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

A kind of engineering bacteria, soluble technology, applied in the field of engineering bacteria, can solve the problems of low protein yield, serious protein loss, and complicated purification process of recombinant strains, and achieve the effect of benefiting enzyme production, ensuring enzyme activity, and simplifying the purification process

Active Publication Date: 2011-01-19
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0004] NotI is a very commonly used restriction endonuclease in routine molecular biology experiments, but in commercial production there are still low protein yields of recombinant strains, The purification process is complicated and other reasons
The previously reported NotI purification process involved a phosphocellulose anion exchange column, a heparin agarose affinity chromatography column, a DEAE-agarose column, and a four-step column elution process on a PEI column, and finally NotI was stored in its storage buffer. Dialysis 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 NotI efficient soluble expression engineering bacteria of the present invention

[0037] 1. Construction of recombinant plasmid pBR322-EagIM

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

[0039] 2. Electric conversion

[0040] After the recombinant plasmid was correctly sequenced, it was electroporated to transform competent cells ER2566 (purchased from NewEngland Biolabs, Inc.) under the conditions of 3000V, 25μF, 200Ω, and 4.6ms in a Bio-RadGenePluser electroporator, and spread on an LB plate containing 100mg / L Ampicillin. Positive clones were screened by inverting overnight culture at 37°C.

[0041] 3. Construction of recombinant plasmid pACYC184-NotI

[0042] The nucleotide sequence of the restriction endonuclease NotI (with BamHI restriction s...

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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 efficient soluble expression of NotI and a construction method thereof. The present invention also relates to the application of the engineering bacterium in the preparation of NotI enzyme, which belongs to the construction of an engineering bacterium expressing a restriction endonuclease NotI 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. NotI is a rare restriction endonuclease that recognizes an eight-base nucleotide sequence (5'-GC / GGCCGC-3'), and it can als...

Claims

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Application Information

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Patent Type & Authority Applications(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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