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Method for separating RAVE compound from Saccharomyces cerevisiae cells by using FLAG label

A technology of Saccharomyces cerevisiae cells and complexes, applied in microorganism-based methods, biochemical equipment and methods, fermentation, etc., can solve the problems of insufficient content, difficult to make substantial progress, difficult to purify RAVE, etc., to achieve concentration and purity. boosted effect

Inactive Publication Date: 2014-10-15
JIANGNAN UNIV
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Problems solved by technology

However, since RAVE is a regulatory protein, its content in cells is not abundant, and the Ravlp and Rav2p subunits are easily degraded, which makes it very difficult to purify RAVE, which also makes it difficult to obtain substantial research on RAVE complexes in recent years. progress, especially its three-dimensional structure

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  • Method for separating RAVE compound from Saccharomyces cerevisiae cells by using FLAG label

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Embodiment 1

[0032] A method utilizing FLAG tags to separate RAVE complexes from Saccharomyces cerevisiae BY4742rav2-FLAG cells, the steps are as follows:

[0033] A. Large-scale cultivation of Saccharomyces cerevisiae recombinant strain BY4742rav2-FLAG:

[0034] (1) Pick a single colony of Saccharomyces cerevisiae BY4742rav2-FLAG from the YPD plate containing antibiotics (200 μg / ml G418), inoculate in 8 bottles of Erlenmeyer flasks each containing 200ml of seed medium, and culture at 30°C and 200rpm To mid-logarithmic phase (OD≈1);

[0035] (2) Transfer 8 bottles of seed culture solution to 8 bottles of Erlenmeyer flasks each equipped with ILYPD medium, and cultivate overnight at 30°C and 200 rpm until the middle of the stationary phase (OD ≈ 3.5);

[0036] B. Collect and disrupt cells:

[0037] (1) Collect the cells by centrifugation at 8000xg for 5 minutes at room temperature, resuspend the cells in sterile water at 25°C, and place at room temperature for 5 minutes;

[0038] (2) Cent...

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Abstract

A method for separating an RAVE compound from Saccharomyces cerevisiae cells by using an FLAG label relates to modification and purification of an RAVE compound protein in Saccharomyces cerevisiae, and belongs to the fields of the molecular biology and proteomics. The invention provides a method for separating the RAVE compound from a Saccharomyces cerevisiae recombinant strain by using the affinity chromatography principle of the FLAG label, and lays a solid foundation for the subsequent researches of the structure of the compound. The affinity chromatography principle of the FLAG label is used to separate easily biodegradable multimeric regulatory protein RAVE compound from a large amount of a cell homogenates supernatant, so the concentration and the purity of the RAVE compound are greatly improved; the FLAG label only contains 8 amino acids, and has a very small influence on the structures and the functions of proteins, so the activity of the separated RAVE compound is maximally guaranteed; and a series of proteins interacting with the RAVE compound by separating through the FLAG label affinity chromatography, and the identification by combining with mass spectrum can enrich the research data of RAVE compound related proteomics.

Description

technical field [0001] The invention relates to the modification and purification of RAVE complex protein in Saccharomyces cerevisiae, and belongs to the fields of molecular biology and proteomics. Background technique [0002] V-ATPases (Vacuolar ATPases) mainly exist in eukaryotic endomembrane systems, such as lysosomes, endosomes, Golgi apparatus, vacuoles, and plasma membranes. V-ATPase can use the energy of hydrolyzing ATP to transfer protons, maintain the acid-base environment balance in different parts of the cell, and then participate in many physiological metabolic activities, such as cell endocytosis, protein secretion and storage, and fusion of intracellular membranes and transport, Na + Physiological processes such as reabsorption of cells, immune response, sperm maturation, autophagy, reabsorption of osteoclasts, and secretion of neurotransmitters. V-ATPase is composed of two structural and functional parts, a total of 14 subunits: one part is water-soluble V ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/02C07K1/22C12R1/865
Inventor 张震宇高堂杰顾春银
Owner JIANGNAN UNIV
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