Synthesis of a gene for sensory rhodopsin I and its functional expression in Halobacterium halobium.
We have designed, synthesized, and expressed in Halobacterium halobium a gene encoding sensory rhodopsin I (SR-I). The gene has been optimized for cassette mutagenesis by incorporating 30 unique restriction sites with uniform spacing throughout the 720-bp coding region. For expression, the coding region was placed downstream of the promoter and translation initiation region of the bacterioopsin gene on a selectable vector. This construct encodes SR-I with an extended N terminus that includes the 13-amino acid leader sequence and the 8-amino acid N terminus of bacterioopsin. To obtain a SR-I- H. halobium strain for expressing the synthetic gene, we used homologous recombination to delete the chromosomal gene encoding SR-I, sopI. The deletion strain was transformed with the synthetic sopI expression vector. Using antibody directed against the C-terminal region of SR-I, we detected in transformant membranes a protein with the electrophoretic mobility expected for SR-I with a processed N-terminal extension. The synthetic gene product was functionally identical to SR-I. Its flash-induced absorption difference spectrum and photochemical reaction cycle in membrane envelope vesicles were characteristic of SR-I. The protein fully restored phototaxis responses in the deletion strain.
Krebs
M P
MP
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Spudich
E N
EN
Khorana
H G
HG
Spudich
J L
JL
D001436:Bacteriorhodopsins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D015139:Blotting, Southern; D002465:Cell Movement; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000138:chemical synthesis / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D005813:Genes, Synthetic; D005822:Genetic Vectors; D019617:Halobacterium salinarum / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D025602:Halorhodopsins; D008565:Membrane Proteins / Q000302:isolation & purification; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010777:Photochemistry; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D025603:Sensory Rhodopsins
D004269:DNA, Bacterial; D025602:Halorhodopsins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C061497:SRI protein, Halobacterium; D025603:Sensory Rhodopsins; D001436:Bacteriorhodopsins