ISSN:
1432-1912
Schlagwort(e):
Key wordsβ2-Adrenoceptor
;
Bacteriorhodopsin
;
Halobacteriumsalinarium
;
Haloferax volcanii
;
Heterologous expression
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Medizin
Notizen:
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 β2-adrenoceptor (β2-AR), a protein with a similar seven-transmembrane-helix topology. An expression vector for BO synthesis was modified to express β2-ARs under the control of BO regulatory elements 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 β2-ARs were detected in Western blots. However, binding of the β-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 β2-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 β-AR ligands isoprenaline and propranolol showed affinities expected for functional β2-ARs. Thus, functional human β2-ARs were expressed in halobacteria, constituting a first approach for expression of a eukaryotic protein in the domain of Archaea.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/PL00004926
Permalink
