Expression, purification and characterization of recombinant C. Elegans UNC-18.
The Caenorhabditis elegans unc-18-encoded protein (UNC-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. To further characterize the properties of this important neural protein, we expressed it at a high level in Spodoptera frugiperda Sf21 cells using a baculovirus expressing system. A cDNA containing the coding sequence for UNC-18 was inserted into the transfer vector pBlueBac to yield the recombinant virus pAcNPV/unc-18. At maximal expression, the recombinant virus produces a protein of 67 kDa, which constitutes about one-third of total cell protein. The UNC-18 protein was highly purified and its biochemical and functional properties were assessed. The protein is globular with an isoelectric point of 6.95. Circular dichroism spectroscopy indicated that the alpha-helix and beta-sheet account for 10.0 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 protein kinase C and binds to the recombinant C. elegans syntaxin in vitro. These findings suggest that in vesicle traffic UNC-18 is a regulator factor associated with the plasma membrane through syntaxin, although intrinsically cytoplasmic.
Ogawa
H
H
Department of Biology, Fujita Health University, Aichi, Japan.
Hayashi
N
N
Hori
I
I
Kobayashi
T
T
Hosono
R
R
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010750:Phosphoproteins; D010766:Phosphorylation; D017433:Protein Structure, Secondary; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins / Q000096:biosynthesis; D017421:Sequence Analysis; D018411:Spodoptera; D013347:Subcellular Fractions / Q000737:chemistry; D033921:Vesicular Transport Proteins
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; D015801:Helminth Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D050765:Qa-SNARE Proteins; D011994:Recombinant Proteins; C082636:Unc-18 protein, C elegans; D033921:Vesicular Transport Proteins