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
CN110331157BActive Publication Date: 2021-06-08湖北凯利特生物科技有限公司
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
湖北凯利特生物科技有限公司
Publication Date
2021-06-08

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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.
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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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