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N glycosyl transferase AaNGT and application thereof

A glycosyltransferase, glycosylation technology, applied in the directions of transferase, application, enzyme, etc., can solve problems such as high cost and complicated steps

Inactive Publication Date: 2017-08-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, glycoproteins were obtained through direct extraction in vivo, or chemical synthesis in vitro, both of which have the disadvantages of complicated steps and high cost.
After searching, there is no report about the N-glycosyltransferase AaNGT derived from Aggregatibacter aphrophilus and its enzymatic properties and its application in N-linked glycosylation modification of polypeptides.

Method used

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  • N glycosyl transferase AaNGT and application thereof
  • N glycosyl transferase AaNGT and application thereof
  • N glycosyl transferase AaNGT and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: Preparation of N-glycosyltransferase AaNGT

[0030] 1. Construction of expression strains

[0031] Transform the plasmid synthesized by GenScript into BL21 competent cells, heat shock at 42°C for 1 min, and spread on Amp plate. Pick a single colony into a 5ml test tube culture medium at 37°C and 200rpm for 12h, and verify the plasmid. The results of plasmid agarose gel electrophoresis figure 1 .

[0032] 2. Expression and purification of AaNGT protein

[0033] Pick a single colony BL21pET45b-AaNGT into 50ml LB medium (containing 50ug / ml ampicillin Amp) and activate at 200rpm at 37°C for 12h. Then expand the culture, transfer the bacterial solution to 1L culture medium (containing 50ug / ml ampicillin Amp), incubate at 200rpm at 37°C for about 3.5h, measure the OD value, when the OD 600 When the value is 0.6, ice-bath for 20 minutes, and then add 400 μL of 0.5 M IPTG to induce protein expression (16° C. 200 rpm). After 20 hours of induction, collect the...

Embodiment 2

[0036] Example 2: Application of N-glycosyltransferase AaNGT in polypeptide glycosylation modification

[0037] 1. Determination of enzyme activity:

[0038] The substrate peptide for the determination of enzyme activity is the fluorescently labeled hexapeptide DANYTK synthesized by Nanjing GenScript Company. Under the catalysis of NGT, it reacts with UDP-Glc or UDP-Gal respectively. The reaction solution is detected by HPLC fluorescence detector. The system is shown in Table 1-3:

[0039] Table 1-3 AaNGT enzyme activity detection reaction system

[0040]

[0041] For the reaction results, see image 3 , found that AaNGT can use UDP-Glc and UDP-Gal to modify polypeptides containing glycosylation sequences, and then perform pristine analysis on the products. The mass spectrometry results are shown in Figure 4 .

[0042] 2. Determination of optimum pH:

[0043]In order to explore the optimal pH of AaNGT, we selected different pH buffers, HAc (5.0, 6.0) PBS (6.0, 7.0, 8....

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Abstract

The invention discloses a N glycosyl transferase AaNGT. An amino acid sequence thereof is shown as SEQ ID NO.1. The invention also discloses an application of the N glycosyl transferase AaNGT in polypeptide galactosylated modification. AaNGT is capable of supplying a simple and convenient method for the forming of glycopeptide; the capacity of AaNGT for utilizing UDP-Glc is obviously higher than that of the reported N glycosyl transferase ApNGT which is sourced from Actinobacillus pleuropneumoniae; the AaNGT also can transfer the special glycosyl donor UDP-GlcNH2 to the polypeptide containing N glycosylation sites, and then the other glycosyl transferases can be utilized to convert the GlcNH2 into the GlcNAc so as to form natural N glycan linking, so that a new method is supplied for the development of the glycoprotein vaccine. The invention is beneficial to the AaNGT becoming a protein modified tool enzyme.

Description

technical field [0001] The invention relates to an N-glycosyltransferase AaNGT derived from Aggregatibacter aphrophilus and its application, belonging to the technical field of glycoengineering in molecular biology. Background technique [0002] Glycoproteins are a class of important physiologically active substances formed by covalently modifying and linking oligosaccharides and polypeptide chains, which widely exist in cell membranes, interstitial cells, plasma and mucus. It plays a very important role in the process of cell signal recognition, neural regulation, information transmission between cells and immune regulation. The glycosylation modification of protein is divided into N-O-P-C- and G-glycosylation in order of importance, among which N-linkage is the most common, and the first sugar of N-linkage is GlcNAc. It is the most thoroughly researched. Studies have found that combining sugar antigens with carrier proteins to form glycoprotein vaccines can stimulate the...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C07K1/107
CPCC07K1/1077C12N9/1048
Inventor 陈敏孔蕴李江
Owner SHANDONG UNIV
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