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Viromembrane protein with site-specific mutagenesis and site-specific decoration, preparation method and applications of viromembrane protein

A technology of site-directed mutagenesis and protein, which is applied in peptide preparation methods, chemical instruments and methods, botanical equipment and methods, etc., can solve the problems of ineffective research on weak and transient protein interactions between viruses and hosts, To achieve the effect of avoiding dissociation

Active Publication Date: 2012-12-26
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Aiming at the problem that the traditional protein-protein interaction research method cannot effectively study the weak and transient interaction between the virus and the host protein, the unnatural amino acid DiZPK was introduced into the VSV G protein at a fixed point through the gene codon extension technology , and realized the intracellular expression of site-directed mutation VSVG. The site-directed mutation unnatural amino acid DiZPK has photocrosslinking properties. Converted into a covalently bonded protein, followed by separation, purification, and mass spectrometry identification to obtain potential proteins that interact with VSV G

Method used

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  • Viromembrane protein with site-specific mutagenesis and site-specific decoration, preparation method and applications of viromembrane protein
  • Viromembrane protein with site-specific mutagenesis and site-specific decoration, preparation method and applications of viromembrane protein
  • Viromembrane protein with site-specific mutagenesis and site-specific decoration, preparation method and applications of viromembrane protein

Examples

Experimental program
Comparison scheme
Effect test

preparation example

[0080] Synthesis and identification of unnatural amino acid DiZPK (Lys-diazirine)

[0081] The chemical synthesis reaction formula of unnatural amino acid DiZPK is as follows.

[0082]

[0083] As shown in the above formula, 15mL of raw material 1 (5-hydroxy-2-pentanone) and 40mL of liquid ammonia were stirred and reacted at -40°C for 5h, then cooled to -60°C, and NH 2 OSO 3 H (20g) in methanol solution was added to room temperature and reacted overnight. The precipitate was filtered off, triethylamine was added to the supernatant, and I was slowly added under ice-bath conditions. 2 , until the color of the reaction solution becomes darker and no more bubbles are generated. After the reaction was complete, the solvent was evaporated, extracted with ether and dried. Diethyl ether was distilled off, and the remaining liquid was distilled under reduced pressure to obtain 25.4 g of product 2 as a colorless viscous liquid.

[0084] The above product 2 was dissolved in pyr...

Embodiment 1

[0088] Example 1: Construction of site-directed mutation of vesicular stomatitis virus envelope glycoprotein expression plasmid

[0089] (1) Mutation site selection

[0090] According to the three-dimensional crystal structure and hydrophobicity analysis of the VSV G protein, the inventors selected 24 sites, because one of the most important structural features of the protein interaction interface, there are some key residues on the interface in the protein interaction It plays a more important role than other residues, and the key amino acid residues are usually: W, Y, I, D, P, and there are 4 Y residues and 2 W residues in the mutation site selected by the present invention , 2 I residues, 3 D residues, 1 P residue. The information of the specific mutation site is as follows, where the amino acid position refers to the position on the sequence shown in SEQ ID NO:1.

[0091] Table 1. Mutation sites

[0092]

[0093]

[0094] Mutation Primer Design

[0095] In order ...

Embodiment 2

[0103] Example 2: Construction of a site-directed mutagenesis plasmid for vesicular stomatitis virus envelope glycoprotein marked with a Flag tag

[0104] The flag tag is a peptide segment of 8 amino acid residues, the sequence is: AspTyrLysAspAspAspAspLys (SEQ ID NO: 47), it is a soluble protein tag, and it is a tag designed for immunoaffinity chromatography analysis. The protein bound to the solid phase through Flag can be eluted in a non-denaturing way, for example, using Flag small peptide to elute the bound protein. Currently, there is a commercial Flag purification method. In order to facilitate the purification or capture of the subsequent site-directed mutant protein, the present invention first introduces a TAG codon into the base sequence of the wild-type VSV G according to Example 1, and then introduces a TAG codon at the C-terminal of the VSV G protein. Add the Flag tag. Specific steps are as follows:

[0105] (1) Amplification primer design:

[0106] Design for...

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Abstract

The invention discloses viromembrane protein with site-specific mutagenesis and site-specific decoration, a preparation method and applications of the viromembrane protein. Amber codon TAG is introduced to the specific site of envelope protein G (VSV G) gene of vesicular stomatitis virus, unnatural amino acid DiZPK with photo-crosslinking property is introduced to the specific site of VSV G by utilizing the orthorhombic aminoacyl-tRNA synthetase-tRNA through site-specific mutagenesis. In the interaction process of virus and host cells, the DiZPK photo-crosslinking reaction is triggered by 365nm ultraviolet light, the covalent bond is formed between the VSV G and interacting protein, the interaction dissociation can be avoided, and the powerful measure is provided for the research of the interaction of the virus and protein of the host cells.

Description

technical field [0001] The invention involves the introduction of the amber codon TAG at a specific site of the envelope protein G (VSV G) gene of vesicular stomatitis virus, and the use of orthogonal aminoacyl tRNA synthetase-tRNA to convert the unnatural amino acid DiZPK with photocrosslinking properties Site-directed mutagenesis to specific sites of VSV G. During the interaction between the virus and the host cell, 365nm ultraviolet light is used to trigger the photocrosslinking reaction of DiZPK, forming a covalent bond between VSV G and the interacting protein, avoiding the dissociation of the interaction, and providing a bridge between the virus and the host protein. The role of research provides a powerful tool. Background technique [0002] Vesicular stomatitis virus and its membrane protein VSVG [0003] Vesicular stomatitis virus (VSV) is a species of rhabdoviridae (Rhabdoviridae), which is a single negative-sense RNA enveloped virus with a genome size of 11,000...

Claims

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Application Information

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IPC IPC(8): C07K14/145C12N15/47C12N15/63C12N1/21C12N5/10C12N15/70C07K1/14
CPCC07K14/005C12N2760/20222
Inventor 周德敏郑永祥赵传科张传领俞飞肖苏龙张礼和
Owner PEKING UNIV
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