Abstract
The aim of this work is to develop a prokaryotic system capable of expressing membrane-bound receptors in quantities suitable for biochemical and biophysical studies. Our strategy exploits the endogenous high-level expression of the membrane protein bacteriorhodopsin (BR) in the Archaeon
Halobacterium salinarum. We attempted to express the human muscarinic acetylcholine (M
1) and adrenergic (a2b) receptors by fusing the coding region of the
m1 and
a2b genes to nucleotide sequences known to direct
bacterio-opsin (
bop) gene transcription. The fusions included downstream modifications to produce non-native carboxyl-terminal amino acids useful for protein identification and purification.
bop mRNA and BR accumulation were found to be tightly coupled and the carboxyl-terminal coding region modifications perturbed both.
m1 and
a2b mRNA levels were low, and accumulation was sensitive to both the extent of the
bop gene fusion and the specific carboxyl-terminal coding sequence modifications included. Functional
a2b adrenergic receptor expression was observed to be dependent on the downstream coding region. This work demonstrates that a critical determinant of expression resides in the downstream coding region of the wild-type
bop gene and manipulation of the downstream coding region of heterologous genes may affect their potential for expression in
H. salinarum.
