A Study On The Mrp Gene Cloning And Expression

This issue selected the Mrp Na+/H+antiporters in alkaliphiles bacillus for the study. In Bacillus subtilis, the Mrp antiporter is membrane protein complexes encoded by an operon consisting of mrpABCDEFG genes. Alkaliphiles membrane protein-mediated Na+efflux intracellular and extracellular is an important basis for the alkalophilic adaptation, and the Na+/H+antiporter is one of the key factors in the process of Na+efflux.The mrpF gene in mrp operon is encoding a membrane protein with a molecular weight of 15kDa. There are indications that MrpF of B. subtilis exhibits cholate transport activity.In the present work, we cloned the mrpF gene from Bacillus subtilis strain 168 and generate an expression vectors harbouring N-terminal MBP tagged MrpF proteins. The MBP-MrpF protein was over-expressed in Escherichia coli strain BL21 (DE3) and purified to homogeneous by affinity chromatography.This study involved a series of molecular biology and genetic engineering experiment content:gene cloning, fusion expression vector construction, the fusion protein expression and purification.We designed research programs as follows:(1) Gene cloning:To obtain a model of Bacillus subtilis bacteria, extract genomic DNA molecule.According to the characteristics of mrp operon design primers both sides of the target gene, the primers containing the appropriate restriction enzyme site point, the gene mrpE, F, G will be amplified by PCR;(2) Fusion vector construction:Using the appropriate restriction enzyme to digest plasmid and amplification gene product, choice a plasmid vector with a tag helping target protein expression and purification, and also a resistance mark. Design connection system, ligase, and finally transform into clone and expression host bacteria, after screening and verification for proper digestion of recombinant plasmid;(3) Expression and purification:Inducting expression of recombinant protein in the host bacteria. Explore the optimization expression conditions to achieve heterologous overexpression to obtain sufficient quantities of recombinant protein. Affinity chromatography to purify recombinant proteins and then study the structure and function.(4) Protein activity study:Select the appropriate substrate, using isothermal titration calorimetry to study the interaction between protein and substrate, speculating the protein functional activity.