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Method for increasing secretion and expression of recombinant human epidermal growth factors by engineering bacteria

A technology of epidermal growth factor, secretion and expression, applied in the field of bioengineering, can solve the problems of hEGF limitation, high extraction cost, low product purity, etc., and achieve the effects of reducing cost, increasing secretion, and shortening fermentation time

Active Publication Date: 2019-12-10
ZHEJIANG NUPTEC BIOMACROMOLECULAR PURIFICATION RESINS MFG INC LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Before the 1980s, the source of hEGF was mainly extracted from tissues and body fluids. Due to the extremely low content of hEGF, the extraction cost was high and the product purity was low, which limited the application of hEGF
[0006] The secretory expression of recombinant protein is beneficial to subsequent purification, but the disadvantage of secretory expression is that some proteins cannot be secreted in the cell

Method used

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  • Method for increasing secretion and expression of recombinant human epidermal growth factors by engineering bacteria
  • Method for increasing secretion and expression of recombinant human epidermal growth factors by engineering bacteria
  • Method for increasing secretion and expression of recombinant human epidermal growth factors by engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Inoculate 100ul of engineering bacteria frozen in glycerol into 50ml LB liquid medium containing 50ug / ml kanamycin, culture at 35°C, 220rpm for 5 hours, measure OD 600 2.3, obtain the seed culture solution;

[0090] The seed culture solution was inoculated into the shake flask containing 50ml MBL medium by 4vol.% inoculation amount, cultivated at 37°C for 4 hours at 220rpm, when the OD 600 At 3, add IPTG with a final concentration of 0.1 mM, glycine with a final concentration of 0.2% by mass, and Triton X-100 with a final concentration of 0.002% by mass to start induction, and induce at 25°C for 10 hours, OD 600 was 5.38, and the fermentation supernatant was obtained. The concentration of recombinant human epidermal growth factor was detected by enzyme-linked immunosorbent assay (ELISA). The concentration of recombinant human epidermal growth factor in the supernatant was 4.51 mg / L, see Table 1 below.

[0091] The components of the MBL medium in the shake flask are: ...

Embodiment 2

[0096] Example 2 was carried out in the same manner as in Example 1 above, except that the final concentration of IPTG added was 0.3 mM. The results of this example are shown in Table 1 below.

Embodiment 3

[0098] Example 3 was carried out in the same manner as in Example 1 above, except that the final concentration of IPTG added was 0.2 mM. The results of this example are shown in Table 1 below.

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Abstract

The invention relates to a method for increasing secretion and expression of recombinant human epidermal growth factors by engineering bacteria. The method for increasing secretion and expression of the recombinant human epidermal growth factors by the engineering bacteria comprises the following steps: (1) inoculating an LB liquid medium containing carnamycin with engineering bacteria frozen in glycerol, and carrying out culturing so as to obtain a seed culture solution; (2) inoculating an MBL medium with the seed culture solution, carrying out culturing, adding IPTG, glycine and Triton X-100, and carrying out induction so as to obtain liquid supernatant after fermentation; and (3) separating recombinant human epidermal growth factors from the liquid supernatant after fermentation, and carrying out purification. During the induction, the added IPTG has a final concentration of 0.1-0.3mM; the added glycine has a final concentration of 0.2-1.5%, percentage by mass; and the added TritonX-100 has a final concentration of 0.002-0.2%, percentage by mass. The method for increasing secretion and expression of the recombinant human epidermal growth factors by the engineering bacteria increases secretion of hEGF with fermentation time shortened; and thus, cost is reduced.

Description

technical field [0001] The invention belongs to the field of bioengineering, and more specifically, the invention relates to a method for producing recombinant human epidermal growth factor by adopting a genetic engineering method. Background technique [0002] Human epidermal growth factor (hEGF) is a small molecular polypeptide composed of 53 amino acids, with a molecular weight of about 6000 Daltons and an isoelectric point of about 4.2-4.6. There are three pairs of disulfide bonds in the molecule, and it is relatively stable to physical and chemical factors such as acid, alkali and heat. [0003] hEGF has a wide range of biological effects and can strongly promote the growth of various epidermal tissues. It has been used in the treatment of burns, ulcers, various wounds and corneal injuries in medicine. hEGF can also promote the metabolism of normal epidermal cells, adding it to beauty and skin care products can achieve the effects of whitening, anti-wrinkle and anti-ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/485C12N15/70
CPCC07K14/485C12N15/70
Inventor 钱永常张旭初宋叶华
Owner ZHEJIANG NUPTEC BIOMACROMOLECULAR PURIFICATION RESINS MFG INC LTD