title: Generation of human PTH1R construct with FLAG epitope located internally: comparison of two-fragment assembly by using PCR overlap extension or ligase.
abstract: Parathyroid hormone (PTH) regulates bone remodeling and calcium and phosphate homeostasis. PTH actions are mediated by type I PTH/PTH-related peptide receptor (PTH1R). There has been no commercially available, specific antibody to detect human PTH1R expression so far. Flag-tagged human PTH1R construct, converting the sequence DKEAPTGS (residues 94-101) in the exon E2 region of PTH1R to DYKDDDDK of Flag epitope, was generated by using PCR overlap extension or ligase enzyme for two-fragment assembly. We found that Flag-tagged PTH1R assembled by ligase was easy to be manipulated, but its efficiency was lower than that of PCR overlap extension. The PTH1R plasmids generated by both techniques were expressed successfully in vitro and in vivo and possessed the same physiological function as wild-type PTH1R. The Flag-tagged PTH1R construct will provide invaluable tools for study of PTH1R signaling and trafficking.
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA. biw5@pitt.edu
authors.forename: Bin
authors.initials: B
authors.lastname: Wang
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Yang
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D002460:Cell Line; D011088:DNA Ligases / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D005091:Exons / Q000235:genetics; D005813:Genes, Synthetic; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000032:analysis / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014176:Protein Biosynthesis; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000502:physiology; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D009842:Oligopeptides; C473449:PTH1R protein, human; D010455:Peptides; D044168:Receptor, Parathyroid Hormone, Type 1; D011993:Recombinant Fusion Proteins; C085706:FLAG peptide; D015252:Deoxyribonucleases, Type II Site-Specific; C029312:GGTACC-specific type II deoxyribonucleases; D000262:Adenylyl Cyclases; D011088:DNA Ligases