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Trans-N-glycosidase BtNGT and application thereof

A glycosyltransferase and glycosylation technology, applied in the field of N-glycosyltransferase BtNGT, can solve the problems of complicated steps and high cost, and achieve the effect of simple synthesis

Active Publication Date: 2017-08-25
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 BtNGT from Bibersteinia trehalosi and its enzymatic properties and its application in N-linked glycosylation modification of polypeptides.

Method used

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  • Trans-N-glycosidase BtNGT and application thereof
  • Trans-N-glycosidase BtNGT and application thereof
  • Trans-N-glycosidase BtNGT and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Preparation of N-glycosyltransferase BtNGT

[0027] 1. Construction of expression strains

[0028] 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 .

[0029] 2. Expression and purification of BtNGT protein

[0030] A single colony BL21pET45b-BtNGT was picked into 50ml LB medium (containing 50ug / ml ampicillin Amp) and activated 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, coll...

Embodiment 2

[0033] Example 2: Application of N-glycosyltransferase BtNGT in polypeptide glycosylation modification

[0034] 1. Determination of enzyme activity:

[0035] 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:

[0036] Table 1-3 AaNGT and BtNGT enzyme activity detection reaction system

[0037]

[0038] See the reaction result image 3 , the experiment proves that BtNGT can use UDP-Glc and UDP-Gal to complete the glycosylation modification of polypeptides. Then carry out simple verification on the product, see the simple result picture Figure 4 .

[0039] 2. Determination of optimum pH:

[0040] In order to explore the optimal pH of BtNGT, we selected buffers with different pH, HAc (5.0, 6.0) PBS...

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Abstract

The invention discloses a trans-N-glycosidase BtNGT, which is derived from bibersteinia trehalosi, and the amino acid sequence is shown as SEQ ID NO.1. The invention further discloses an application of the trans-N-glycosidase BtNGT in carrying out N-linked glycosylation modification on polypeptide, the trans-N-glycosidase BtNGT can serve as a tool enzyme for polypeptide glycosylation modification to simply and rapidly utilize UDP-Glc or UDP-Gal to glycosylate polypeptide containing N-X-S / T sequence, other glycosyltransferases can then be utilized to modify the carbohydrate chain of glycopeptide, and thereby a target glycopeptide is obtained. A new approach to the glycosylation modification of polypeptide and protein is provided, and a simple method is provided for the synthesis of glycoprotein vaccines.

Description

technical field [0001] The invention belongs to the technical field of glycoengineering in molecular biology, and relates to an N-glycosyltransferase BtNGT derived from Bibersteinia trehalosi and an application thereof. 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 its research is the most thorough. Studies have found that combining sugar antigens with carrier proteins to form glycoprotein vaccines can stimulate the body to produce immune memory, so the re...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12P21/00
CPCC12N9/1048C12P21/005
Inventor 陈敏李江
Owner SHANDONG UNIV
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