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Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems

A technology of fucosyl and Pichia pastoris, which is applied in the field of genetic engineering and protein engineering, can solve the problems of no core fucosyltransferase, etc., and achieve the effects of short cycle, easy technical operation and low cost

Inactive Publication Date: 2016-09-28
DALIAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there have been no reports on the successful expression of core fucosyltransferases in Pichia pastoris for large-scale synthesis

Method used

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  • Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems
  • Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems
  • Method for secreting and producing human-derived core fucose-base transferases by aid of pichia pastoris expression systems

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Construction of recombinant human core fucosyltransferase FUT8 secretion expression system

[0057](1) According to the protein data bank (UniProtKB-Q9BYC5) human core fucosyltransferase structural information design fusion protein sequence, in order to realize the secretion and expression of core fucosyltransferase using Pichia pastoris. Remove the transmembrane region where the N-terminus of the core fucosyltransferase binds to the Golgi membrane, and introduce an alpha signal peptide (Native saccharomyces cereisiae secretion signal) at the N-terminus of the remaining sequence, and introduce 6 His amino acids at the C-terminus for separation Purification label. Sequence listing SEQ ID No.1;

[0058] The nucleotide sequence list SEQ ID No.2 of the recombinant human core fucosyltransferase FUT8 gene was synthesized according to the codon preference of Pichia pastoris;

[0059] Construct the above fusion gene on the expression vector of Pichia pastoris, and t...

Embodiment 2

[0060] Example 2 Construction of inducible recombinant expression plasmid pPICZαA-FUT8

[0061] The double digestion is to use the restriction endonucleases XhoI and XbaI to combine the core fucosyltransferase FUT8 gene synthesized in step (1) with the inducible expression vector pPICZαA after double digestion and use T4DNA ligase Ligate the DNA fragments overnight at 16°C, heat-shock at 42°C to transform Escherichia coli TP10 competent cells, coat LB medium plates containing 25ug / ml Zeocin, culture at 37°C overnight, randomly pick single clones, and extract Recombinant expression plasmid pPICTαA-FUT8, identified positive clones by double enzyme digestion, such as figure 2 As shown, it is a schematic diagram of the constructed recombinant core fucosyltransferase protein FUT8 inducible expression plasmid vector pPICTαA-FUT8;

[0062] The above-mentioned LB medium is made of 5 g of yeast powder, 10 g of peptone, 5 g of sodium chloride, adjusting the pH value to 7.2-7.4, and ad...

Embodiment 3

[0063] Embodiment 3 Pichia pastoris strain transformation and screening

[0064] Use the restriction endonuclease PmeI to digest the inducible expression vector pPICZαA-FUT8, take 5-20ug of the plasmid to transform the Pichia pastoris X-33 competent cells by electric shock, the parameters of the eppendorf-Eporator electrotransformer used are: voltage 1.5KV , capacitive resistance 200Ω, discharge time 4.8-5.2ms, add 1.0ml ice-cold 1.0M sorbitol immediately after electric shock, shake and culture at 30°C for 1-2h, then spread YPDS plate containing 100ug / ml Zeocin, place at 28-30°C Transformants grew out after 2-4 days of culture.

[0065] The YPDS plate is made of: 10g yeast powder, 20g peptone, 20g-D glucose, 15g agar, 10g sorbitol and add water to 1000ml;

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Abstract

The invention provides a method for producing, efficiently secreting and expressing core fucose-base transferases FUT8 in pichia pastoris expression systems, and belongs to the field of genetic engineering and the field of protein engineering. The method mainly comprises constructing the pichia pastoris secretion and expression systems for the recombinant human-derived core fucose-base transferases FUT8; separating and purifying the recombinant human-derived core fucose-base transferases FUT8; detecting the enzymatic activity of the recombinant human-derived core fucose-base transferases FUT8. The method has the advantages that the core fucose-base transferases FUT8 synthesized, secreted and expressed by the aid of the method can be widely applied to protein inhibitor, biological function and in-vitro oligosaccharide research; the method is low in synthesis cost, short in synthesis period and easy to technically implement, and the core fucose-base transferases FUT8 obtained by the aid of the method are high in purity.

Description

technical field [0001] The invention relates to oligosaccharide biosynthesis, in particular to a method for secreting and producing recombinant human core fucosyltransferase FUT8 by using a Pichia pastoris expression system, belonging to the fields of genetic engineering and protein engineering. Background technique [0002] Most glycosyltransferases are membrane proteins with two transmembrane domains assembled on the endoplasmic reticulum and Golgi membranes, and glycosyltransferases catalyze each monosaccharide to the corresponding sugar chain in the endoplasmic reticulum and Golgi Formation of biologically active sugar chains. [0003] So far, there are 11 known fucosyltransferases, and the core fucosyltransferase Fut8 is the oldest gene in the fucosyltransferase gene family. GDP-L-fucose: N-acetyl-β-D-glucosamine core fucosyltransferase (Fut8) transfers guanosine diphosphate fucose (GDP-Fuc) to mixed or complex N – The 6-position carbon atom of the innermost N-acetylg...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/81C12N15/56
CPCC12N9/1051C07K2319/02C07K2319/21C12N15/815C12N2800/102C12N2800/22C12Y204/01068
Inventor 胡学军杨岩李文哲丁宁杨春光孙慎侠
Owner DALIAN UNIV
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