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Method for high-efficiency expression of l-asparaginase Ⅱ in recombinant Escherichia coli

A technology of recombinant Escherichia coli and asparaginase, which is applied in the biological field and can solve the problem of low enzyme activity

Active Publication Date: 2015-10-28
徐东
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in the prior art, there are very few studies on extracellular expression and fed-batch culture in the fermentation process of Escherichia coli, and the existing enzyme activity rate is not high

Method used

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  • Method for high-efficiency expression of l-asparaginase Ⅱ in recombinant Escherichia coli
  • Method for high-efficiency expression of l-asparaginase Ⅱ in recombinant Escherichia coli

Examples

Experimental program
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Effect test

Embodiment 1

[0037] For the method for highly expressing L-asparaginase II in recombinant Escherichia coli, see figure 1 shown, including the following steps:

[0038] S11. Obtaining recombinant Escherichia coli expressing L-asparaginase II:

[0039] (1) Amplify gene fragments: design and synthesize primers, where the upstream primer is 5'TGC GGATCC CAT TAC CCA ATA TCA-3', downstream primer is 5'GAG CTCGAG GTA CTG ATT GAA CT3', extract DNA from Escherichia coli JM109 as a template, mix the above primers and amplification templates into a set of reaction tubes, perform PCR amplification, and obtain amplified gene fragments;

[0040] (2) Construction of recombinant plasmid: select plasmid pET-32a (+) as the transformation vector, use endonucleases BamHI and SacⅠ to digest the amplified gene fragment and plasmid pET-32a (+) at the same time, and then use T4 ligase to combine The same double-digested amplified gene fragment was connected with the plasmid pET-32a(+) to obtain the recombin...

Embodiment 2

[0051]For the method for highly expressing L-asparaginase II in recombinant Escherichia coli, see figure 1 shown, including the following steps:

[0052] S21. Obtain recombinant Escherichia coli expressing L-asparaginase II, the method is the same as that of S11 in Example 1, and will not be repeated here.

[0053] S22. Cultivate the seed liquid, the method is the same as that of S12 in Example 1, and will not be repeated here.

[0054] S23, fermentation culture: the engineered bacterium obtained after cultivating the seed liquid obtained in step S12 overnight is used as a fermentation liquid, and 7% of the total inoculum is inserted into the medium two, and put into a fermenter for step-by-step fermentation culture:

[0055] (1) The initial parameter setting of the fermenter reaction is the same as that of S13(1) in Example 1, and will not be repeated here;

[0056] (2) After culturing for 5 hours, set the culture temperature to 32°C, and the conditions for lactose inductio...

Embodiment 3

[0061] For the method for highly expressing L-asparaginase II in recombinant Escherichia coli, see figure 1 shown, including the following steps:

[0062] S31. Obtain recombinant Escherichia coli expressing L-asparaginase II, the method is the same as that of S11 in Example 1, and will not be repeated here.

[0063] S32. Cultivate the seed solution, the method is the same as that of S12 in Example 1, and will not be repeated here.

[0064] S33, fermentation culture: the engineered bacterium obtained after cultivating the seed liquid obtained in step S12 overnight is used as a fermentation liquid, and 7% of the total inoculum is inserted into medium two, and put into a fermenter for step-by-step fermentation:

[0065] (1) The initial parameters of the fermenter reaction are set to be the same as those in S13(1) of Example 1, and will not be repeated here;

[0066] (2) When culturing for 5 hours, keep the culturing temperature at 37°C, and the conditions for lactose induction ...

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Abstract

The invention provides a method for efficiently expressing L-asparaginase II by recombinant escherichia coli, and an L-asparaginase II. The method for efficiently expressing L-asparaginase II by the recombinant escherichia coli comprises the steps of acquiring the recombinant escherichi for expressing L-asparaginase II; culturing a seed solution; carrying out fermenting cultivation; and acquiring the L-asparaginase II, wherein the fermenting cultivation is carried out by step by step. The method for efficiently expressing L-asparaginase II by the recombinant escherichia coli is simple in process and convenient for operations, has high output and reduces production cost.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for expressing L-asparaginase II efficiently in recombinant Escherichia coli. Background technique [0002] L-asparaginase (E.C.3.5.1.1) is a enzyme that can catalyze the hydrolysis of L-asparagine into L-aspartic acid and ammonia, and can also catalyze the similar reaction of L-glutamate. The asparaginase produced by Escherichia coli is divided into type Ⅰ and type Ⅱ. Asparaginase type Ⅰ exists in the cytoplasm and has no anticancer activity; asparaginase type Ⅱ is secreted into the periplasm and has anticancer activity. [0003] The relative molecular weight of L-asparaginase Ⅱ is 140KD, and it is an important protein antitumor drug. The mechanism of action of L-asparaginase II is to reduce the concentration of L-asparagine and L-glutamine in the human body. These two amino acids are important components for the synthesis of purine and pyrimidine rings. Tumor...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/82C12N15/55C12N15/70C12N1/21C12R1/19
Inventor 徐东颜林春
Owner 徐东
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