INVITRO EXPRESSION OF BOVINE OPSIN USING RECOMBINANT BACULOVIRUS - THE ROLE OF GLUTAMIC ACID-(134) IN OPSIN BIOSYNTHESIS AND GLYCOSYLATION

Expression levels of functional bovine opsin in the insect cell line IPLB-Sf9 using recombinant baculovirus were shown not to depend on the use of novel transfer vectors (pAcRP23, pAcDZ1) that were reported to improve biosynthesis levels of other proteins in this system. A production of 5-mu-g opsin per 10(6) cells (approx. 1.5% of total cell protein) was achieved by batch fermentation of infected cells in spinner cultures. Infection of the cells in the presence of the glycosyltransferase inhibitor tunicamycin led to the synthesis of the complete protein, which, however, now migrated with a substantially lower M(r). This demonstrates that opsin in insect cells also undergoes N-linked glycosylation and allowed partial purification (10-fold) of the resulting rhodopsin by affinity chromatography over Concanavalin A-Sepharose. Through site-directed mutagenesis (rhod)opsin mutants have been obtained allowing dissection of functional domains of opsin. Amino acid substitutions that involved Glu-134 and/or Arg-135 affected the normal biosynthetic process leading in part to nonglycosylated, to a small extent even incomplete, protein. A number of mutations, that involve other charged residues within the second and third transmembrane domain of the protein, had no effect on the biosynthetic processing of the protein. We therefore suggest that the charge-pair Glu-134-Arg-135 is part of an important internal signal sequence and that alterations in this region may result in incorrect membrane translocation and/or folding of the protein.