Study On The Cloning And Expression Of The Bop Gene Of A Halophil Ic Archaea Strain Isolated From A Salt Lake In Inner Mongolia

Bacteriorhodopsin (BR) encoded by the bop gene is a unique membrane protein with seven membrane-spanning a-helixes which function as a light-driven proton pump. Under the light conditions, protons can be transfered by proton pump action from the plasma site to the extracellular side of the cytoplasma membrane using light energy, and produce transmembrane proton gradient. In this way the original light energy was conversed into electrochemical energy which provides energy for the life activities of Halophilic Archaea.In this study the complete bop gene sequence of an extreme halophilic archaea strain isolated from Eejnoor Lake in Inner Mongolia named Natrinema IM-1 has been cloned. According to the bop gene sequences in the GenBank, a pair of specific primer has been digsined for the amplification of the complete sequence of bop gene of Natrinema IM-1, and the complete sequence of bop gene encoding Bacteriorhodopsin has been amplified by PCR using genomic DNA of Natrinema IM-1 as template. The complete sequence of the bop gene has been registered in GenBank, and its accession number is KT 873301.The complete sequence of bop gene has been inserted into the expression vector pET-28a(+), and the recombinant expression vector named pET-28a-IM-1 has been constructed. The recombinant BR protein of Natrinema IM-1 has been successfully expressed in Escherichia coli strain BL21 (DE3), and the expression conditions have been optimized. Results show that the expression achieves the highest level at 25 ℃, induced with 0.50mM of IPTG for 4 hours. But a part of the expressed protein can form inclusion bodies in the process of expression. The expression and the purification of the BR have been analized using SDS-PAGE. The purified BR protein has been scanned by photometre under light and dark conditions, and the measurement shows a characteristic absorption peak at 568nm under light and a characteristic absorption peak at 555nm under dark condition. This result is identical with the reported Bacteriorhodopsin absorption peak in refrences. The result demonstrates that the BR encoded by the bop gene of Natrinema IM-1 is capable to switch the light reaction and the dark reaction, and able to carred out the light cycling. This indicates that the expressed BR protein encoded by Natrinema IM-1 bop gene has great application potential as a photoelectric material. This study laid the foundation for the large scale expression and the application of the BR in many fields.