Abstract
Halobacteria are halophilic representatives of the recently defined
domain, the Archaea. Halobacterium salinarium belongs to this group
of microorganisms and contains large amounts of bacteriorhodopsin
in its membrane. Bacteriorhodopsin is a seven-transmembrane protein
that consists of bacterio-opsin (BO), and the chromophore retinal,
which is covalently attached to BO. We have investigated whether
the expression machinery for BO can be utilized for synthesis of
the human beta 2-adrenoceptor (beta 2-AR), a protein with a similar
seven-transmembrane-helix topology. An expression vector for BO synthesis
was modified to express beta 2-ARs under the control of BO regulatory
clements in H. salinarium. Homologous recombination into the genome
was verified by polymerase chain reactions. Northern blots revealed
transcripts of the calculated size and significant amounts of epitope-tagged
beta 2-ARs were detected in Western blots. However, binding of the
beta-AR antagonist 125I-cyanopindolol revealed low levels of functional
receptors, and the ligand binding properties of these receptors were
altered when compared to native receptors. Expression of chimeras
containing larger amino terminal portions of BO did not result in
higher receptor levels. Expression of beta 2-AR in Haloferax volcanii,
another member of halobacteria, was achieved with a vector carrying
the ferredoxin promoter. The levels of functional receptor as determined
by 125I-cyanopindolol binding were 180 fmol/mg protein. The beta-AR
ligands isoprenaline and propranolol showed affinities expected for
functional beta 2-ARs. Thus, functional human beta 2-ARs were expressed
in halobacteria, constituting a first approach for expression of
a eukaryotic protein in the domain of Archaea.
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