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Escherichia coli genetic engineering strain for exocytosis expression of Ulp1 protease and application of escherichia coli genetic engineering strain

A technology of genetically engineered strains and Escherichia coli, applied in the field of protein expression, can solve the problems of slow growth rate, low secretion and expression yield, and expensive medium, and achieve the effects of low cost, high cutting activity and high cutting efficiency.

Pending Publication Date: 2022-05-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Ulp1 has been reported to be secreted and expressed in Bacillus subtilis and Pichia pastoris, there are still many shortcomings such as low yield of secreted expression, slow growth rate and expensive medium.

Method used

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  • Escherichia coli genetic engineering strain for exocytosis expression of Ulp1 protease and application of escherichia coli genetic engineering strain
  • Escherichia coli genetic engineering strain for exocytosis expression of Ulp1 protease and application of escherichia coli genetic engineering strain
  • Escherichia coli genetic engineering strain for exocytosis expression of Ulp1 protease and application of escherichia coli genetic engineering strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Construction of the Escherichia coli BL21 (DE3) bacterial strain of embodiment 1 pal gene deletion

[0053] The gene knockout of Escherichia coli BL21(DE3) uses the Red homologous recombination technology, and the plasmids used include pKD46, pKD13 and pCP20.

[0054] 1. Construction of Escherichia coli BL21(DE3)△pal:kan strain

[0055] (1) According to the upstream and downstream sequences of the pal gene in the Escherichia coli BL21 (DE3) genome published by NCBI, two pairs of primers were designed using pKD13 as a template: 5′pal-FRT-1 / 3′pal-FRT-1 and 5′pal- FRT-2 / 3'pal-FRT-2.

[0056] (2) Use pKD13 as a template, 5'pal-FRT-1 and 3'pal-FRT-1 as primers for the first round of PCR; use the recovered product of the first round of PCR as a template, 5'pal-FRT-2 and 3'pal-FRT-2 and 3' 'pal-FRT-2 is used as a primer for the second round of PCR. Through these two rounds of PCR reactions, a 70bp homology arm sequence at both ends and a PCR product containing a kanamycin re...

Embodiment 2

[0073] Example 2 Escherichia coli BL21 (DE3) △ pal strain extracellular secreted method for expressing Ulp1 protease

[0074] 1. Construction of extracellular secreted expression Ulp1 vector and strain

[0075] According to the nucleotide sequence of the 403-621 amino acid region of the Ulp1 gene published by NCBI, the nucleotide sequence encoding the SX signal peptide, the nucleotide sequence encoding the Linker whose amino acid sequence is RGSGG, and 6 histidine residues were introduced into its N-terminus. Base sequence, fusion gene structure such as Figure 4 As shown, the size of the fusion gene is 747bp, and the size of the fusion protein is 28.7kDa. The fusion gene was cloned into the downstream of the RBS of pET22b with NdeI and XhoI, named pET22b-SX-6His-Linker-Ulp1, see the vector structure Figure 5 . The expression vector pET22b-SX-6His-Linker-Ulp1 was transformed into Escherichia coli BL21(DE3)△pal strain to obtain the Escherichia coli strain expressing Ulp1 by...

Embodiment 3

[0080] Example 3 Detection of the expression of Ulp1 protease secreted extracellularly

[0081] Whether the Ulp1 protease expressed extracellularly in Example 2 is expressed or not is determined by SDS-PAGE. The extracellular secreted Ulp1 protease contains a 6×His tag, and Ni NTAbeads can be added to the expression supernatant for specific enrichment and concentration, which is helpful for protein detection. Specific steps are as follows:

[0082] (1) Take 4ml of the bacterial liquid after induced expression and centrifuge at 13000rpm for 10min at 4°C.

[0083] (2) Collect the supernatant after centrifugation, filter it with a 0.45 μm filter membrane, add 20 μl Ni NTAbeads, and rotate at 4° C. for 1 h.

[0084] (3) After the supernatant is fully combined with the beads, centrifuge at 3000 rpm for 5 min at 4°C and discard the supernatant. Add 100 μl of protein loading buffer to the remaining beads, cook at 95°C for 5 minutes.

[0085] (4) SDS-PAGE electrophoresis was perfo...

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Abstract

The invention relates to the technical field of protein expression, in particular to an escherichia coli genetic engineering strain for exocytosis expression of Ulp1 protease and application of the escherichia coli genetic engineering strain. According to the fusion protein of the Ulp1 protease and the carrier and the escherichia coli engineering strain for expressing the Ulp1 protease, the carrier and the strain can achieve efficient extracellular secretory expression of the Ulp1 protease, the Ulp1 protease can be subjected to secretory expression in a large amount within a short time, and efficient SUMO label cutting activity can be achieved without purification. The preparation method of the Ulp1 protease, provided by the invention, has the advantages of simple preparation process, rapidness, high efficiency, low cost and the like, and has a relatively good application value.

Description

technical field [0001] The invention relates to the technical field of protein expression, in particular to an Escherichia coli genetically engineered strain expressing Ulp1 protease by extracellular secretion and application thereof. Background technique [0002] Small ubiquitin-like modifier (small ubiquitin-like modifier, SUMO) is a member of the ubiquitin-like protein family, it has the function of binding lysine side chain to modify the target protein (Sumoylation, SUMOylation). SUMOylation is a reversible post-translational modification process that plays an extremely important role in eukaryotic cells, such as transcription, DNA recombination, DNA repair, protein transport, and cell cycle regulation. SUMO is currently developed as a fusion tag to promote the soluble expression of recombinant proteins, and is used to express a variety of active proteins, polypeptides, cytokines and antimicrobial peptides. SUMO tags can promote the folding of recombinant proteins, enha...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/60C12N15/62C12N15/70C12N1/21C12R1/19
CPCC12N9/60C12N15/70C12Y304/22068C07K2319/02C07K2319/21
Inventor 董娜付灵龙吴迪
Owner CHINA AGRI UNIV
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