Abstract
The human bradykinin B2 receptor (B2R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast Pichia pastoris. In the expression vector, B2R gene was driven under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and Western blot analysis, it was proved that B2R recombinant receptor proteins were expressed at a high level in the yeast. Furthermore, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B2R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy.
Similar content being viewed by others
References
Hall, J.M., Bradykinin Receptors, Gen. Pharmacol., 1997, vol. 28, pp. 1–6.
Marceau, F. and Regoli, D., Bradykinin Receptor Ligands: Therapeutic Perspectives, Nat. Rev. Drug. Discov., 2004, vol. 3, pp. 845–852.
Eggerickx, D., Raspe, E., Bertrand, D., et al., Molecular Cloning, Functional Expression and Pharmacological Characterization of a Human Bradykinin B Receptor Gene, Biochem. Biophys. Res. Commun., 1992, vol. 187, pp. 1306–1313.
Guadalupe, R.C., Jose, V.R., Werner, M.E., et al., Regulation of the Human Bradykinin B Receptor Expressed in sf21 Insect Cells: A Possible Role for Tyrosine Kinases, J. Cell Biochem., 2000, vol. 76, pp. 658–673.
Hess, J.F., Borkowski, J.A., Young, G.S., et al., Cloning and Pharmacological Characterization of a Human Bradykinin (BK-2) Receptor, Biochem. Biophys. Res. Commun., 1992, vol. 184, pp. 260–268.
Zhang, S.P., Wang, H.Y., Lovenberg, T.W., et al., Functional Studies of Bradykinin Receptors in Chinese Hamster Ovary Cells Stably Expressing the Human B2 Bradykinin Receptor, Int. Immunopharmacol., 2001, vol. 1, pp. 955–965.
Reiländer, H. and Wei, H.M., Production of G-Protein-Coupled Receptors in Yeast, Curr. Opin. Biotechnol., 1998, vol. 9, pp. 510–517.
Weiß, H.M., Haase, W., and Michel, H., Expression of Functional Mouse 5-HT5A Serotonin Receptor in the Methylotrophic Yeast Pichia pastoris: Pharmacological Characterization and Localization, FEBS Lett., 1995, vol. 377, pp. 451–456.
Boettner, M., Prinz, B., Holz, C., et al., High-Throughput Screening for Expression of Heterologous Proteins in the Yeast Pichia pastoris, J. Biotechnol., 2002, vol. 99, pp. 51–62.
Cereghino, J.L. and Cregg, J.M., Heterologous Expression in the Methylotrophic Yeast Pichia pastoris, FEMS Microbiol., 2000, vol. 24, pp. 45–66.
Sarramegna, V., Talmont, F., Roch, M.S., et al., Green Fluorescent Protein as a Reporter of Human μ-Opioid Receptor Overexpression and Localization in the Methylotrophic Yeast Pichia pastoris, J. Biotechnol., 2002, vol. 99, pp. 23–39.
Feng, W.K., Cai, J., Pierce, W.M., et al., Expression of CB2 Cannabinoid Receptor in Pichia pastoris, Protein Expres. Purif., 2002, vol. 26, pp. 496–505.
Prasher, D.C., Eckenrode, V.K., Ward, W.W., et al., Primary Structure of the Aequorea Victoria Green Fluorescent Protein, Gene, 1992, vol. 111, pp. 229–233.
Chalfie, M., Tu, Y., Eukirchen, G., et al., Green Fluorescent Protein as a Marker for Gene Expression, Science, 1994, vol. 263, pp. 802–805.
Barak, L.S., Ferguson, S.S., Zhang, J., et al., Internal Trafficking and Surface Mobility of a Functionally Intact Beta2-Adrenergic Receptor-Green Fluorescent Protein Conjugate, Mol. Pharmacol., 1997, vol. 51, pp. 177–184.
Tarasova, N.I., Stauber, R.H., Choi, J.K., et al., Visualization of G Protein-Coupled Receptor Trafficking with the Aid of the Green Fluorescent Protein: Endocytosis and Recycling of Cholecystokinin Receptor Type A, J. Biol. Chem., 1997, vol. 272, pp. 14817–17824.
Li, Z.Y., Beker, J., and Noe, C.R., Functional Expression of Recombinant N-Methyl-D-Aspartate Receptors in the Yeast Saccharomyces cerevisiae: Localization and Pharmacological Characterization, Eur. J. Biochem., 1998, vol. 252, pp. 391–399.
Pringle, J.R., Adams, A.E.M., Drubin, D.G., et al., Immunofluorescence Methods for Yeast, Methods Enzymol., 1991, vol. 194, pp. 565–602.
Hecquet, C., Becker, R.P., Tan, F., et al., Kallikreins When Activating Bradykinin B2 Receptor Induce Its Redistribution on Plasma Membrane, Int. Immunopharmacol., 2002, vol. 2, pp. 1795–1806.
Author information
Authors and Affiliations
Additional information
The text was submitted by the authors in English.
Rights and permissions
About this article
Cite this article
Yang, G.X., Liu, T.L., Zhang, H. et al. Expression and localization of recombinant human B2 receptors in the methylotrophic yeast Pichia pastoris . Russ J Genet 42, 728–731 (2006). https://doi.org/10.1134/S1022795406070040
Received:
Issue Date:
DOI: https://doi.org/10.1134/S1022795406070040