Department
Biological Sciences
Major
Biochemical Engineering
Research Advisor
Aronstam, Robert
Advisor's Department
Biological Sciences
Funding Source
UMR cDNA Resource Center
Abstract
Epitope tagging of G-protein coupled receptors (GPCRs) typically involves the addition of a specific antigenic amino acid sequence (e.g., hemagglutinin) to the N-terminus, a region not involved in receptor ligand binding or signal transduction functions. However, with certain GPCRs this method fails due to posttranslational cleavage of the N-terminal tag or inhibition of membrane isertion. To overcome this, we modified a cloning vector so that it would insert an artificial cleavage site at the N-terminus of the protein before the epitope. This ensures that a functional receptor will be expressed and properly inserted into the membrane. Clones of an opioid and calcitonin-like receptor (the tagged versions of which are subject to improper post-translational processing) were tagged using this new vector. The modified clones were expressed in a mammalian cell line and visualized using immunofluorescence. This method offers an efficient means to create immunologically identifiable constructs of human GPCRs.
Biography
Amanda Sutterer is a sophomore in Biochemical Engineering from Jackson Missouri who is active in RHA and plans to either go to graduate school or enter the field of research and development.
Research Category
Natural Sciences
Presentation Type
Poster Presentation
Document Type
Poster
Location
Havener Center, Carver-Turner Room
Presentation Date
11 April 2007, 1:00 pm - 3:00 pm
Novel Method to Express Functional Epitope-Tagged GPCRs
Havener Center, Carver-Turner Room
Epitope tagging of G-protein coupled receptors (GPCRs) typically involves the addition of a specific antigenic amino acid sequence (e.g., hemagglutinin) to the N-terminus, a region not involved in receptor ligand binding or signal transduction functions. However, with certain GPCRs this method fails due to posttranslational cleavage of the N-terminal tag or inhibition of membrane isertion. To overcome this, we modified a cloning vector so that it would insert an artificial cleavage site at the N-terminus of the protein before the epitope. This ensures that a functional receptor will be expressed and properly inserted into the membrane. Clones of an opioid and calcitonin-like receptor (the tagged versions of which are subject to improper post-translational processing) were tagged using this new vector. The modified clones were expressed in a mammalian cell line and visualized using immunofluorescence. This method offers an efficient means to create immunologically identifiable constructs of human GPCRs.
