Replica filter assay of human beta-adrenergic receptors expressed in E.coli.
We have developed a replica filter assay that permits the direct identification of bacterial colonies expressing membrane receptors. E.coli transformed with appropriate phage or plasmid vectors containing human beta-adrenergic receptor cDNAs were grown on LB/agar plates. Bacterial colonies transferred onto nitrocellulose filters showed specific [125I]-iodocyanopindolol binding. beta-adrenergic receptors expressed in bacteria retained their pharmacological properties when transferred onto filters. This strategy, which is considerably simplified and more rapid compared to similar methods based upon expression of receptor genes in eukaryotic cells, may be a useful tool for cloning membrane receptors.
Galli
G
G
Laboratori di Biotecnologie, ENICHEM, Monterotondo, Roma, Italy.
Matteoni
R
R
Bianchi
E
E
Testa
L
L
Marazziti
D
D
Rossi
N
N
Tocchini-Valentini
G
G
D001923:Brain Chemistry; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D020120:Iodocyanopindolol; D008168:Lung / Q000737:chemistry; D008851:Micropore Filters; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000096:biosynthesis / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011943:Receptors, Adrenergic, beta; D011994:Recombinant Proteins; D020120:Iodocyanopindolol; D004247:DNA; D010869:Pindolol