Research article summary (published 30 Aug 2009):

Generation of human PTH1R construct with FLAG epitope located internally: comparison of two-fragment assembly by using PCR overlap extension or ligase.

Full 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.

Author information

Author/s: Wang, Bin (B); Yang, Yanmei (Y);

Affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA. biw5(-atsign-)pitt.edu

Journal and publication information

Publication Type: Comparative Study; Evaluation Studies; Journal Article

Journal: Journal of biomolecular techniques : JBT (J Biomol Tech), published in United States. (Language: eng)

Reference: 2009-Sep; vol 20 (issue 4) : pp 195-200

Dates: Created 2009/09/01; Completed 2009/11/04;

PMID: 19721821, status: MEDLINE (last retrieval date: 11/4/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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MeSH headings (categories)

This article was linked to the MESH Headings shown below.

Adenylate Cyclase - metabolism Amino Acid Sequence Amino Acid Substitution Bacteriophage T4 - enzymology Base Sequence Cell Line DNA Ligases - metabolism Deoxyribonucleases, Type II Site-Specific Exons - genetics Genes, Synthetic

Humans Kidney Molecular Sequence Data Peptides - analysis; genetics Polymerase Chain Reaction - methods Protein Biosynthesis Receptor, Parathyroid Hormone, Type 1 - genetics; physiology Recombinant Fusion Proteins - genetics; physiology Sequence Deletion Transfection

Associated Chemicals: PTHR1 protein, human (0) ; Peptides (0) ; Receptor, Parathyroid Hormone, Type 1 (0) ; Recombinant Fusion Proteins (0) ; FLAG peptide (98849-88-8) ; Deoxyribonucleases, Type II Site-Specific (EC 3.1.21.4) ; GGTACC-specific type II deoxyribonucleases (EC 3.1.21.4) ; Adenylate Cyclase (EC 4.6.1.1) ; DNA Ligases (EC 6.5.1.-)

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