کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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2563374 | 1127523 | 2010 | 32 صفحه PDF | دانلود رایگان |
The Sf9 cell/baculovirus expression system is widely used for high-level protein expression, often with the purpose of purification. However, proteins may also be functionally expressed in the defined Sf9 cell environment. According to the literature, the pharmacology of G-protein-coupled receptors (GPCRs) functionally reconstituted in Sf9 cells is similar to the receptor properties in mammalian cells. Sf9 cells express both recombinant GPCRs and G-proteins at much higher levels than mammalian cells. Sf9 cells can be grown in suspension culture, providing an inexpensive way of obtaining large protein amounts. Co-infection with various baculoviruses allows free combination of GPCRs with different G-proteins. The absence of constitutively active receptors in Sf9 cells provides an excellent signal-to background ratio in functional assays, allowing the detection of agonist-independent receptor activity and of small ligand-induced signals including partial agonistic and inverse agonistic effects. Insect cell Gαi-like proteins mostly do not couple productively to mammalian GPCRs. Thus, unlike in mammalian cells, Sf9 cells do not require pertussis toxin treatment to obtain a Gαi-free environment. Co-expression of GPCRs with Gαi1, Gαi2, Gαi3 or Gαo in Sf9 cells allows the generation of a selectivity profile for these Gαi/o-isoforms. Additionally, GPCR–G-protein combinations can be compared with defined 1:1 stoichiometry by expressing GPCR–Gα fusion proteins. Sf9 cells can also be employed for ligand screening in medicinal chemistry programs, using radioligand binding assays or functional assays, like the steady-state GTPase- or [35S]GTPγS binding assay. This review shows that Sf9 cells are a versatile model system to investigate the pharmacological properties of GPCRs.
Journal: Pharmacology & Therapeutics - Volume 128, Issue 3, December 2010, Pages 387–418