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Method for constructing and producing restriction endonuclease Ecop15I

A restriction endonuclease and enzyme linking technology, applied in the field of bioengineering, can solve the problems of poor expression effect and affect the biological activity of protein complexes, and achieve the effect of good application value

Active Publication Date: 2009-09-23
BIOMICS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If the two segments of genes are simply fused to express a fusion protein with a higher molecular weight, the expression effect is not good, and the fusion of the two segments of the protein monomer may cause a change in the folding structure and affect the biological activity of the protein complex.

Method used

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  • Method for constructing and producing restriction endonuclease Ecop15I
  • Method for constructing and producing restriction endonuclease Ecop15I
  • Method for constructing and producing restriction endonuclease Ecop15I

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, material and reagent

[0048] 1. Materials:

[0049] Plasmids and cell lines pUC19 and pUC57 are products of Biotech (Nantong) Co., Ltd. (Nantong Biotech). Root Biotechnology Ltd., based on Molecular Cloning CaCl 2 Self-made competent state, guarantee titer above 106 (CFU / ug pUC19).

[0050] 2. Reagents:

[0051] (1) Tool enzymes: Taq DNA polymerase, Pfu DNA polymerase (Nantong Biolabs); restriction enzymes, SmaI, BamHI, etc. (New England Biolabs); T4 DNA ligase (New England Biolabs);

[0052] (2) Biochemical reagents: Yeast, Typtone, Tris base, glycine, SDS, agarose, etc. (Shanghai Sangong);

[0053] (3) DNA purification kit: common type plasmid recovery kit, agarose gel DNA recovery kit (Nantong Biotech);

[0054] (4) Chromatography packing: Ni sepharose affinity packing; DEAE sepharose anion exchange packing; CM sepharose anion exchange packing (Beijing Webster Bohui).

Embodiment 2

[0055] Embodiment 2, experimental method

[0056] 1. Artificial synthesis of genes

[0057] The Ecop15I gene sequence (Genbank No. AJ634453) was obtained from the NCBI website, and the fusion expression sequence was designed. Considering the convenience of downstream enzyme protein purification requirements, 6×His Tag was fused to the C-terminus of the Res subunit and the N-terminus of the Mod subunit of Ecop15I, respectively.

[0058] Dozens of pairs of primers were designed according to the principle of overlapping oligonucleotides. KpnI, XhoI, BamHI, and SalI restriction sites were added to the head and tail of the Res and Mod fragments of the primers at both ends, and a G was added after the BamHI restriction site. bases to ensure that the enzyme sequence is in the correct reading frame during translation. For the nearly 3kb Res gene fragment, the Res fragment is divided into two segments through the only BglII restriction site in the middle, and then digested with KpnI ...

Embodiment 3

[0069] Embodiment 3, experimental result

[0070] (1) Construction results of pACYC_P15 co-expression plasmid

[0071] The Res subunit and the Mod subunit of Ecop15I synthesized 3 genes by artificial PCR, respectively Eco_Mod, Eco_ResI and Eco_ResII (see attached Figure 3 Show). The three genes were respectively blunt-ended cloned and loaded into pUC57 to obtain three plasmids, pMod, pRes_I and pRes_II; pRes_I and pRes_II were digested with BglII and NotI, and ligated with T4 ligase to obtain the complete Res fragment plasmid.

[0072] The pMod plasmid was digested with the pACYCDuet-1 plasmid BamHI and SalI and then enzyme-ligated to obtain the pACYC_Mod plasmid containing the Mod subunit, and then combined with the pRes_All plasmid KpnI and XhoI to obtain the two complete Ecop15I subunits pACYC_P15 plasmid (see attached Figure 4 Show).

[0073] (2) Expression and purification of Ecop15I

[0074] Using the induction time gradient, IPTG induction concentration gradient,...

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Abstract

The invention provides a method for constructing and producing recombination III-type restriction endonuclease Ecop15I. The III-type restriction endonuclease Ecop15I plays an important role in serial analysis of gene expression, and the prior method is difficult to realize large-scale expression and purification. By utilizing a gene recombination method, two subunit fragments of the Ecop15I fused with a purification tag are artificially synthesized and are cloned into two independent expression units of a prokaryotic co-expression vector pACYCDuet-1 respectively; under IPTG induction, the expression units with independent T7 promoters, ribosome binding sites, start codon and stop codon simultaneously and independently express two subunits of enzyme in Escherichia coli, and are folded into a composite structure with biological activity; purification is performed through Ni affinity chromatography and DEAE ion-exchange chromatography; and the recombination-type Ecop15I restriction endonuclease with high yield and good activity and stability is finally obtained. Therefore, the method provides an effective way for mass production.

Description

technical field [0001] The invention relates to the construction, expression and purification of recombinant type III restriction Ecop15I endonuclease co-expression plasmid, belonging to the field of bioengineering. Through the method provided by the invention, the recombinant Ecop15I restriction endonuclease with higher purity and better activity is obtained, which provides an effective way for large-scale production. At the same time, the Ecop15I endonuclease can be used as a tool for gene expression sequence analysis and related gene identification, and has good application value. Background technique [0002] "Restriction endonuclease" is generally considered to be a general term for endonucleases that can recognize a specific sequence of 4 to 8 nucleotides on double-stranded DNA and can cut the sequence. At present, more than 2,900 restriction-modification (R / M) systems have been reported to be identified. According to their subunit composition, catalytic mechanism, cl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/55C12N9/16C12N1/21C12R1/19
Inventor 陆毅祥陈莉黄兵孙云成戴雪朱远源
Owner BIOMICS BIOTECH
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