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Engineering bacteria of soluble expression Not I, and construction method and application thereof

An engineering bacteria and soluble technology, applied in the field of engineering bacteria, can solve the problems of low protein yield of recombinant strains, serious protein loss, complicated purification process, etc., and achieve the effects of being beneficial to enzyme yield, ensuring enzyme activity, and simplifying purification process.

Active Publication Date: 2011-11-30
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] Not I is a very commonly used restriction endonuclease in routine molecular biology experiments, but in commercial production there are still reasons such as low protein yield of recombinant strains and complicated purification process
The previously reported Not I purification process involved a phosphocellulose anion exchange column, a heparin agarose affinity chromatography column, a DEAE-agarose column, and a four-step elution process on the PEI column, and finally Not I was added to its storage buffer. Dialysis in solution overnight, the entire purification process takes about 3-4 days, the process is cumbersome and the protein loss is serious

Method used

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  • Engineering bacteria of soluble expression Not I, and construction method and application thereof
  • Engineering bacteria of soluble expression Not I, and construction method and application thereof
  • Engineering bacteria of soluble expression Not I, and construction method and application thereof

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Embodiment 1

[0036] Embodiment 1 Construction of Not I efficient soluble expression engineering bacteria of the present invention

[0037] 1. Construction of recombinant plasmid pBR322-Eag I M

[0038] The nucleotide sequence of the methylase Eag I M (respectively cited Nhe I and Sph I restriction sites at both ends) was cloned into the pBR322 vector (purchased from TaKaRa Company) to construct the recombinant plasmid pBR322-Eag I M.

[0039] 2. Electric conversion

[0040] After the recombinant plasmid was correctly sequenced, the competent cell ER2566 (purchased from NewEngland Biolabs, Inc.) was transformed by electroporation under the conditions of 3000V, 25μF, 200Ω, 4.6ms in the Bio-Rad Gene Pluser electroporator, and coated on a medium containing 100mg / L Ampicillin On the LB plate, cultivate overnight at 37°C upside down to screen for positive clones.

[0041] 3. Construction of recombinant plasmid pACYC184-Not I

[0042] The nucleotide sequence of the restriction endonuclease Not...

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Abstract

The invention discloses engineering bacteria of soluble expression Not I, and a construction method and application thereof. The engineering bacteria of the invention comprises methylase Eag I M gene, restriction enzyme Not I gene and escherichia coli (E. coli) ER2566 (mcrC-mrr). After separating and identifying the Not I gene, the invention obtains the engineering bacteria of efficient soluble expression Not I through the reconstruction of the recombinant. In the invention, the Not I restriction enzyme with high purity can be prepared through groping the inducement conditions of recombining the engineering bacteria and optimizing a protein purification process.

Description

technical field [0001] The present invention relates to an engineering bacterium, in particular to an engineering bacterium for highly soluble expression of Not I and a construction method thereof. The present invention also relates to the application of the engineering bacterium in the preparation of Not I enzyme, which belongs to the expression restriction endonuclease Not I Engineering bacteria construction field. Background technique [0002] Restriction endonuclease is an indispensable and important tool for contemporary genetic engineering research, and its purity directly affects the experimental results of gene cloning, nucleotide sequence analysis, and enzymatic property research. Since the first restriction endonuclease was proposed in the late 1960s, more and more restriction endonucleases have been discovered, purified and applied. Not I is a rare restriction enzyme that recognizes an eight-base nucleotide sequence (5'-GC / GGCCGC-3'), and it can also recognize (5...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/52C12N15/55C12N15/63C12N9/16C12R1/19
Inventor 任桂萍李德山张巧叶贤龙
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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