Abstract
The receptor for human endothelial differentiation gene-1 protein (EDG-1) was C-terminally tagged with green fluorescent protein and expressed in the methylotrophic yeast, Pichia pastoris. EDG-1 expression was driven by the highly inducible alcohol oxidase 1 promoter. Expression of EDG-1 recombinant protein was detected by Western blot analysis and confocal microscopy. The recombinant EDG-1 receptor protein was located in the plasma membrane. Radioligand binding assays demonstrated that the␣EDG-1 receptors expressed in Pichia pastoris␣have specific and saturation binding of 32P-labeled sphingosine 1-phosphate.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Beer MS, Stanton JA, Salim K, Rigby M, Heavens RP, Smith D, McAllister G (2000) EDG receptors as a therapeutic target in the nervous system. Ann. N. Y. Acad. Sci. 905:118–131
Boettner M, Prinz B, Holz C, Stahl U, Lang C (2002) High-throughput screening for expression of heterologous proteins in the yeast Pichia pastoris. J. Biotechnol. 99:51–62
Cereghino JL, Cregg JM (2000) Heterologous expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24:45–66
Feng WK, Cai J, Pierce WM, Song ZH (2002) Expression of CB2 cannabinoid receptor in Pichia pastoris. Protein Expres. Purif. 26:496–505
Kluk MJ, Hla T (2002) Signaling of sphingosine-1-phosphate via the S1P/EDG-family of G-protein-coupled receptors. Biochim. Biophys. Acta 1582:72–80
Koichi S, Michio U, Fumikazu O (2000) Differential roles of Edg-1 and Edg-5, sphingosine 1-phosphate receptors, in the signaling pathways in C6 glioma cells. Mol. Brain Res. 85:151–160
Lee MJ, Van BJR, Thangada S, Liu CH, Hand AR, Menzeleev R, Spiegel S, Hla T (1998) Sphingosine-1-phosphate as a ligand for the G protein-coupled receptor EDG-1. Science 279:1552–1555
Li ZY, Beker J, Noe CR (1998) Functional expression of recombinant N-methyl-d-aspartate receptors in the yeast Saccharomyces cerevisiae Localization and pharmacological characterization. Eur. J. Biochem. 252:391–399
Liu CH, Thangada S, Lee MJ, Brocklyn JRV, Spiegel S, Hla T (1999) Ligand-induced trafficking of the sphingosine-1-phosphate receptor EDG-1. Molec. Biol. Cell 10:1179–1190
Reiländer H, Weiss HM (1998) Production of G-protein-coupled receptors in yeast. Curr. Opin. Biotechnol. 9:510–517
Saba JD, Hla T (2004) Point-counterpoint of sphingosine 1-phosphate metabolism. Circ. Res. 94:724–734
Sarramegna V, Talmont F, Roch MS, Milon A, Demange P (2002) Green fluorescent protein as a reporter of human μ-opioid receptor overexpression and localization in the methylotrophic yeast Pichia pastoris. J. Biotechnol. 99:23–39
Pitson SM, D’andrea RJ, Vandeleur L, Moretti PA, **a P, Gamble JR, Vadas MA, Wattenberg BW (2000) Human sphingosine kinase: purification, molecular cloning and characterization of the native and recombinant enzymes. Biochem. J. 350:429–441
Weiss HM, Haase W, Michel H, Reilander H (1995) Expression of functional mouse 5-HT5A serotonin receptor in the methylotrophic yeast Pichia pastoris: pharmacological characterization and localization. FEBS Lett. 377:451–456
Weiss HM, Haase W, Michel H, Reilander H (1998) Comparative biochemical and pharmacological characterization of the mouse 5HT5A 5-hydroxytryptamine receptor and the human β2- adrenergic receptor produced in the methylotrophic yeast Pichia pastoris. Biochem. J. 330:1137–1147
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Yang, G., Liu, T., Peng, W. et al. Expression and localization of recombinant human EDG-1 receptors in Pichia pastoris . Biotechnol Lett 28, 1581–1586 (2006). https://doi.org/10.1007/s10529-006-9125-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-006-9125-4