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Super respiratory chain compound protein

A complex and respiratory chain technology, applied in the field of mammalian cell respiratory chain super complex protein, can solve the problem of unclear mechanism of action of timosaponin

Active Publication Date: 2018-03-20
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the mechanism of action of timosaponin is still unclear

Method used

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  • Super respiratory chain compound protein
  • Super respiratory chain compound protein
  • Super respiratory chain compound protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0124] Example 1. Respiratory Chain Supercomplex Protein I 1 III 2 IV 1 purification of

[0125] Extraction of mitochondria

[0126] 100g of fresh porcine heart tissue was cut into 2cm×2cm×2cm pieces and suspended with 100mL buffer A (100mM Tris pH7.4, 225mM sorbitol, 60mM KCl, 1mM EGTA and 0.1% bovine serum albumin). Use a large-capacity tissue homogenizer to crush for 300 seconds. Transfer the homogenate to a 50mL centrifuge tube, centrifuge at 3000g for 10 minutes (min), discard cell debris and other precipitates, and retain the supernatant. Transfer the supernatant to a high-speed centrifuge tube and centrifuge at 20,000 for 30 minutes to obtain a precipitate, which is the crudely extracted mitochondria. Prepare buffer B (100mM Tris pH 7.4, 250mM sucrose, 60mM KCl, 40% Percoll (silica particle suspension treated with polyvinylpyrrolidone) and 0.1mM EGTA), resuspend the crudely extracted mitochondria with 100mL buffer B, Centrifuge at 60,000 for 50 minutes, and then...

Embodiment 2

[0137] Example 2. Respiratory Chain Supercomplex Protein I 1 III 2 IV 1 Collection of cryo-EM data and analysis of protein structure

[0138] Electron Microscopy Sample Preparation

[0139] 4 μL of the supercomplex protein soaked with small molecule drugs was dropped onto a 400-mesh Quantifoil R1.2 / 1.3 copper grid (purchased from Quantifoil, Micro Tools GmbH, Germany) covered with a carbon film in advance, and allowed to stand for 1.5 seconds. Frozen samples were then prepared using an FEI Mark IV Vitrobot sample preparation machine.

[0140] Electron Microscopy Data Acquisition

[0141] 300kVTitan Krios is used to collect high-resolution image data, and the detector is Falcon IIdirect electron detector (FEI Company). The control software for data acquisition is EPU (FEI Company). The magnification of the electron microscope is 75,000×, the pixel size is 1.08 angstroms, the defocus value is -1.3 μm to -2.3 μm, the electron dose is 35 / pixel / s, the exposure time is 1.5...

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Abstract

The invention relates to a mammalian cell super respiratory chain compound protein and in particular to a super respiratory chain compound protein of a high atom resolution electron cryomicroscopy structure, and a preparation method of the protein. By adopting the protein structure, the respiratory action mechanism of mammalian animals and diseases related to respiratory action can be further studied.

Description

technical field [0001] The invention relates to a mammalian cell respiratory chain super complex protein, especially a cryo-electron microscope structure with high-resolution atomic resolution, and a preparation method of the protein. Background technique [0002] Respiration occurs in almost all living organisms, and the protein complexes that perform respiration in higher organisms become more complex with biological evolution. In mammals, the site of respiration is the mitochondria. In the mitochondrial matrix, stable chemical energy through the tricarboxylic acid cycle (TCA cycle) is converted into unstable chemical energy and stored in NADH or FADH 2 In the process, these molecules form a proton gradient on both sides of the mitochondrial inner membrane by oxidizing the respiratory chain, and finally the ATP synthase on the mitochondrial inner membrane uses the electrochemical potential energy formed by the proton gradient to synthesize the high-energy molecule ATP. P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12N9/04C07K14/80C07K1/36C07K1/16C07K1/14
CPCC07K14/80C12N9/0006C12N9/0053C12Y101/01C12Y109/03001
Inventor 杨茂君谷金科吴萌
Owner TSINGHUA UNIV
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