Expression of a cannabinoid receptor in baculovirus-infected insect cells.
A cannabinoid receptor recombinant baculovirus (AcNPV-THCR) has been constructed and employed to express rat neural cannabinoid receptors. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-THCR revealed novel hyper-production of a 3.3 kb transcript when probed with nick-translated rat cannabinoid receptor cDNA. Optimal viral protein expression was observed in 35S-metabolically labeled AcNPV-THCR-infected Sf9 cells at a multiplicity of infection of 2.5. Transmission electron microscopy of AcNPV-THCR-infected Sf9 cells showed extensive membrane perturbation and electron-dense cytoplasmic perinuclear accumulation, indicative of receptor glycoprotein expression. Immunofluorescence staining using antiserum produced to a fusion protein consisting of the external domain of the cannabinoid receptor and hepatitis B core antigen revealed cannabinoid receptor expression in AcNPV-THCR-infected Sf9 cells. Scatchard-Rosenthal analysis of [3H]CP55,940 receptor binding indicated a Kd of 3.4 nM and a Bmax equal to 3.17 pmol/mg protein. Western immunoblotting performed on AcNPV-THCR-infected Sf9 cell lysates revealed immunoreactive bands with relative molecular weights ranging from 45 to 79 kDa. The predominant species (55 kDa) exhibited a relative molecular weight consistent with that predicted for the translational product obtained from the cannabinoid receptor cDNA coding sequence. In vitro translation using AcNPV-THCR mRNA also yielded a 55 kDa immunoreactive species. These data indicate that the baculovirus expression system is a viable means of expressing relatively large quantities of cannabinoid receptor recombinant protein.
Pettit
D A
DA
Department of Microbiology/Immunology, Medical College of Virginia/Virginia Commonwealth University, Richmond 23298-0678.
Showalter
V M
VM
Abood
M E
ME
Cabral
G A
GA
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line / Q000648:ultrastructure; D018076:DNA, Complementary / Q000032:analysis; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D051381:Rats; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018411:Spodoptera; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis*
D018076:DNA, Complementary; D043882:Receptors, Cannabinoid; D011955:Receptors, Drug; D011994:Recombinant Proteins; D014764:Viral Proteins