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A method for establishing a receptor protein model for n-glycosylation efficiency detection in Escherichia coli using the skeleton protein fn3

A technology of Escherichia coli and skeleton protein, applied in the direction of microorganism-based methods, chemical instruments and methods, biochemical equipment and methods, etc., can solve problems such as poor flexibility, achieve improved physical and chemical properties, high-efficiency and low-cost large-scale production, elucidation The effect of structure-activity relationship

Active Publication Date: 2020-05-22
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While BCloop is shorter and less flexible than FGloop

Method used

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  • A method for establishing a receptor protein model for n-glycosylation efficiency detection in Escherichia coli using the skeleton protein fn3
  • A method for establishing a receptor protein model for n-glycosylation efficiency detection in Escherichia coli using the skeleton protein fn3
  • A method for establishing a receptor protein model for n-glycosylation efficiency detection in Escherichia coli using the skeleton protein fn3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. In this example, human fibronectin type III domain (Fn3) protein was used as a model gene (EMBL accession number AJ320527). At the 5′ end of the gene, 6 histidine residues are introduced, and at the FGloop region, a base sequence encoding the glycosylation site DQNAT is introduced. See the structure of the fusion gene. figure 1 . The gene was constructed into pIG6H by gene recombination, see the vector structure figure 2 .

[0019] 2. After the human fibronectin type III domain mutant Fn3-Gly-loop gene expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), EcoRV and HindIII were used to construct an E. coli expression vector On pIG6H, the recombinant vector pIG6H-Fn3-Gly-loop was obtained.

[0020] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (per liter of culture) cont...

Embodiment 2

[0024] 1. In this example, human fibronectin type III domain (Fn3) protein was used as a model gene (EMBL accession number AJ320527). Introduce 6 histidine residues encoding at the 5′ end of the gene, and introduce the nucleotide sequence encoding the glycosylation site DQNAT in the FG loop region. The structure of the fusion gene is shown in figure 1 . The gene was constructed into pIG6H by gene recombination, see the vector structure figure 2 .

[0025] 2. After the human fibronectin type III domain mutant Fn3-Gly-loop gene expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), EcoRV and HindIII were used to construct an E. coli expression vector On pIG6H, the recombinant vector pIG6H-Fn3-Gly-loop was obtained.

[0026] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (per liter of...

Embodiment 3

[0030] 1. In this example, human fibronectin type III domain (Fn3) protein was used as a model gene (EMBL accession number AJ320527). At the 5′ end of the gene, 6 histidine residues are introduced, and at the FGloop region, a base sequence encoding the glycosylation site DQNAT is introduced. See the structure of the fusion gene. figure 1 . The gene was constructed into pIG6H by gene recombination, see the vector structure figure 2 .

[0031] 2. After the human fibronectin type III domain mutant Fn3-Gly-loop gene expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), EcoRV and HindIII were used to construct an E. coli expression vector On pIG6H, the recombinant vector pIG6H-Fn3-Gly-loop was obtained.

[0032] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (per liter of culture) cont...

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Abstract

The invention belongs to the field of biotechnologies, and relates to a method for applying recombinant expression separated and purified human-derived protein Fn3 (fibronectin type III domain) mutants as N-glycosylation efficiency detection model proteins. The method includes steps of constructing Fn3-Gly-loop recombinant protein gene expression vectors of the Fn3 mutants; jointly transforming the constructed expression vectors into Escherichia coli engineering strains CLM37 by means of electric shock processes; screening the expression vectors by the aid of antibiotics to obtain positive clones. Recombinant proteins contain Fn3-Gly-loop proteins which are about to be modified by recombinant glycosyl, and the Fn3-Gly-loop fusion protein glycosylation efficiency can be detected by the aid of Western Blot processes. The method has the advantages that the skeleton proteins Fn3 in Escherichia coli are used as receptor proteins, accordingly, the model receptor proteins suitable for N-glycosylation modification efficiency research can be constructed, and the recombinant protein glycosylation efficiency can be easily, quickly and efficiently detected in the Escherichia coli.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an application method of recombinantly expressing, separating and purifying human Fn3 protein mutant as a model protein for N-glycosylation efficiency detection. Background technique [0002] N-glycosylation modification of proteins is an important post-processing process of proteins in organisms. N-glycosylation modification of protein is an important method to improve the physicochemical properties of drug protein, improve pharmacokinetic properties, and improve drug efficacy. The E. coli expression system itself cannot perform N-glycosylation modification on foreign proteins. In 2002, Wacker of the Swiss Federal Institute of Technology (Professor Markus Aebi's research group) successfully introduced the N-glycosylation mechanism of Campylobacter jejuni into E. coli for the first time, and realized N-glycosylation modification of foreign proteins in E. coli, marking the "prokaryote ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C07K19/00C12R1/19
Inventor 胡学军丁宁马君燕孙慎侠杨春光李梦阳张嘉宁
Owner DALIAN UNIV
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