Functional expression of human cone pigments using recombinant baculovirus: compatibility with histidine tagging and evidence for N-glycosylation.
Mammalian color vision is mediated by light-sensitive pigments in retinal cone cells. Biochemical studies on native mammalian cone visual pigments are seriously hampered by their low levels and instability. We describe a novel approach for their functional expression, employing the baculovirus system in combination with histidine tagging to allow future purification and structural analysis. The human red and green cone pigments are produced in relatively large amounts and can be detected by immunocytochemistry as well as by immunoblotting. Histidine tagging has no significant effect on the absorbance maxima. The first evidence is presented that these pigments are N-glycosylated.
Vissers
P M
PM
Institute of Cellular Signalling, Department of Biochemistry, University of Nijmegen, The Netherlands.
DeGrip
W J
WJ
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D006031:Glycosylation; D006639:Histidine / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000737:chemistry*; D012168:Retinal Pigments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013057:Spectrum Analysis / Q000379:methods; D018411:Spodoptera / Q000166:cytology / Q000235:genetics / Q000821:virology; D014466:Ultraviolet Rays
C020932:11-cis-retinal-binding protein; D002352:Carrier Proteins; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012168:Retinal Pigments; D006639:Histidine