Replica filter assay of human β-adrenergic receptors expressed in E. coli

The human β2‐adrenergic receptor expressed in Schizosaccharomyces pombe retains its pharmacological properties

“…Previous papers have reported the use of microbial hosts for the expression of neurotransmitter receptors [ 19,28,29]. Human fl2-adrenergic receptors expressed from fusion genes in E. coli and S. cerevisiae retain the 'native' ligand binding profiles [6,17,19].…”

“…Human fl2-adrenergic receptors expressed from fusion genes in E. coli and S. cerevisiae retain the 'native' ligand binding profiles [6,17,19]. In particular, in the S. cerevisiae cells it was observed that maximal expression required several manipulations includ-ing: (i) coexpression of a modified hufl2AR gene and a transcription transactivator protein (the LAC9 gene product) [9]; (ii) growth to late exponential phase; (iii) induction by galactose for an additional 36 hours.…”

“…The human fl2AR gene was also expressed in E. coli [6,19] and in S. cerevisiae [7]. The hufl2AR expressed in E. coli retains its pharmacological profile and is capable of coupling to a G protein in an in vitro reconstitution system [8].…”

Topological analysis of the human beta 2-adrenergic receptor expressed in Escherichia coli.

“…hu(AR expressed in E. coli displays affinities for adrenergic ligands similar to those observed in eukaryotic cells (11)(12)(13) and is able to couple with Ga (14) (20) or antibody to alkaline phosphatase or to (-galactosidase was added and the samples were incubated at 4TC for 1 hr. Samples were centrifuged, washed twice with 0.5 ml of STEB, resuspended in the same buffer, and incubated with horseradish peroxidaseconjugated anti-mouse immunoglobulin antibodies (for 9E10) or peroxidase-conjugated protein A (for antibody to alkaline phosphatase or (3-galactosidase) at 4TC for 1 hr.…”

“…Antigen-antibody complexes were then quantified by measuring OD450 after one-step Turbo ELISA (Pierce). (12).…”