Research On The Cloning Bop Gene And Its Promoter From The Genome DNA Of The H.salinarium S9, Constructing BR Mutants And Studying Their Photochromic Properties

In our present study, the bop gene and the promoter were amplified directly from the genomic DNA of Halobactrium salinarium S9 by recombinant-PCR method. The length of the sequence is 1200 base pair, which is the same with the sequence of bop gene in the NCBI database. Then, we constructed a recombinant plasmid pXLNovR-bop and expressed bop gene in the L33 cells, The molecular weight of the BR protein expressed from the cloned bop gene is also the same with the BR protein of the Halobactrium salinarium S9 which shows a special absorption peak located at 568nm.This method is easier and reliable than other methods.The single mutant BR_E204Q and tri-mutant BR_I119T/T121S/A126T of BR protein were constructed by directed-mutation method. The absorption peak of the BR_E204Q located between 552nm and 572nm, BR_I119T/T121S/A126T' located between 552nm and 572nm, and BR_E204Q/I119T/T121S/A126T' located at 410nm and 572nm.The M₄12 Flash-dynamics spectrum indicates that the tetra-mutant BR_E204Q/I119T/T121S/A126T has the significantly prolonged M intermediate life (19.85ms) compared with wild-type BRwt (6.23ms), single mutant BR_E204Q (7.10ms) and the tri-mutant BR_I119T/T121S/A126T (8.23ms), Single mutant BR_E204Q and the tri-mutant BR_I119T/T121S/A126T were already proved have a prolonged O intermediate life. Our research shows that the life time of the M intermediate of the BR protein has a close relation to the life time of the O intermediate. Furthermore, the mutant shows the similar changed character could gain the cumulate effect by combine the mutated sites together. At the same time, the research on proton-pump function of the tetra-mutant bacteriorhodopsin illustrates that it transfer proton in an opposite direction in contrast to BRwt, the single mutant BR_E204Q and the tri-mutant BR_I119T/T121S/A126T. Both BR_E204Q and BR_I119T/T121S/A126T illustrates that their proton-pump functions declined compare with the wild-type BR, and the proton-pump function of the tri-mutant BR_I119T/T121S/A126T is weaker than the single mutant BR_E204Q.