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Method for cloning and expressing nitrilre hydratase regulatory protein

A technology for nitrile hydratase and regulatory proteins, which is applied in the field of expressing nitrile hydratase regulatory proteins, can solve problems such as incorrect reporting and difficult detection of regulatory proteins

Inactive Publication Date: 2012-06-13
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The reason is that the ORF part of the nitrile hydratase regulatory gene sequence in Pseudomonas putida is currently reported incorrectly, and even if the ORF of the regulatory protein is correctly identified, the regulatory protein is difficult to detect on SDS-PAGE

Method used

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  • Method for cloning and expressing nitrilre hydratase regulatory protein
  • Method for cloning and expressing nitrilre hydratase regulatory protein
  • Method for cloning and expressing nitrilre hydratase regulatory protein

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Embodiment 1

[0035] 1) Obtain the correct regulatory protein gene sequence and identify the correct ORF

[0036] The primers were designed to correctly amplify the mature enzyme gene sequence and the regulatory protein gene sequence by referring to the nitrile hydratase gene sequence in Pseudomonas putida. Because the ORF part of the nitrile hydratase regulatory gene sequence in Pseudomonas putida is currently reported incorrectly (the regulatory protein gene sequence has 3 possible ORFs), and the regulatory protein is difficult to detect on SDS-PAGE, so the correct Identifying the ORFs of regulatory proteins adds to the difficulty. By trying various ORFs and adding His-tag protection at the N-terminal of the regulatory protein, the ORF that successfully expresses the active regulatory protein is finally determined to be the correct regulatory protein ORF;

[0037] 2) Add His-tag to the N-terminus of the regulatory protein gene sequence

[0038] Escherichia coli BL21 expresses the comple...

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Abstract

The method discloses a method for cloning and expressing nitrilre hydratase regulatory protein, which is characterized in that His-tag is added in the N end of a gene sequence of the nitrilre hydratase regulatory protein and is serially connected with a nitrilre hydratase maturase gene, and the His-tag and the nitrilre hydratase maturase gene are co-expressed in escherichia coli Bl21. The invention belongs to the technical field of gene cloning. The method is simple in operation, and has higher efficiency and success rate.

Description

Technical field: [0001] A method for cloning and expressing a nitrile hydratase regulatory protein, in particular to a method for expressing a nitrile hydratase regulatory protein in Escherichia coli BL21. Background technique: [0002] Nitrile hydratase (NHase for short, EC 4.2.1.84) is a metalloenzyme that catalyzes the transformation of nitrile compounds into amido compounds. The acrylamide produced by this enzyme is nearly one million tons, accounting for one-third of the total acrylamide output. Compared with traditional chemical methods, biotechnology has the advantages of low cost, less energy consumption and less pollution. At present, in developed countries such as the United States, Japan, and France, this biotechnology is replacing traditional chemical methods. In my country, although the biotechnology of nitrile hydratase to synthesize acrylamide started late, it developed rapidly. According to statistics in 2008, acrylamide produced by biological methods in my...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/70C12R1/19C12R1/40
Inventor 周哲敏刘义崔文璟
Owner JIANGNAN UNIV
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