Fermentation medium and fermentation process for promoting secretory expression of HER2 affinity protein

A technology of fermentation medium and protein secretion, which is applied in the direction of fermentation, chemical instruments and methods, and methods based on microorganisms, to achieve the effects of reducing production costs, simplifying process steps, and improving product indicators

Pending Publication Date: 2022-04-12
YUANBEN (ZHUHAI HENGQIN) BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] At present, there are no related patents and literatures on the use of E. coli to secrete, infiltrate, or extracellularly express HER2 affinity body proteins

Method used

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  • Fermentation medium and fermentation process for promoting secretory expression of HER2 affinity protein
  • Fermentation medium and fermentation process for promoting secretory expression of HER2 affinity protein
  • Fermentation medium and fermentation process for promoting secretory expression of HER2 affinity protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Using gene design software, the codons of the HER2 affibody encoding gene with the pel signal peptide gene were optimized to make it suitable for recombinant expression in Escherichia coli. The HEHEHE amino acid sequence was added to the amino terminus of the HER2 Affibody, and the GGGC sequence was added to the carboxyl terminus. The length of the synthetic gene was identified by HindⅢ digestion, and the sequence of the synthetic gene was confirmed by gene sequencing. The HER2 affibody gene was cloned into pET22b(+) plasmid, and transformed into competent BL21(DE)3 Escherichia coli. The relative molecular mass of the expressed product was identified by SDS-PAGE after IPTG induced the recombinant expressing bacteria to be about 7.7kd.

[0049] The nucleotide sequence of the gene encoding the HER2 Affibody protein without the pel signal peptide is (SEQ ID No.1):

[0050] CCATGGCCCATGAACACGAGCACGAGGCGGAAAACAAATTCAACAAAGAAATGCGCAACGCGTACTGGGAAATTGCCCTGCTGCCGAACCTGACCAACC...

Embodiment 2

[0063] The secretory expression of HER2 protein comprises the following steps:

[0064] 1) Plate culture: After sterilizing the plate inoculated with the plate medium, add kanamycin at a final concentration of 50 ppm at about 50°C before cooling and solidifying, and pour the plate. Take the HER2 glycerol tube strain, smear it on the Kana-resistant plate in four divisions, and culture it overnight for 16 hours;

[0065] 2) Activated shake flask: After sterilizing and cooling the activated shake flask inoculated with the activated shake flask medium, inoculate the monoclonal and add kanamycin at a final concentration of 50ppm; pick one monoclonal on the plate, and insert kanamycin resistance In 25mL medium / 100mL shake flask, culture for 4h, OD 600 up to 1.0;

[0066] 3) Shake flasks into tanks: After sterilizing and cooling the shake flasks into tanks inoculated with the culture medium of shaker flasks into tanks, add kanamycin at a final concentration of 50 ppm at the same ti...

Embodiment 3

[0076] The secretory expression of HER2 protein comprises the following steps:

[0077] 1) 2) 3) 4) Same as Example 2, only adjusting the induction temperature to 30°C.

[0078] 5) Fermentation induction and feeding: after 5 hours of fermentation, detect OD 600 In 14.1, set the induction temperature to 30°C, start adding IPTG to start the induction, and the final concentration of the inducer is 0.5mM; and once the DO starts to rise, start feeding, and adjust the feeding speed to 35-50mL / H according to the dissolved oxygen degree, Basically control the dissolved oxygen range at 25±5% by feeding speed; for example, if the dissolved oxygen is higher than 40%, increase the feeding speed to about 50mL / H; if the dissolved oxygen is lower than 20%, reduce the feeding Speed ​​to about 35mL / H. Induce for 8h, detect OD 600 Basically no growth after 37.3, then the induction ends and the tank is put. After induction 4h-6h-8h, samples were taken respectively, samples were prepared and ...

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Abstract

The invention provides a fermentation medium for promoting secretory expression of HER2 (human epidermal growth factor receptor 2) affinity protein and a fermentation process. The HER2 affinity protein is an HER2 affinity recombinant protein with a gel signal peptide, the amino acid sequence of the HER2 affinity recombinant protein is as shown in SEQ ID No. 6, and the nucleotide sequence of the coding gene of the HER2 affinity recombinant protein is as shown in SEQ ID No. 5. The fermentation medium for promoting the secretory expression of the HER2 affinity protein comprises a base material and a supplementary material, and the base material is prepared from 8-15 g/L of peptone, 5-8 g/L of yeast extract powder, 5-10 g/L of glycerin, 0.7-2 g/L of anhydrous sodium sulfate, 0.7-2 g/L of anhydrous magnesium sulfate, 4-6 g/L of anhydrous monopotassium phosphate, 8-10 g/L of anhydrous dipotassium phosphate and 0.3-0.5 g/L of a defoaming agent; the formula of the supplementary material comprises 40-60 g/L of peptone, 20-30 g/L of yeast extract powder and 300-400 g/L of glycerin, and ammonia water with the concentration of 10-15% is additionally prepared. According to the invention, by continuously comparing and optimizing the fermentation formula and process, the secretory expression of 80-90% or more of target protein is realized; only the target protein secreted and expressed in the fermentation supernatant needs to be purified at the downstream, and the bacterial sludge can be abandoned.

Description

technical field [0001] The invention belongs to the field of recombinant protein expression, and in particular relates to a fermentation medium and a fermentation process for promoting the secretion and expression of HER2 affinity body protein. Background technique [0002] Escherichia coli (Escherichia coli) expression system is currently one of the most widely used and most thoroughly researched expression systems, which has significant advantages such as short fermentation cycle, high production efficiency, and easy operation. At present, the target protein expression in E. coli is generally expressed intracellularly, and there are very few reports of the target protein being secreted and expressed in the fermentation medium. The secretion performance of the E. coli expression system is poor because of the complex components of the E. coli secretion system, the fine regulation and the inherent double-membrane structure. [0003] The target protein is secreted and express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C07K19/00C12N15/62C12N15/70C12P21/02C12R1/19
Inventor 刘毅蔡炯肖凯刘明霞丁国中宋旭付玉洁祁丽丽张晋肖阳
Owner YUANBEN (ZHUHAI HENGQIN) BIOTECHNOLOGY CO LTD
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