Mp. Krebs et al., SYNTHESIS OF A GENE FOR SENSORY RHODOPSIN-I AND ITS FUNCTIONAL EXPRESSION IN HALOBACTERIUM-HALOBIUM, Proceedings of the National Academy of Sciences of the United Statesof America, 90(8), 1993, pp. 3486-3490
We have designed, synthesized, and expressed in Halobacterium halobium
a gene encoding sensory rhodopsin I (SR-I). The gene has been optimiz
ed for cassette mutagenesis by incorporating 30 unique restriction sit
es with uniform spacing throughout the 720-bp coding region. For expre
ssion, the coding region was placed downstream of the promoter and tra
nslation initiation region of the bacterioopsin gene on a selectable v
ector. This construct encodes SR-I with an extended N terminus that in
cludes the 13-amino acid leader sequence and the 8-amino acid N termin
us of bacterioopsin. To obtain a SR-I- H. halobium strain for expressi
ng the synthetic gene, we used homologous recombination to delete the
chromosomal gene encoding SR-I, sopI. The deletion strain was transfor
med with the synthetic sopI expression vector. Using antibody directed
against the C-terminal region of SR-I, we detected in transformant me
mbranes 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 differenc
e spectrum and photochemical reaction cycle in membrane envelope vesic
les were characteristic of SR-I. The protein fully restored phototaxis
responses in the deletion strain.