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Extraction method of periplasmic protein of escherichia coli

A technology of Escherichia coli and periplasmic protein, applied in the field of protein extraction, can solve the problems of low extraction efficiency of periplasmic protein, need to be further improved, and affect separation and purification, so as to achieve short extraction cycle, improve extraction purity, extraction rate, and cost cheap effect

Active Publication Date: 2022-04-26
天津鸿宇泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The operation process of the above method is simple, but the long-term stirring (at least 12h) in the extraction process will easily lead to the rupture of the bacteria, which will cause the obtained extract to be too viscous, which will affect the separation and purification in the later stage; and the extraction efficiency of the periplasmic protein by this method Not high, needs to be further improved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0039] The preparation examples 1-5 provide the preparation method of the treatment liquid, and the preparation example 1 is taken as an example to illustrate below.

[0040] Prepare an 8mM silver nitrate solution in the dark, add hydroxyapatite micropowder into the silver nitrate solution, mix it with ultrasonic vibration for 1.5h, centrifuge at 3500rpm for 15min, collect the micropowder, wash with deionized water, and dry to obtain the desired loaded nano silver;

[0041] 15g of acetic acid solution, 8g of chitosan and 0.8g of loaded nano-silver were uniformly mixed to prepare the required treatment solution with a pH value of 4.5.

preparation example 2-5

[0042] Preparation examples 2-5 are the same as preparation example 1, except that the preparation parameters of loaded nano-silver and the quality of the raw materials of the treatment solution are different, see Table 1 for details.

[0043] Table 1:

[0044] Condition parameters / raw material dosage Preparation Example 1 Preparation example 2 Preparation example 3 Preparation Example 4 Preparation Example 5 Silver nitrate solution preparation concentration 8mM 5mM 6mM 9mM 10mM ultrasonic mixing time 1.5h 1h 1.2h 1.8h 2h Centrifugal speed 3500rpm 3000rpm 3200rpm 3800rpm 4000rpm centrifugation time 15min 20min 18min 12min 10min Acetic acid solution dosage 15g 10g 12g 18g 20g Chitosan dosage 8g 5g 6g 9g 10g Loaded Nano Silver Dosage 0.8g 0.5g 0.6g 1g 1.2g pH value 4.5 3.8 4.2 4.8 5.2

preparation example 6

[0045] Preparation examples 6-10 provide methods for cultivating Escherichia coli, and preparation example 6 is taken as an example below to illustrate.

[0046] Preparation Example 6

[0047](1) Take 100 μL of Escherichia coli competent cells and put them on ice to melt, take 1 μL of the plasmid containing the protein to be expressed and mix with the melted Escherichia coli competent cells. min, after another 1.5 min in ice bath, add 100 mL of LB liquid medium, incubate at 37°C for 50 min on a shaker, then centrifuge at 7000 rpm for 5 min, discard the supernatant, collect the remaining bacteria and resuspend to obtain the bacterial liquid;

[0048] (2) Take 10 μL of the bacterial solution resuspended in step (1) on the LB solid medium, spread it evenly with a spreading stick, and finally place it in a 37°C incubator for 8-16h;

[0049] (3) Pick a suitable colony from the LB solid medium, add 100mL of LB liquid medium, place it in a shaker at 37°C for 15h, then centrifuge at ...

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Abstract

The invention relates to the technical field of protein extraction, and particularly discloses an escherichia coli periplasmic protein extraction method which comprises the following steps: preparing a treating fluid, pretreating escherichia coli, centrifuging, and collecting to obtain pretreated thalli; the pretreated thalli are treated with a sucrose hypertonic solution and then centrifuged, finally, the pretreated thalli are treated with a magnesium sulfate solution and then centrifuged, and the required periplasmic protein is obtained; according to the method, the treating fluid is firstly adopted to interfere the outer membrane of the escherichia coli, so that the outer membrane loses integrity and loses a protective effect on osmotic pressure, then the high-concentration sucrose solution is utilized to cause high osmotic pressure to shrink cells, and finally the low-concentration magnesium sulfate is utilized to treat the cells, so that the cell concentration is reduced on the premise of ensuring the integrity of the inner membrane of the escherichia coli. And the periplasmic protein is induced to be fully released, so that the extraction purity and the extraction rate of the periplasmic protein are remarkably improved.

Description

technical field [0001] The application relates to the technical field of protein extraction, more specifically, it relates to a method for extracting E. coli periplasmic proteins. Background technique [0002] Escherichia coli (Escherichia coli) has a clear genetic background, is easy to operate, and is convenient for large-scale cultivation. Therefore, it is often used to express recombinant proteins for genetic engineering. It is widely used in medicine, agriculture and other industries. It is the most scientific research and application. Preferred host for protein expression. The periplasm (Periplasm) is a double membrane structure composed of the inner and outer membranes of Escherichia coli, and its oxidative environment can provide an environment suitable for the correct folding of foreign proteins. Expressing recombinant proteins in the periplasmic space of Escherichia coli has many advantages. Due to the low protein content in the periplasmic space, the protease act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/61C07K1/14C12N1/21C12R1/19
CPCC07K14/61C12N1/20Y02A50/30
Inventor 董晓宁伍波邹国英李勇吴智广李超辉南洋
Owner 天津鸿宇泰生物科技有限公司
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