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Method for building N-glycosylation receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain)

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

Active Publication Date: 2015-12-16
DALIAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
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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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  • Method for building N-glycosylation receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain)
  • Method for building N-glycosylation receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain)
  • Method for building N-glycosylation receptor protein models in Escherichia coli by aid of skeleton proteins Fn3 (fibronectin type III domain)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1. In this example, the human framework protein fibronectin type III domain (Fn3) protein is used as the model gene (EMBL accession number AJ320527). At the 3' end of the gene, 6 histidine residues are introduced, and at the 3' end, a base sequence encoding the glycosylation site DQNAT is introduced. For the structure of the fusion gene, see 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 recombinant gene Fn3-Gly expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), it was constructed on the Escherichia coli expression vector pIG6H with EcoRV and HindIII, The recombinant vector pIG6H-Fn3-Gly 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 (...

Embodiment 2

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

[0034] 2. After the human fibronectin type III domain recombinant gene Fn3-Gly expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), it was constructed on the Escherichia coli expression vector pIG6H with EcoRV and HindIII, The recombinant vector pIG6H-Fn3-Gly was obtained.

[0035] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (...

Embodiment 3

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

[0043] 2. After the human fibronectin type III domain recombinant gene Fn3-Gly expression cassette sequence shown in the sequence table was synthesized (Nanjing GenScript Biotechnology Co., Ltd.), it was constructed on the Escherichia coli expression vector pIG6H with EcoRV and HindIII, The recombinant vector pIG6H-Fn3-Gly was obtained.

[0044] 3. the expression vector constructed and pACYCpgl electric shock transformation CLM37 escherichia coli bacterial strain, then transformants were inoculated to the LB solid medium (...

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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 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 Escherichia coli to express N-glycosylated homogeneous glycoprotein has become one of the current research hotspots. Currently, the Campylobacter jejuni glycosylation modification system pgl (protein lycosylation) is used to successfully produce uniform N-glycosylated proteins in Escherichia coli. In addition, studies have found that Campylobacter jejuni oligosaccharide transferase pglB and oligosaccharide flippase pglK have relaxed substrate recognition specificity, making it possible to customize target glycoproteins in E. coli according to requirements. However, at present, the efficienc...

Claims

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Application Information

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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 UNIVERSITY
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