Preparation method using Escherichia coli to express oligopeptide-1
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A technology of Escherichia coli and oligopeptide, applied in the field of bioengineering, can solve the problems of high operation cost, complicated extraction process and low content.
Inactive Publication Date: 2017-01-11
YANTAI HUAXIN BIOPHARM TECH CO LTD
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[0005] Although oligopeptide-1 is widely present in human body fluids, its content is extremely low, and the extraction method is far from meeting the needs of the market
Although there are some genetic engineering production methods of oligopeptide-1 at present, these methods all have the disadvantages of low product concentration in the fermentation broth, complicated extraction process, high operating cost and low yield. Therefore, so far, there is no one Mature genetic engineering downstream production technology to produce oligopeptide-1
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Embodiment 1
[0017] ⑴ Engineering bacteria fermentation:
[0018] Pick a single colony from the strain tube stored at -80°C and insert it into 2-10ml seed medium, add ampicillin at the same time to make the concentration in the medium reach 10-200ug / ml, 28-32°C, 50 —Cultivate on a shaker at 250rpm for 8-12 hours, insert the previous culture solution into the seed medium again according to the inoculum size of 1-8%, add ampicillin at the same time, make it at 10-200ug / ml, 28-32°C, 50 -250rpm shaker culture for 8-12 hours, then add the activated seed solution to the fermentation medium according to the inoculation amount of 1-8%, 28-32°C, 100-200rpm, maintain the pH value at 7.0, and cultivate for 9-11 hours, Then temperature induction, continue to cultivate for 2-8 hours. Oligopeptide-1 was expressed in the strain.
[0019] (2) Separation and purification of expression products of host cells:
[0020] Use a refrigerated centrifuge to centrifuge the fermentation broth at a speed of 6000-1...
Embodiment 2
[0025] ⑴ Engineering bacteria fermentation:
[0026] Pick a single colony from the strain tube stored at -80°C and insert it into 8ml seed medium, add ampicillin at the same time to make the concentration in the medium reach 50ug / ml, culture at 31°C, 100rpm shaker for 10 hours, Insert the previous culture solution in the seed medium again by 3% inoculum size, add ampicillin simultaneously, make it at 50ug / ml, 31 ℃, 100rpm shaker culture 10 hours, press the activated seed again by 3% inoculum size The solution was added to the fermentation medium, maintained at 31° C., 200 rpm, and the pH value was 7.0, and cultivated for 10 hours, then induced by temperature, and continued to cultivate for 4 hours. Oligopeptide-1 was expressed in the strain.
[0027] (2) Separation and purification of expression products of host cells:
[0028] Use a refrigerated centrifuge to centrifuge the fermentation broth at 8000 rpm for 15 min at 4°C, discard the supernatant and save the precipitate at...
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Abstract
The invention discloses a preparation method using Escherichia coli to express oligopeptide-1. The method comprises the following steps: 1, carrying out engineering bacteria fermentation: picking single colony from a preserved strain tube, inoculating a culture medium with the single colony, adding ampicillin, and carrying out shaking culture; 2, carrying out expanded bed adsorption: centrifuging a fermentation liquid by using a refrigerated centrifuge, removing the obtained precipitate, reserving the fermentation liquid, adding the fermentation liquid to an expanded bed, eluting, collecting a human oligopeptide-1-containing eluate at the elution peak, and preserving the eluate for later use; 3, carrying out gel separation: adding the eluate to a gel chromatography column through a chromatographic system, eluting with a buffer solution, and reserving the eluate obtained at an absorption peak; and 4, freeze-drying the eluate to obtain oligopeptide-1. The method has the advantages of simplicity, low cost and high yield. The prepared oligopeptide-1 can be used in drugs, healthcare foods and cosmetics as an active component or additive.
Description
[0001] field of invention [0002] The invention relates to a preparation method for expressing oligopeptide-1 by Escherichia coli, belonging to the field of bioengineering. [0003] Background of the invention [0004] Oligopeptide-1 is a single-chain polypeptide composed of 53 amino acids, with a molecular weight of 6216 Daltons, and there are no alanine, phenylalanine and lysine in the polypeptide. Oligopeptide-1 causes a series of biochemical changes in cells through signal transmission, activates genes related to cell division, makes static cells enter the cell division cycle, and repairs and proliferates skin surface cells, thereby enabling cell proliferation and treating injured and damaged skin. The epidermis skin has excellent curative effect. A very small amount of oligopeptide-1 can strongly promote cell division and growth, and its biggest feature is that it can promote cell proliferation and differentiation, thereby replacing aging and dead cells with new cells. ...
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