Ligand binding analysis of human neuropeptide Y1 receptor mutants expressed in E. coli.
Site-directed mutants of the human neuropeptide Y1 (NPY Y1) receptor expressed as a maltose binding protein fusion protein in E. coli show identical ligand binding parameters compared with the same mutants expressed in mammalian cells using a vaccinia virus expression system. However, it was remarkable that two receptor mutants, which were initially classified as non-binding when expressed in an eukaryotic expression system, could actually be revealed to have wild-type binding activity when expressed in E. coli. Re-expression and retesting of these mutants in mammalian cells confirmed this result. This shows that bacterial expression can be used as a fast, versatile and valuable alternative to mammalian expression systems for the analysis of ligand binding sites in G-protein coupled receptors.
Münch
G
G
Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, NSW, Australia.
Walker
P
P
Shine
J
J
Herzog
H
H
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D006801:Humans; D008024:Ligands; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017354:Point Mutation; D011485:Protein Binding; D017476:Receptors, Neuropeptide Y / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D017476:Receptors, Neuropeptide Y; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C097212:neuropeptide Y-Y1 receptor