Method for efficiently expressing L-asparaginase II by 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: 2013-12-11
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 cultu

Method used

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  • Method for efficiently expressing L-asparaginase II by recombinant escherichia coli
  • Method for efficiently expressing L-asparaginase II by recombinant escherichia coli

Examples

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Embodiment 1

[0037] Recombinant Escherichia coli for efficient expression of L-asparaginase Ⅱ, see figure 1 As shown, including the following steps:

[0038] S11. Obtain the recombinant E. 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 E. coli JM109 as a template, mix the above primers and amplification template 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 vector for transformation, and simultaneously digest the amplified gene fragment and plasmid pET-32a (+) with endonucleases BamHI and Sac I, and then use T4 ligase to The amplified gene fragments digested with the same double enzymes were ligated with plasmid pET-32a(+) to obtain recombinant plasmid pET32a(+)-LA, and th...

Embodiment 2

[0051] Recombinant Escherichia coli for efficient expression of L-asparaginase Ⅱ, see figure 1 As shown, including the following steps:

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

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

[0054] S23. Fermentation culture: The engineered bacteria obtained after culturing the seed liquid obtained in step S12 overnight is used as the fermentation liquid, and 7% of the total inoculation amount is connected to medium 2 and put into the fermentation tank for stepwise fermentation culture:

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

[0056] (2) When the culture reaches 5h, set the culture temperature to 32℃, the conditions for lactose induction are the ...

Embodiment 3

[0061] Recombinant Escherichia coli for efficient expression of L-asparaginase Ⅱ, see figure 1 As shown, including the following steps:

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

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

[0064] S33: Fermentation culture: The engineered bacteria obtained after culturing the seed liquid obtained in step S12 overnight is used as the fermentation liquid, and 7% of the total inoculation amount is connected to medium 2 and put into the fermentor for stepwise fermentation culture:

[0065] (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;

[0066] (2) When cultured to 5h, keep the culture temperature at 37℃, the conditions of lactose induction are the same as S13(2)...

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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 specifically relates to a method for efficiently expressing L-asparaginase II in recombinant Escherichia coli. Background technique [0002] L-asparaginase (E.C.3.5.1.1) is a kind of enzyme that can catalyze the hydrolysis of L-asparagine into L-aspartic acid and ammonia, and it can also catalyze the similar reaction of L-glutamate amine. Asparaginase produced by Escherichia coli is divided into type I and type II. Asparaginase type I exists in the cytoplasm and has no anti-cancer activity; asparaginase type II is secreted into the periplasm of the cell and has anti-cancer activity. [0003] The relative molecular weight of L-asparaginase II is 140KD, which is an important protein anti-tumor drug. The mechanism of action of L-asparaginase II is to reduce the concentration of L-asparagine and L-glutamine in the human body, both of which are important components for the synthesis of purine and pyrimidine...

Claims

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Application Information

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IPC IPC(8): C12N9/82C12N15/55C12N15/70C12N1/21C12R1/19
Inventor 徐东颜林春
Owner 徐东
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