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A Study on the Mrp Gene Cloning and Expression

Author: ChenYanKe
Tutor: XieHao；WangHongZuo
School: Wuhan University of Technology
Course: Environmental Science
Keywords: Basophilic bacteria Na ~ / H to reverse transporters Mrp system Cloning and expression Purification
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 63
Quote: 1
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Abstract


The topics selected basophilic bacteria Mrp (multiple resistance and pH adaptation) Na / H reverse vector for the study. The Mrp Na / H vectors reverse allogeneic protein complex is composed by six to seven genes encoding membrane proteins, the system of the a secondary transporter protein located on the cell membrane, plays a key role in regulating alkalophilic bacterial cytoplasm pH balanced steady . The basophils plaque protein-mediated extracellular and intracellular Na circulating basophils adaptive based on Na / H reverse carrier is one of the key factors in the Na cycle. Which MrpF gene encodes a molecular weight of 15KD membrane protein, and the relationship between the relationship between MrpF sodium ion-coupled transporters and its voltage-gated sodium ion channel has attracted widespread attention. Although there are indications that MrpF acid transport activity, but there is no experimental evidence that the coupling between the mediated Na and bile acid transit. This study Bacillus subtilis Bacillus subtilis was successfully amplified Mrp Systems gene, and to build the N-side with the maltose binding protein (MBP) tag expression vector, wherein our gene MrpF reached the heterologous overexpression and affinity and chromatography to obtain purified. The study involved the construction of gene cloning, fusion expression vector, fusion protein expression and purification of the protein's active range of molecular biology and genetic engineering experiment content, design research program: (1) cloning: Get Bacillus subtilis mode bacteria, extraction of genomic DNA molecules, in both sides of the target gene primers were designed according to the characteristics of the mrp operon, primers were designed to contain suitable restriction enzyme cleavage sites, the gene was amplified by PCR technology mrpE F G; (2) Fusion Expression vector: plasmid vector with the appropriate restriction endonuclease and amplified target gene product, the requirements of the selected plasmid vector with a help of the target protein expression and purification of the pins and a resistance marker, the design of the connection system, the connection ligase, final conversion Ru Kelong and expression host strain, to obtain the correct recombinant plasmid is filtered and digested authentication; (3) Expression and Purification: inducible expression of recombinant protein in the expression host bacteria . Groping to optimize the expression conditions, in order to achieve the heterologous overexpression, so as to obtain a sufficient amount of recombinant protein. Affinity chromatography purification of recombinant protein, further study of the function and structure. (4) protein activity: Select the appropriate substrate, using isothermal titration calorimetry protein substrate interactions between, suggesting that the functional activity of the protein.

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