Functional expression of rat VPAC1 receptor in Saccharomyces cerevisiae.
The yeast Saccharomyces cerevisiae was examined as host for heterologous expression of the G protein-coupled VPAC1 receptor. Rat VPAC1 receptor cDNA and two chimeric constructs encoding the yeast mating factor pre-pro alpha-leader peptide fused in-frame to rat VPAC1 receptor were expressed in yeast cells under control of a galactose inducible promoter. The rat VPAC1 receptor was fused to the HSV tag epitope to ensure proper immunological detection of the receptor. Crucial conditions for high-level expression of active rat VPAC1 receptor included growth in amino acid supplemented minimal medium, fusion to the yeast alpha-leader peptide and a temperature shift from 30 degrees C to 15 degrees C before promoter induction. Western blotting showed that the expressed receptor was highly glycosylated and a band of 47 kDa was obtained upon endoglycosidase H treatment. Binding with radioiodinated vasoactive intestinal polypeptide revealed a KD of 2.5 nM and an IC50 of 15 nM when displacing with unlabeled vasoactive intestinal polypeptide. VPAC1 receptor density quantified by Western blotting was 510 pmol/mg membrane protein of which only 66 pmol/mg were able to bind vasoactive intestinal polypeptide.
Department of Clinical Biochemistry, Bispebjerg Hospital, University of Copenhagen, Denmark.
M K
MK
Hansen
J W
JW
Tams
J
J
Fahrenkrug
P A
PA
Pedersen
D000818:Animals; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003470:Culture Media / Q000494:pharmacology; D018076:DNA, Complementary / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D019204:GTP-Binding Proteins / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D006031:Glycosylation; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics; D011499:Protein Processing, Post-Translational; D051381:Rats; D018005:Receptors, Vasoactive Intestinal Peptide / Q000096:biosynthesis* / Q000235:genetics; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D003470:Culture Media; D018076:DNA, Complementary; D005656:Fungal Proteins; C048632:MF(ALPHA)1 protein, S cerevisiae; D011498:Protein Precursors; D018005:Receptors, Vasoactive Intestinal Peptide; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014660:Vasoactive Intestinal Peptide; D019204:GTP-Binding Proteins