High efficient expression method for actinomyces-based nitrile hydratase gene in escherichia coli

A technology of Escherichia coli and nitrile hydratase, applied in the field of microbial genetic engineering, can solve the problems of slow progress of nitrile hydratase gene research and other problems

Active Publication Date: 2013-09-25
JIANGNAN UNIV
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Problems solved by technology

[0005] Because the nitrile hydratase gene derived from some actinomycetes is expressed in a system with Escherichia coli as the host, the regulatory mechanism it receives is quite different from that of wild bacteria, and the expressed protein is easily aggregated to form inclusion bodies, so actinomycetes The source of heterologous expression of the nitrile hydratase gene has been slowly studied over the past 10 years

Method used

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  • High efficient expression method for actinomyces-based nitrile hydratase gene in escherichia coli
  • High efficient expression method for actinomyces-based nitrile hydratase gene in escherichia coli
  • High efficient expression method for actinomyces-based nitrile hydratase gene in escherichia coli

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Embodiment 1

[0030] 1) Obtain the full-length gene of nitrile hydratase by PCR

[0031] Primers (BAE up, BAE down) were designed to amplify the full-length nitrile hydratase gene sequence by referring to the nitrile hydratase gene sequence in Rhodococcus.

[0032] The upstream primer BAE up (containing the Nde I restriction site) is shown in SEQ ID NO.2, and the downstream primer BAE down (containing the HindIII restriction site) is shown in SEQ ID NO.3;

[0033] 2) Design a pair of primers with Escherichia coli SD sequence and spacer sequence

[0034] The complete sequence of the natural nitrile hydratase in Rhodococcus expressed in Escherichia coli BL21 has been reported internationally, but none of them are ideal. By replacing the upstream spacer sequence of the nitrile hydratase α gene and the regulatory protein e gene with SD sequence (as shown in SEQ ID 6) and a spacer sequence (as shown in SEQ ID 7), a large amount of soluble nitrile hydratase can be successfully expressed.

[003...

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Abstract

The invention discloses a high efficient expression method for an actinomyces-based nitrile hydratase gene in escherichia coli, and belongs to the field of microbial genetic engineering technology. The method processes high efficiency and safety. It is benefit for large scaled nitrile hydratase extraction and purification, and further theoretical research about nitrile hydratase that a large amount of soluble nitrile hydratase can be obtained in a short expression period by applications of the method.

Description

Technical field: [0001] A method for efficiently expressing nitrile hydratase gene derived from actinomycetes in Escherichia coli, the invention relates to a method for successfully expressing nitrile hydratase in Escherichia coli BL21 (DE3), belonging to the field of microbial genetic engineering. Background technique: [0002] Nitrile hydratase (NHase for short, EC 4.2.1.84) is a metalloenzyme capable of hydrating nitriles into more valuable amides. NHase is widely distributed in microorganisms, and has been found in Rhodococcus, Pseudomonas, Pseudonocardia, Bacillus, Nocardia ( Nocardia), Comamonas (Comamonas) in the middle can get the gene sequence of nitrile hydratase. [0003] Researchers from various countries obtain the nitrile hydratase gene in basically the same way. Generally, they extract the genomic DNA of wild bacteria, and then determine the gene sequence of nitrile hydratase in the strain by consulting various gene libraries, design primers according to the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/88C12R1/01
Inventor 周哲敏余越春崔文璟周丽何琛辉
Owner JIANGNAN UNIV
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