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Method for rapidly adding FLAG marker to C end of Saccharomyces cerevisiae protein Rav2p

A technology of Saccharomyces cerevisiae and labeling, which is applied in the related fields of molecular biology to achieve the effect of eliminating enzyme cleavage and ligation

Inactive Publication Date: 2013-07-10
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are few studies on RAVE and the regulation mechanism of RAVE on V-ATPase activity, but some studies have found that RAVE interacts with V-ATPase V 1 Part of the E subunit and G subunit function to regulate the activity of V-ATPase

Method used

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  • Method for rapidly adding FLAG marker to C end of Saccharomyces cerevisiae protein Rav2p
  • Method for rapidly adding FLAG marker to C end of Saccharomyces cerevisiae protein Rav2p
  • Method for rapidly adding FLAG marker to C end of Saccharomyces cerevisiae protein Rav2p

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Embodiment 1

[0044] A method for rapidly adding a FLAG tag on the C-terminus of Saccharomyces cerevisiae protein Rav2p, the steps are as follows:

[0045] Find the gene sequence of Rav2p from NCBI, the Genebank sequence number is NC 001136.

[0046] A. Genome extraction of Saccharomyces cerevisiae BY4742 and Saccharomyces cerevisiae SF838-5A RAV1-Myc13:

[0047] (1) Pick a single colony of Saccharomyces cerevisiae BY4742 from the YPD solid medium and inoculate it in 5ml of YPD liquid medium; pick a single colony of Saccharomyces cerevisiae SF838-5A RAV1-Myc13 from the YPD solid medium and inoculate it in 5ml of YPD Liquid medium, cultivated at 30°C and 200rpm until OD≈3;

[0048] (2) Suspend Saccharomyces cerevisiae with 500 μl of sorbitol buffer, add 40 μl of 1000 U / ml lyticase and 1 μl of β-mercaptoethanol, mix well and incubate at 37°C for 3 hours to destroy the cell wall;

[0049] (3) Centrifuge at 8000rpm for 1min to discard the supernatant (acceleration should be slow), add 500μl T...

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Abstract

The invention discloses a method for rapidly adding an FLAG marker to the C end of a Saccharomyces cerevisiae protein Rav2p, relates to reconstruction and modification of an Rav2p gene in Saccharomyces cerevisiae, and belongs to the molecular biology related field. The method for rapidly adding the FLAG marker to the C end of the Saccharomyces cerevisiae protein Rav2p is characterized in that the C end of the Saccharomyces cerevisiae protein Rav2p has the FLAG marker, and an antibiotic G418 resistant gene KanMX6 is inserted, so it is in favor of screening recombinant bacteria. Linear DNA constructed by the inventor of the invention is utilized to realize the reconstruction and modification of the Rav2p gene in order to construct the Saccharomyces cerevisiae recombinant bacteria, wherein the C end of the Rav2p of the recombinant bacteria contains the FLAG marker.

Description

technical field [0001] The invention relates to the transformation and modification of the Rav2p gene in Saccharomyces cerevisiae and belongs to the related field of molecular biology. Background technique [0002] V-ATPase (VacuolarATPase) is a protein macromolecule commonly found in the inner membrane system of eukaryotes, which can hydrolyze ATP to transfer protons and maintain the normal physiological environment of cells. V-ATPase transports protons in cells, balances the pH of organelles, and plays an important role in biological processes such as cell membrane transport, protein degradation, and coupling transport of small molecules. [0003] V-ATPase is composed of two structural and functional parts, a total of 14 subunits: one part is water-soluble V 1 , composed of 8 subunits, the main function is to hydrolyze ATP; the other part is V embedded in the membrane 0 , composed of 6 subunits, the main function is to transfer protons. A shank-like structure is formed ...

Claims

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Application Information

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IPC IPC(8): C12N15/62C12N1/19C12R1/865
Inventor 张震宇顾春银李忠浩杨冬美
Owner JIANGNAN UNIV
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