Production and purification of formyl peptide receptor : explorations of protein-protein interactions
Year: 1997
Type: dissertation
Abstract: Neutrophils play an essential role in fighting bacterial infections.Many neutrophil functions are the result of an interplay between cell surface receptors and their ligands.Formyl peptide receptor (FPR), through the action of its ligand, fMLF, has been shown to mediate neutrophil migratory, as well as cytotoxic functions.This receptor is believed to be organized into seven-transmembrane regions and is coupled to guanosine triphosphate-binding protein (G protein), which is a key component in FPR-mediated signal transduction.Previously, numerous attempts have been made to purify the receptor.In this work, we describe the expression and partial purification of FPR expressed in insect cells infected with recombinant baculovirus.The receptor expressed in these cells had a significantly decreased ligand binding affinity with a dissociation constant of 70 nM, as compared to the receptor on neutrophils with dissociation constant of 3 nM, suggesting that the processing of the receptor may be different.We, therefore, shifted our effort to purify FPR from Chinese hamster ovary (CHO) cells, which were found to bind ligand with a similar affinity as human neutrophils.The purified receptor, as well as intact CHO cells expressing FPR (CHO-FPR), was used in the selection of phage peptide library in order to identify sequences that bind to the receptor.Although no consensus sequence was identified, the use of CHO-FPR cells in affinity purification of phage peptide library allowed the selection of phage that had binding characteristics different from the phage selected using wild-type CHO cells.This was demonstrated by flow cytometry, which proved to be a rapid and efficient method for screening the selected phage.Finally, to further explore the protein-protein binding sites on FPR, and more specifically the interaction between the receptor and G protein, two cytoplasmic tail deletion mutants of FPR were constructed and analyzed for their signal transduction capabilities.Partial calcium release and suppressed chemotaxis by the deletion mutants suggest that the deleted regions are not absolutely necessary in eliciting the FPR-mediated response.Our results support the notion that there are several binding sites between FPR and G protein.
Author: Maria Renata Kohler
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W149171070