by the sulfuric acid phenol method. Mannose dried to constant weight under reduced pressure was used as a reference substance, and control solutions of different concentrations were prepared. The appropriately diluted test samples were colorized according to the sulfuric acid phenol method and the absorbance was measured. The standard curve was fitted with the absorbance and the concentration of the reference substance to obtain the mannose content of the sample. The total protein content of the sample was determined by the Kjeldahl method. Finally, the ratio of the mannose content in the total protein in the sample was calculated [W / W, mannose mg•g (Pro) -1 ], the results are shown in Table 1.

[0154] Table 1 Determination of mannose content in recombinant protein by sulfuric acid phenol method

[0155]

[0156] The results in Table 1 verify that the method of the present invention, i.e., changing the inoculation amount of the strain, the bacterial concentration of the seed liquid, the temperature, and the pH fermentation conditions during the fermentation process, can increase the growth rate of the strain and reduce the mannosylation of the Pichia pastoris recombinant protein, and under certain conditions, the O-glycosylation modification level of the expression product is inversely proportional to the growth rate of the strain.

[0157] The applicant declares that, in the description of the above specification:

[0158] The descriptions of the terms "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 are not necessarily for the same embodiment or example, and the specific features, structures, materials or characteristics described may be combined or combined in a suitable manner in any one or more embodiments or examples; in addition, a person of ordinary skill in the art may combine or combine the different embodiments or examples and the features of the different embodiments or examples described in this specification without causing any contradiction.

[0159] Finally, it should be noted that:

[0160] The above embodiments are only used to illustrate the technical solutions of the present invention, but not to limit them.

[0161] 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 make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention. The 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

1. A method for preparing recombinant human albumin with low O-glycosylation level, characterized in that: The method comprises taking at least one measure during the fermentation process to increase the growth rate of the strain and reduce the protein O-glycosylation level; The method comprises the following steps: S1. Seed solution preparation: Take the preserved strains and streak them on the plate, pick a single colony and inoculate it into YPD liquid medium for cultivation, and when the OD600 of the shake flask seeds reaches 10-12, use it as the seed solution for tank loading; S2, put the BSM culture medium into the fermentation tank, adjust the pH to 5.0-5.8, sterilize, calibrate the dissolved oxygen after cooling, set the temperature and ventilation volume, add the sterilized PTM1 solution to the culture medium, and adjust the pH of the culture medium to 5.0-5.8 again; S3, discard part of the culture medium in the fermentation tank through the sampling port, connect the shake flask seeds to the fermentation tank, calibrate the dissolved oxygen, set the speed, and maintain the dissolved oxygen above 20% by adjusting the speed and ventilation during the fermentation; S4, when the dissolved oxygen increases suddenly, the glycerol feeding stage is entered, and glycerol feeding is added at a uniform rate; S5, after 10-12 hours of glycerol feed addition, the mixed feed stage is entered, glycerol feed and methanol feed are added simultaneously, the pH is adjusted to 6.0-6.5, and the temperature is lowered; S6, after 2-3 hours of feed addition, the methanol feed period begins, methanol feed is added, and various fermentation parameters are monitored in real time; S7. After the fermentation is completed, the tank is placed, the fermentation product is centrifuged, and the supernatant is collected; 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 is shown in SEQ ID NO.1; R2, connect the sequence to the pPICZα A vector, with restriction sites PmII and XbaI, transfer the ligation product into E. coli DH5α competent cells, spread on LB plates containing zeocin, pick a single clone and transfer it into LB liquid culture medium containing zeocin, re-extract the plasmid, and confirm that the expression plasmid is correctly constructed by restriction digestion and sequencing verification, and is named pPICZα A-rHSA; R3, linearize the plasmid vector by Sac I digestion and then electro-transform into CBS7435 competent cells, spread the transformed strain on YPD resistance plate containing zeocin, pick the positive single clone, and obtain the recombinant human albumin expression strain CBS7435-rHSA; In step S1, the culture temperature of the strain in the YPD liquid medium is 28-30°C; In step S1, the OD600 of the seeds in the shake flask was 10; In step S2, the pH value is adjusted to 5 twice.

2. A method for preparing recombinant human albumin with low O-glycosylation level as claimed in claim 1, characterized in that: In step S1, each 1 L of YPD liquid culture medium includes the following components: 5-10 g of yeast extract, 10-20 g of peptone, and 10-20 g of glucose.

3. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 1, characterized in that: In step S2, each 20 L of BSM culture 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.

4. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 1, characterized in that: In step S2, each 500 mL of PTM1 solution includes the following components: 3-5 g of copper sulfate pentahydrate, 0.044-0.1 g of potassium iodide, 1.5-2 g of manganese sulfate monohydrate, 0.1-0.2 g of sodium molybdate dihydrate, 0.01-0.03 g of boric acid, 0.25-0.35 g of cobalt chloride, 10-12 g of zinc chloride, 32.5-35 g of ferrous sulfate heptahydrate, 0.1-0.2 g of biotin, and 2.5-3.0 mL of sulfuric acid.

5. The method for preparing recombinant human albumin with low O-glycosylation level according to claim 4, characterized in that: The glycerol feed includes: glycerol, purified water, defoamer and PTM1; the methanol feed includes: methanol and defoamer.

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