Functional Expression of pharaonis Phoborhodopsin in Escherichia coli
Kazumi SHIMONO, Masayuki IWAMOTO, Masato SUMI, and Naoki KAMO
Laboratory of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan
Pharaonis phoborhodopsin (ppR) (1-6), the photoreceptor of the negative phototaxis of archaebacterial Natronobacterium pharaonis was expressed in a heterologous system, Escherichia coli. Flash-photolysis on millisecond time scale showed as follows: (i) The photocycling rate is slower than that of bacteriorhodopsin. (ii) The rate is slower at alkaline solution. (iii) The absoftion spectrum of ppR has its maximum at 498 nm with a pronounced shoulder at 470 nm. (iv) For the time range of ms or longer, there exist two photo-intermediates whose absoftion maxima are 390 and 560 nm. (v) The order of the formation of these intermediates was determined and was the same as ppR. (vi) The photocycling rate was accelerated by addition of azide. These indicated that photochemical properties of ppR expressed in E. coli were the same as those of native ppR in N. pharaonis. We concluded that the integral membrane protein ppR is correctly folded in vivo in the eubacterial E. coli membrane. Since the cell conditions (e.g. salt concentration, membrane structure or pH) are greatly differ between E. coli and N. pharaonis, it is generally doubtful whether the membrane protein expressed in E. coli is incorporated into its membrane. As a matter of fact, bacteriorhodopsin is expressed as an inclusion body in E. coli and a certain treatment was necessary to take a proper conformation. Thus, the functional expression of ppR in E. coli is interesting. The present results open the investigation relating the structure to the photochemical properties of ppR using the site-directed mutagenesis, and for this investigation, E. coli ppR has an advantage because of its much higher growth rate than that of N. pharaonis.
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