Degree Type
Honors Capstone Project
Date of Submission
Spring 5-1-2011
Capstone Advisor
Martin B. Forstner Ph.D.
Honors Reader
Rebecca A. Bader Ph.D
Capstone Major
Physics
Capstone College
Arts and Science
Audio/Visual Component
no
Capstone Prize Winner
no
Won Capstone Funding
yes
Honors Categories
Sciences and Engineering
Subject Categories
Atomic, Molecular and Optical Physics | Physics
Abstract
Lipid membranes or bilayers serve as barriers for the cell and its organelles. A distinguishing feature of cellular function is the ability to monitor and process the biochemical information between the cells’ intra and extracellular environments in order to maintain the homeostatic reactions that are essential to life. Surface membrane proteins play a key role in mediating this information exchange by functionalizing the bilayer. As a result of their importance in living systems, proteins are integrated into biomimetic membrane systems to replicate their natural functions in biomedical technologies. Current protein integration methods are limited by the selection of commercially available lipids for attachment onto the target protein and by the location along the protein in which successful coupling to a lipid anchor may occur. The recent developments of an “aldehyde tag” have shown that the insertion of a 6 amino acid consensus sequence into a protein is sufficient to target it for post-translational modification by formylglycine-generating enzyme (FGE). FGE will enzymatically transform the cysteine in the consensus sequence into a formylglycine, thus leading to the site-specific introduction of an aldehyde side chain for further chemical modification such as lipidation. We introduced the consensus sequence on the C-terminus of an enhanced green fluorescence protein (EGFP) and co-expressed it with FGE in E. Coli to maximize the yield of aldehyde conversion. Lipids were chemically modified to bear a reactive aminooxy group and then covalently attached to the aldehyde tagged EGFP in aqueous solution. The resulting EGFP-lipid constructs were successfully incorporated into a solid supported lipid bilayer as verified by fluorescence microscopy. Membrane integrity as well as protein and lipid mobility was analyzed using fluorescence recovery after photo-bleaching (FRAP). This site-specific lipidation strategy promises to allow for the use of a variety of different lipid anchors and to provide unprecedented freedom in the choice of the lipidation site on the target protein.
Recommended Citation
Zhen, ChaoJie, "A Biosynthetic Membrane-Anchor/Protein System Based on a Genetically Encoded "Aldehyde Tag"" (2011). Renée Crown University Honors Thesis Projects - All. 253.
https://surface.syr.edu/honors_capstone/253
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