Heterologous expression of functional G‐protein‐coupled receptors inCaenorhabditis elegans

Autor: Wenyu Sun, Hui Jin, Kristopher Kramp, David Salom, Zhaoyang Feng, Krzysztof Palczewski, Beata Jastrzebska, Pengxiu Cao
Rok vydání: 2011
Předmět:
Zdroj: The FASEB Journal. 26:492-502
ISSN: 1530-6860
0892-6638
DOI: 10.1096/fj.11-197780
Popis: New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A2A subtype receptor [(h)A2AR] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6–1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A2AR were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.—Salom, D., Cao, P., Sun, W., Kramp, K., Jastrzebska, B., Jin, H., Feng, Z., Palczewski, K. Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans.
Databáze: OpenAIRE
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