EXPRESSION, PURIFICATION AND CHARACTERIZATION OF RECOMBINANT C-ELEGANS UNC-18

The Caenorhabditis elegans unc-18-encoded protein (UNC-18) is implicat ed in the processes of vesicle targeting, docking, and/or fusion. To f urther characterize the properties of this important neural protein, w e expressed it at a high level in Spodoptera frugiperda Sf21 cells usi ng a baculovirus expressing system. A cDNA containing the coding seque nce for UNC-18 was inserted into the transfer vector pBlueBac to yield the recombinant virus pAcNPV/unc-18. At maximal expression, the recom binant virus produces a protein of 67 kDa, which constitutes about one third of total cell protein. The UNC-18 protein was highly purified a nd its biochemical and functional properties were assessed. The protei n is globular with an isoelectric point of 6.95. Circular dichroism sp ectroscopy indicated that the alpha-helix and beta-sheet account for 1 0.0 and 59.0%, respectively. Immunolabeling the Sf21 cells expressing UNC-18 showed that the expressed UNC-18 is predominantly localized in the cytoplasm as a soluble monomer. The protein is phosphorylated by p rotein kinase C and binds to the recombinant C. elegans syntaxin in vi tro. These findings suggest that in vesicle traffic UNC-18 is a regula tor factor associated with the plasma membrane through syntaxin, altho ugh intrinsically cytoplasmic. Copyright (C) 1996 Elsevier Science Ltd