MdrP mutant gene, amino acid, and protein function and drug accumulation activity detection

A technology of mutants and proteins, applied in the field of proteins, can solve problems such as difficult to achieve

Active Publication Date: 2019-10-22
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Abstract
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Problems solved by technology

[0015] The main difficulty in solving the above problems lies in how to eliminate the electrochemical gradient potential generated by the respiratory chain on the membrane, that is, how to obtain Na alone + or K + The driving level of , because inhibition of the respiratory chain is a fatal blow to living cells, it is difficult to achieve, and solving this problem will help us study non-H + Driven drug pump proteins, also critical for discovering other drivers of proteins

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  • MdrP mutant gene, amino acid, and protein function and drug accumulation activity detection
  • MdrP mutant gene, amino acid, and protein function and drug accumulation activity detection
  • MdrP mutant gene, amino acid, and protein function and drug accumulation activity detection

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Embodiment Construction

[0047] In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

[0048] Measuring norfloxacin drug accumulation experiment in the prior art can only confirm that the protein is active and transmembrane H + Gradients can serve as driving forces, but other cations (Na + 、K + )'s effect, not to mention H + Relatively quantitative comparative analysis of the driving effect, the more commonly used method for different ion binding is to explain at the protein level, but it is impossible to determine the binding and effectively play a role in transport in a single experiment, and the experiment is time-consuming and laborious .

[0049] In order to solve the above technical problems, the prese...

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Abstract

The invention belongs to the technical field of protein, and discloses an MdrP mutant gene, amino acid, and protein function and drug accumulation activity detection. According to the invention, CCCPis added to the Buffer containing cations (Na<+>, K<+>) to remove the transmembrane H<+> gradient, only transmembrane cation gradient is retained to drive protein translocation, or Monensin or Nigericin is added in the buffer to remove the cation gradient, and only the transmembrane H<+> gradient is retained to drive the protein translocation, and a relative quantitative comparison between different driving effects can be realized. The important function of highly conserving amino acid site of the MdrP protein can be clarified, and the MdrP protein can be proved as an H<+> and Na<+> driven drug efflux protein; N146 is the H<+> binding site, D223 and Q242 are Na<+>, H<+> competitive binding sites, or can be a favorable theoretical support for understanding the MFS family of drug resistanceproteins.

Description

technical field [0001] The invention belongs to the technical field of protein, and in particular relates to the detection of gene, amino acid, protein function and drug accumulation activity of a MdrP mutant. Background technique [0002] At present, the existing technologies commonly used in the industry are as follows: [0003] In the analysis of existing bifunctional proteins, the key functionally decisive amino acid sites have not yet been clarified, that is, an important site that determines a functional activity. More interpretation is at the protein level, that is, visually confirming related proteins in crystallized proteins, or performing ITC biochemical experiments on the purified protein level of amino acid site-directed mutants to determine the substrate binding effect, so as to study the effect of specific sites on substrates. (ions or molecules) binding. [0004] Most of the existing technical schemes are norfloxacin drug accumulation experiments. + 、K + A...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/00C12N15/11G01N33/68
CPCC07K14/00G01N33/68
Inventor 姜巨全张瑞邹俏郑秀桃郭思伽王玉婷
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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