Expression method of AEP cyclase in pichia pastoris and application thereof

A Pichia pastoris, expression method technology, applied in the field of genetic engineering, can solve the problem that the expression amount cannot meet industrial production and the like, and achieve the effects of improving stability and prolonging half-life

Active Publication Date: 2019-10-29
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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  • Expression method of AEP cyclase in pichia pastoris and application thereof
  • Expression method of AEP cyclase in pichia pastoris and application thereof
  • Expression method of AEP cyclase in pichia pastoris and application thereof

Examples

Experimental program
Comparison scheme
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 PCR amplification of AEP fragments, the total PCR system is 100 μL, including 10 μL of 10×pfu Buffer, 4 μL of dNTPs (10 mmol / L), 4 μL of forward primer and reverse primer, 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 instrum...

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 particularly relates to an expression method of AEP cyclase in pichia pastoris and application thereof. The AEP cyclase is expressed in pichia pastoris, the gene tandem copy number of the AEP cyclase reaches up to 5 copies by a gene tandem way, and the expression amount of the AEP cyclase is highest up to 0.89+ / -0.03 mg / mL when the gene copy number reaches 3. The invention verifies the cyclization capacity of the AEP cyclase in a novel mode, constructs a tandem substrate, selects a protein as the substrate which can be better observed in SDS-PAGE and has the cyclization possibility, constructs a tandem substrate containing the substrate sequence of the TEV protease; and identifies whether the AEP cyclase is cyclized or not according to the size change of the substrate after the protease is cut or the number of bands. The method is simple and convenient to operate and strong in practicability.

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 Applications(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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