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Method for clarifying Escherichia coli lysate
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A technology of Escherichia coli and lysate, applied in the direction of biochemical equipment and methods, hydrolytic enzymes, microorganism dissolution, etc., can solve the problem of low specificity, and achieve the effect of stable and firm combination and good clarification effect
Active Publication Date: 2019-02-22
普健生物(武汉)科技有限公司
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Problems solved by technology
Some nucleases can only act on RNA, some nucleases can only act on DNA, and some nucleases are less specific and can act on both RNA and DNA
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Embodiment 1～4
[0029] Following examples 1~4, comparative example 1,2, in the blank control, the preparation method of Escherichia coli lysate is as follows:
[0031] Prepare 4 liters of LB medium (tryptone 10g / L, yeast extract 5g / L, sodiumchloride 10g / L, pH 7.0), and after the medium is prepared, sterilize it at 121°C for 30 minutes. After the sterilization, add Amp to the ultra-clean bench to make the final concentration of 100mg / L when the culture medium is cooled and not hot to the hands, and put it in the refrigerator at 4°C after cooling for later use.
[0032] Under aseptic operation conditions, single pET-22a-NU-nc colony was picked from the plate and placed in 100ml LB medium (containing Aamp), and then cultured overnight at 37°C on a shaker at 180rpm. The seed culture cultivated overnight was transferred to LB medium at 2% inoculum, and cultured at 37°C and 250 rpm for 4 hours. The OD600 of the culture medium was about 0.8, and then the inducer IPTG was ...
Embodiment 1
[0042] B1. Add 5.8 g of immobilized nuclease per 100 mL of E. coli lysate, and keep stirring in a constant temperature water bath at 37°C for 4 hours;
[0043] B2. Use a magnetic stand to perform magnetic separation for 5 minutes to obtain solution II;
[0044]B3. Filter solution II, take the filter residue and wash it with ultrapure water, pour the washing water into the filtrate and mix evenly. The washed filter residue and the mixed filtrate are respectively the recovered immobilized nuclease and the clarified E. coli lysate liquid.
Embodiment 2
[0046] B1. Add 8 g of immobilized nuclease per 100 mL of E. coli lysate, and keep stirring in a constant temperature water bath at 37°C for 2 to 4 hours;
[0047] B2. Use a magnetic stand to perform magnetic separation for 5 minutes to obtain solution II;
[0048] B3. Filter solution II, take the filter residue and wash it with ultrapure water, pour the washing water into the filtrate and mix evenly. The washed filter residue and the mixed filtrate are respectively the recovered immobilized nuclease and the clarified E. coli lysate liquid.
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Abstract
The invention discloses a method for clarifying Escherichia coli lysate. The method comprises the steps of firstly preparing a silica nanoparticle coated with a layer of Fe3O4 magnetic nanoparticles by taking tetraethyl orthosilicate and the Fe3O4 magnetic nanoparticles as raw materials, carrying out graft modification on the surface and immobilizing a nuclease on the surface to obtain a special immobilized nuclease. Compared with a common magnetic beadnuclease on the market, the immobilized nuclease has the advantages that when the immobilized nuclease is used for clarifying the Escherichiacoli lysate, the hydrolysis ability is stronger; the viscosity of the hydrolyzed Escherichia coli lysate is greatly reduced after hydrolysis; the stability can be good; the storage time can be up to more than 2 years; and the activity is high and maintains at high level after repeated use.
Description
technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for clarifying Escherichia coli lysate. Background technique [0002] In the field of biotechnology, the Escherichia coli recombinant protein expression system has become the most commonly used recombinant protein expression system due to its clear genetic background, fast cellreproduction, high yield, easy fermentation and amplification, and low price. The recombinant protein expressed in the cell needs to be filtered and clarified after E. coli celllysis before it can be used for the next step of chromatographic purification. In order to be able to extract the components (DNA, protein, etc.) Various substances are released to form a cell lysate. [0003] After the biological tissue or bacteria are lysed, due to the release of a large amount of nucleic acid, the viscosity of the lysate increases rapidly. This is caused by the suspension of a large amount of nucleic acid...
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