LARGE-SCALE PRODUCTION AND PURIFICATION OF THE HUMAN GREEN CONE PIGMENT - CHARACTERIZATION OF LATE PHOTO-INTERMEDIATES

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment, Marked differences from the same pathway in the rod pigm ent were observed. The histidine-tagged human green cone pigment was f unctionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was exte nsively purified in a single step by immobilized metal affinity chroma tography and displays the expected spectral characteristics. The purif ied pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovin e retina lipid proteoliposomes, identification and analysis of the pho to-intermediates Meta I, Meta II and Meta III was accomplished. Simila r to the rod pigment, our results indicate the existence of a Meta I-M eta II equilibrium, but we find no evidence for pH dependence. Replace ment of native Cl- by NO3- in the anion-binding site of the cone pigme nt affected the spectral position of the pigment itself and of the Met a I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and N O3- state. However, in qualitative agreement with results reported bef ore for chicken cone pigments, the rate of Meta II decay was significa ntly higher in the human cone pigment than in the rod pigment.