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Method for labeling heterologous protein expressed by insect protein or insect expression system

A protein and glycoprotein technology, applied in the field of labeling insect proteins or exogenous proteins expressed by insect expression systems, can solve the problems of long implementation period, unfavorable promotion, complicated operation, etc.

Active Publication Date: 2018-11-02
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is complicated to operate and has a long implementation cycle, which is not conducive to popularization

Method used

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  • Method for labeling heterologous protein expressed by insect protein or insect expression system
  • Method for labeling heterologous protein expressed by insect protein or insect expression system
  • Method for labeling heterologous protein expressed by insect protein or insect expression system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0194] Embodiment 1 utilizes Ac 4 GlcNAz, DBCO-PEG4-biotin and FITC-streptavidin labeling of glycoproteins in insect cells

[0195] (i) Add Ac at final concentrations of 0 mM, 0.05 mM, 0.1 mM, 0.2 mM and 0.4 mM to insect cell culture medium in five 125 mL culture flasks 4 DMSO solution of GlcNAz, SF9 cells (purchased from Wuhan Hanlinbo Biotechnology Co., Ltd.) were mixed with 2×10 5 The concentration of cells / mL was inoculated into the medium. The cells were shaken at 120rpm / min at 27°C for 72 hours to obtain a cell suspension, so that the unnatural sugar Ac 4 GlcNAz is metabolically integrated into the cell.

[0196] (ii) Collect the cell suspensions obtained in step (i) respectively, centrifuge at 1000 rpm for 10 minutes, and remove the supernatant to obtain cell pellets. The cell pellet was washed twice with PBS solution containing 1 wt% bovine serum albumin, and centrifuged at 1000 rpm for 10 min to remove unused Ac 4 GlcNAz, to obtain the cells to be treated.

[01...

Embodiment 2

[0206] Example 2 Marking of the N8 protein expressed by the insect baculovirus system

[0207] (a) Prepare two bottles of 600mL each, culture in suspension in HF medium to the logarithmic growth phase, and the density is about 1.5-2.5×10 6 The liquid culture of High five cells (purchased from Wuhan Hanlinbo Biotechnology Co., Ltd.) of cells / ml. Add unnatural sugar Ac to 50 ml of fresh HF medium 4 The DMSO solution of GlcNAz, so that the final concentration of the unnatural sugar is 200 μM, is filtered through a 0.22 μM filter to sterilize. Control without Ac 4 HF medium for GlcNAz. Will add or not add Ac 4 The HF medium of GlcNAz was added to the liquid culture of the above-mentioned High five cells, and the prepared baculovirus was added respectively, and cultured on a shaker at 27° C. and 120 rpm. The baculovirus adopts Bac-to- The expression system was constructed according to the manufacturer's instructions provided by thermofisher, and its genome carries a gene cap...

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Abstract

The invention relates to a method for labeling heterologous protein expressed by insect protein or an insect expression system. The method comprises the following steps: doping azido-doped peracetylated N-acetylglucosamine into glycosyl of protein expressed by insects under the assistance of a metabolic pathway of the insects; subsequently, carrying out click chemistry reaction, and coupling a detachable marker to the protein, thereby realizing labeling of the protein from the insects. The method can be used for in-situ labeling of insect cells or labeling of the heterologous protein expressedby the insect expression system.

Description

technical field [0001] The present invention relates to a method for labeling proteins of insect origin by bioorthogonal reactions. The method can realize the in-situ labeling of the protein in the insect cell, and can also realize the labeling of the exogenous protein expressed by the insect baculovirus expression system. Background technique [0002] Protein is the most abundant biomacromolecule in cells, and it participates in almost all life processes. Therefore, the study of the structure, function and interaction of proteins is an important task in the field of life sciences. In many cases, proteins need to be labeled for real-time and / or quantitative observation. Currently, the most commonly used methods for labeling proteins include fluorescent protein, radioactive isotopes, and non-specific modification of fluorescent groups to lysine or tyrosine in proteins through chemical reactions. However, these methods are often complicated to operate, and may also affect t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07C12N15/866C07K14/435G01N33/68
CPCG01N33/68C12N5/0601C12N15/86C07K14/4354G01N2333/43552C12N2500/34C12N2710/14043
Inventor 李学兵张振宁傅立峰
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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