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Sf9 cells: a versatile model system to investigate the pharmacological properties of G protein-coupled receptors.
MedLine Citation:
PMID:  20705094     Owner:  NLM     Status:  In-Process    
Abstract/OtherAbstract:
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 Gα(i)-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 Gα(i)-free environment. Co-expression of GPCRs with Gα(i1), Gα(i2), Gα(i3) or Gα(o) in Sf9 cells allows the generation of a selectivity profile for these Gα(i/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 [(35)S]GTPγS binding assay. This review shows that Sf9 cells are a versatile model system to investigate the pharmacological properties of GPCRs.
Authors:
Erich H Schneider; Roland Seifert
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-08-10
Journal Detail:
Title:  Pharmacology & therapeutics     Volume:  128     ISSN:  1879-016X     ISO Abbreviation:  Pharmacol. Ther.     Publication Date:  2010 Dec 
Date Detail:
Created Date:  2010-11-01     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7905840     Medline TA:  Pharmacol Ther     Country:  England    
Other Details:
Languages:  eng     Pagination:  387-418     Citation Subset:  IM    
Copyright Information:
Copyright © 2010 Elsevier Inc. All rights reserved.
Affiliation:
Laboratory of Molecular Immunology, NIAID/NIH, Bethesda, MD 20892-1886, USA. schneidere@mail.nih.gov
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