(purple membrane/proton pump/oligonucleotide purification/site-sp4

To study the mechanism of light-dependent proton translocation by bacteriorhodopsin, we have intro- duced single-codon changes in the gene so as to produce the following specific amino acid substitutions in the protein: Tyr- 185 to Phe, Pro-186 to Leu, Trp-189 to Phe, Ser-193 to Ala, and Glu-194 to Gln. The strategy involved replacement of a 62-base-pair restriction fragment by synthetic DNA duplexes containing the modified nucleotide sequences. This required a unique restriction site (Xho I) at Ile-203 which was created by oligonucleotide-directed point mutagenesis. The six DNA du- plexes corresponding to the modified native and mutant re- striction fragments were all prepared by DNA ligase-catalyzed joining of chemically synthesized deoxyribooligonucleotides. The bacterioopsin expression plasmids reconstructed by using the synthetic DNA fragments were characterized by restriction analysis and DNA sequence determination. An extremely rap- id, efficient, and general method for purification of the syn- thetic oligonucleotides and of DNA fragments was developed.