Extensive proteolysis inhibits high-level production of eukaryal G protein-coupled receptors in the archaeon Haloferax volcanii.
In this study the usage of the halophilic archaeon Haloferax volcanii as a production system for eukaryal G protein-coupled receptors (GPCRs) was characterized. The genes of four GPCRs were fused to the dihydrofolate reductase gene of H. volcanii. In Northern blots both 5' fragments and full-length fusion transcripts were found. In contrast, only C-terminal fusion protein fragments could be detected in Western blot analyses. Ligand binding experiments revealed that a minor amount of correctly folded human beta 2 adrenergic receptor was inserted into the membrane. The introduction of different modifications at the 5' and the 3' end of the receptor genes did not significantly increase the production level. Determination of the subcellular localization showed that fusion protein fragments containing one or more receptor helices were located in the membrane. The results indicate that neither transcription, translation nor membrane translocation but the activity of one or more proteases limits the level of GPCR production in H. volcanii.
Patenge
N
N
Max-Planck-Institut für Biochemie, Martinsried, Germany.
Soppa
J
J
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D019677:Artificial Gene Fusion; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D005822:Genetic Vectors; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018343:Receptors, Adrenergic, beta-2 / Q000096:biosynthesis / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D018042:Receptors, Neurokinin-3 / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics