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

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.

Claims

1. 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 during the fermentation process to increase the protein expression rate and reduce the protein O-glycosylation level; 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.

2. The method for preparing recombinant human albumin with high expression and low O-glycosylation level according to claim 1, characterized in that: The following steps are involved: 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 complete, the tank is placed in a centrifuge and the fermentation product is centrifuged to collect the supernatant.

3. A method for preparing recombinant human albumin with high expression and low O-glycosylation level as claimed in claim 2, characterized in that: The strain used 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. The plasmid vector was linearized by Sac I and then electroporated into CBS7435 competent cells. The transformed strain was spread on YPD resistance plates containing zeocin, and positive single clones were picked to obtain the recombinant human albumin expression strain CBS7435-rHSA.

4. A method for preparing 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, mixed fed-batch fermentation is also included: glycerol feed and methanol feed are added simultaneously, the temperature is 24° C., and the pH is adjusted to 5.

85.

5. The method for preparing 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.

6. The method for preparing 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 above 30%.

7. The 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.

8. The method for preparing recombinant human albumin with high expression and low O-glycosylation level according to claim 2 or 4, characterized in that: In step S4, dissolved oxygen and methanol feeding are coupled, and the coupling correlation is inversely correlated. 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.

9. 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.

10. The method for preparing recombinant human albumin with high expression and low O-glycosylation level according to claim 9, characterized in that: The glycerol feed includes: glycerol, purified water, defoamer and PTM1; the methanol feed includes: methanol and defoamer.

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