Br Gene Site-directed Mutagenesis And Expression

The halophilic archaeon Halobacterium produces the unique membrane protein bacteriorhodopsin (BR). BR is composed of two components: bacerio-opsin, a 248 amino acid integral membrane protein that spans the membrane seven times and retinal, the chromophore, which is covalently bound to the opsin. It forms a two-dimensional crystalline lattice, called the purple membrane, in the cell membrane of Halobacterium species and acts as a light-driven proton pump. The electrochemical proton gradient generated across the membrane is used by the cells for ATP synthesis Absorption of a photon by BR initiates a catalytic cycle that leads to vectorial transport of a proton out of the cell. After absorption, the all-frans-retinal isomerizes to a 13-c/s configuration and BR undergoes a photocycle:BR570â†'K590â†'L550â†'M410â†'N520â†'O640â†'BR570Bacteriohodopsin is a promising biological photoelectric material. We intent to conduct a more thermostable Br by Site-directed mutagensis. A point (No.274 T, No.274 A turn to C G) mutation was introduced to br gene by successive PCR technique, and cloned into pUC-19 vector. After its sequence was determined, the mutant gene was cloned into a shuttle vector pNov-R and transformed into BR-deficient halobacteria (L33). Purple clones were picked out from the plate, which show BR was expressed. PCR analysis told us that the mutant br gene was transformed into L33. Western-blot result indicated that the protein could bind to anti-BR antibody specifically. Thus, a BR mutant (Tyr79-籄rg) was successively conducted. The mutant protein was also analysesed by some bioinfomnatic software.