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A kind of expression method and application of aep cyclase in Pichia pastoris

An expression method, the technology of Pichia pastoris, applied in the field of genetic engineering, can solve the problem that the expression level cannot meet the requirements of 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

AI Technical Summary

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 kind of expression method and application of aep cyclase in Pichia pastoris
  • A kind of expression method and application of aep cyclase in Pichia pastoris
  • A kind of expression method and application of aep cyclase in Pichia pastoris

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Expression of AEP cyclase in Pichia pastoris

[0039] S1: Construction of pBDM-AEP vector

[0040] Construction principle see figure 1 .

[0041] First, the amino acid sequence of AEP was obtained from the literature, and the sequence was handed over to Wuhan Jinkairui Bioengineering Co., Ltd. for gene synthesis. Using the plasmid containing the AEP sequence provided by Wuhan Jinkairui Bioengineering Co., Ltd. as a template, BDM-AEP-F: GTCAGCACGTGATGGTGATTATCTTCATTTACCG, see SEQ ID NO: 1 and BDM-AEP-R: GGCCATTAAGGAATAGAAGCGCATGCCTGAGAGG, see SEQ ID NO: 2, for Forward and reverse primers for AEP fragment PCR amplification, the total PCR system is 100μL, including 10×pfu Buffer 10μL, dNTPs (10mmol / L) 4μL, forward primer and reverse primer 4μL each, plasmid as template: 20-30ng, and finally add pfu DNA polymerase 4μL, and finally add water to make up to 100μL. After configuring the components required for the PCR reaction, preheat the PCR instrument at 95°C f...

Embodiment 2

[0066] Example 2 Identification of cyclization ability of AEP cyclase

[0067] Step 1: Obtain the cyclization substrate of AEP cyclase

[0068] 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:5. Construct tandem substrates by overlap extension PCR, the forward primer is

[0069] SUMO-TEV-F: aactttaagaaggagatataccatgggcctgcaactgaaaggtt...

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Abstract

The invention belongs to the technical field of genetic engineering, and in particular relates to an expression method and application of AEP cyclase in Pichia pastoris. In the present invention, by expressing in Pichia pastoris, the number of copies of the AEP cyclase gene in series can reach up to 5 copies through the method of gene tandem, and when the gene copy number reaches 3, the expression of AEP cyclase is the highest, reaching 0.89± 0.03mg / mL. 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 an expression method and application of AEP cyclase in Pichia pastoris. 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 butelase1 from butterfly pea can effectively cyclize the macrocyclic oligopeptide Kal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/50C12N15/81C12N15/66
CPCC12N9/63C12N15/66C12N15/815C12N2800/22C12Y304/22034
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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