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

A skeleton protein, Escherichia coli technology, applied in the biological field, can solve the problems of low efficiency of recombinant protein, increased difficulty of recombinant oligosaccharide analysis, increased difficulty of separation and purification of glycoprotein, etc., and achieves improved solubility and high efficiency in vitro glycosylation modification. Effect

Active Publication Date: 2020-06-02
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the efficiency of N-glycosylation modification of exogenous recombinant proteins in this system is low, generally reaching about 50-70%, which makes it difficult to separate and purify glycoproteins in the later stage, and further increases the difficulty of analyzing recombinant oligosaccharides on glycoproteins

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. In this example, the human scaffold protein fibronectin type III domain (Fn3) protein was used as the model gene (EMBL accession number AJ320527). At the 3' end of the gene, the nucleotide sequence encoding 6 histidine residues was introduced, and the nucleotide sequence encoding the glycosylation site DQNAT was introduced at the 3' end. For the structure of the fusion gene, see figure 1 . The gene was constructed into pIG6H by genetic recombination, see the vector structure figure 2 .

[0025] 2. After synthesizing the Fn3-Gly expression cassette sequence of the human fibronectin type III domain recombinant gene shown in the sequence table (Nanjing GenScript Biotechnology Co., Ltd.), use EcoR V and HindIII to construct on the E. coli expression vector pIG6H , the recombinant vector pIG6H-Fn3-Gly was obtained.

[0026] 3. The constructed expression vector and pACYCpgl were electroporated into E. coli strain CLM37, and then the transformants were inoculated into L...

Embodiment 2

[0033] 1. In this example, the human scaffold protein fibronectin type III domain (Fn3) protein was used as the model gene (EMBL accession number AJ320527). At the 3' end of the gene, the nucleotide sequence encoding 6 histidine residues was introduced, and the nucleotide sequence encoding the glycosylation site DQNAT was introduced at the 3' end. For the structure of the fusion gene, see figure 1 . The gene was constructed into pIG6H by genetic recombination, see the vector structure figure 2 .

[0034] 2. After synthesizing the Fn3-Gly expression cassette sequence of the human fibronectin type III domain recombinant gene shown in the sequence table (Nanjing GenScript Biotechnology Co., Ltd.), use EcoR V and HindIII to construct on the E. coli expression vector pIG6H , the recombinant vector pIG6H-Fn3-Gly was obtained.

[0035] 3. The constructed expression vector and pACYCpgl were electroporated into E. coli strain CLM37, and then the transformants were inoculated into L...

Embodiment 3

[0042] 1. In this example, the human scaffold protein fibronectin type III domain (Fn3) protein was used as the model gene (EMBL accession number AJ320527). At the 3' end of the gene, the nucleotide sequence encoding 6 histidine residues was introduced, and the nucleotide sequence encoding the glycosylation site DQNAT was introduced at the 3' end. For the structure of the fusion gene, see figure 1 . The gene was constructed into pIG6H by genetic recombination, see the vector structure figure 2 .

[0043] 2. After synthesizing the Fn3-Gly expression cassette sequence of the human fibronectin type III domain recombinant gene shown in the sequence table (Nanjing GenScript Biotechnology Co., Ltd.), use EcoR V and HindIII to construct on the E. coli expression vector pIG6H , the recombinant vector pIG6H-Fn3-Gly was obtained.

[0044] 3. The constructed expression vector and pACYCpgl were electroporated into E. coli strain CLM37, and then the transformants were inoculated into L...

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Abstract

The invention belongs to the field of biotechnologies, and relates to a method for building efficient N-glycosylation receptor protein models in Escherichia coli by the aid of skeleton proteins Fn3 (fibronectin type III domain). The human-derived proteins Fn3 with N-glycosylation recognition sites are used as receptor proteins. The method includes steps constructing Fn3-Gly recombinant protein gene expression vectors; forming recombinant genes of the N-glycosylation recognition sites carried by the human-derived proteins Fn3; cloning the genes onto the expression vectors capable of secreting the recombinant genes onto pericoel. Recombinant proteins expressed by the vectors can be used as the prokaryote research N-glycosylation receptor protein models for N-glycosylation recombinant research. The method has the advantages that the nearly 100% N-glycosylation recombinant proteins which are good in heat stability, high in expression quantity and convenient to separate and purify can be obtain, excellent protein receptors can be provided for N-glycosylation recombinant engineering, the receptor protein models can be provided for prokaryote N-glycosylation recombinant engineering, and foundation can be laid for efficiently carrying out oligosaccharide chain analysis and function research in late periods of the N-glycosylation recombinant engineering.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for establishing an efficient N-glycosylation receptor protein model in Escherichia coli in vivo with a human Fn3 protein carrying an N-glycosylation recognition site as a receptor protein. Background technique [0002] With the in-depth research and development of glycobiology, the application of E. coli to express N-glycosylated homogeneous glycoproteins has become one of the current research hotspots. At present, the production of uniform N-glycosylated proteins has been successfully achieved in E. coli by using the glycosylation modification system pgl (protein glycosylation) of Campylobacter jejuni. In addition, it was found that Campylobacter jejuni oligosaccharide transferase pglB and oligosaccharide flippase pglK have loose substrate recognition specificities, making it possible to customize target glycoproteins in E. coli according to requirements. However, at presen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C07K14/47C12R1/19
Inventor 胡学军丁宁马君燕杨春光孙慎侠李梦阳张嘉宁
Owner DALIAN UNIV
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