A fusion expression method of aep cyclase in Escherichia coli, a method for identifying the cyclization ability of aep cyclase and its application

A technology of fusion expression and Escherichia coli, applied in the field of genetic engineering, can solve the problem that the expression level cannot meet industrial production

Active Publication Date: 2021-06-08
湖北凯利特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Studies have found that AEP cyclase can be expressed recombinantly in Escherichia coli, and its expression level is 1.8 mg / L, which still cannot meet the requirements of industrial production

Method used

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  • A fusion expression method of aep cyclase in Escherichia coli, a method for identifying the cyclization ability of aep cyclase and its application
  • A fusion expression method of aep cyclase in Escherichia coli, a method for identifying the cyclization ability of aep cyclase and its application
  • A fusion expression method of aep cyclase in Escherichia coli, a method for identifying the cyclization ability of aep cyclase and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1 Fusion expression of AEP cyclase in Escherichia coli

[0034] In this example, five fusion tags of MBP, NusA, SUMO, GFP, and Ubiquitin were used to fuse and express AEP, and vectors containing corresponding fusion tags were constructed, which were pET28a-GFP-AEP-6*His, pET28a-MBP-AEP- 6*His, pET28a-SUMO-AEP-6*His, pET28a-NusA-AEP-6*His, and pET28a-Ubiquitin-AEP-6*His. The specific experimental plan is as follows:

[0035] S1: Link the AEP sequence containing the protease polypeptide substrate recognition sequence with the fusion tag to construct a recombinant DNA sequence.

[0036] Use the AEP fragment synthesized by the gene as a template for PCR amplification. The sequence of the AEP fragment is shown in SEQ ID NO.16. The total PCR system is 100 μL, including 10 μL of 10×pfu Buffer, 4 μL of dNTPs (10 mmol / L), forward primer and reverse 4 μL of each primer, plasmid as template: 20-30ng, finally add 4 μL of pfu DNA polymerase, and finally add water to mak...

Embodiment 2

[0078] Example 2 Identification of cyclization ability of AEP cyclase

[0079] Step 1: Obtain the cyclization substrate of AEP cyclase

[0080] Since the SUMO protein is 11.0kDa in size, it can be well observed in SDS-PAGE and has the possibility of cyclization, so it is used as the cyclization substrate of AEP cyclase. Add GGGGSGGGGS to the N-terminal of the SUMO protease substrate, and add a longer Linker1 (GSGS), TEV protease substrate sequence (ENLYFQ / S), and a longer Linker2 (DVGGGGSEGGGSGGPGSGGEGSAGGGSAGGGS) to the C-terminal of the SUMO protease substrate in sequence and 6*His(HHHHHH), and finally add the recognition sequence of AEP cyclase at both ends to form a tandem substrate (GLP-SUMO protease substrate-Linker1-TEV protease substrate-Linker2-STRN / GLP-6*His ), the recognition sequence of AEP cyclase is shown in SEQ ID NO:13. Tandem substrates were constructed by overlap extension PCR, and the primer sequences were as follows:

[0081] SUMO-TEV-F: aactttaagaaggaga...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to a fusion expression method of AEP cyclase in Escherichia coli, a method for identifying the cyclization ability of AEP cyclase and an application thereof. The present invention uses different fusion tags for fusion expression in Escherichia coli, and then removes the fusion tag to obtain AEP cyclase with a higher expression level, and its expression level is 0.36±0.05mg / mL, which is higher than that reported in the previous literature 1.8mg / L. The present invention adopts a new method to verify the cyclization ability of AEP cyclase. By constructing a tandem substrate, a protein that can be better observed in SDS-PAGE and has the possibility of cyclization is selected as the substrate , and construct a tandem substrate containing the substrate sequence of TEV protease, and identify whether it is cyclized according to the change in the size of the substrate after protease cleavage or the number of bands, the operation is simple, convenient, and practical.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a fusion expression method of AEP cyclase in Escherichia coli, a method for identifying the cyclization ability of AEP cyclase and an application thereof. Background technique [0002] Endoasparaginases (AEPs) are a class of key enzymes that can catalyze short peptides to form cyclized peptides. They are widely distributed in plants. The macrocyclic oligopeptides they catalyze form have a cyclic backbone and a typical cysteine Amino acid knot (six conserved cysteine ​​residues form three disulfide bonds), the structure is exceptionally stable, making macrocyclic oligopeptides have a variety of biological activities and thus have great application potential as a framework for drug design. However, it is difficult to extract natural AEP cyclase from plant tissue, and the yield of the enzyme is low. In 2014, Nguyen GK found that the asparagine endonuclease bu...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N9/50C12Q1/37
CPCC12N9/63C12N15/70C12N2800/22C12Q1/37C12Y304/22034
Inventor 彭文舫胡晓韵易犁范贤马立新
Owner 湖北凯利特生物科技有限公司
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