CELL-FREE SYNTHESIS, FUNCTIONAL REFOLDING, AND SPECTROSCOPIC CHARACTERIZATION OF BACTERIORHODOPSIN, AN INTEGRAL MEMBRANE-PROTEIN

Bacteriorhodopsin (bR) is an integral membrane protein which functions as a light-driven proton pump in Halobacterium halobium (also known a s Halobacterium salinarium). The cell-free synthesis of bR in quantiti es sufficient for FTIR and NMR spectroscopy and the ability to selecti vely isotope label bR using aminoacylated suppressor tRNAs would provi de a powerful approach for studying the role of specific amino acid re sidues. However, no integral membrane protein has yet been expressed i n a cell-free system in quantities sufficient for such biophysical stu dies. We report the cell-free synthesis of bacterioopsin, its purifica tion, its refolding in polar lipids from H. halobium, and its regenera tion with all-trans-retinal to yield bacteriorhodopsin in a form funct ionally similar to bR in purple membrane. Importantly, the yields obta ined from in vitro and in vivo expression are comparable. Functionalit y of the cell-free expressed bR is established using static and time-r esolved absorption spectroscopy and FTIR difference spectroscopy.