A biosynthetic Membrane-Anchor/Protein System Based on a Genetically Encoded “Aldehyde Tag”

Abstract

The covalent binding of an aldehyde side-chain containing protein to a lipid with an aminooxy -modified head group opens a versatile avenue to bio-functionalize lipid membranes without compromising function and dynamic properties of both the protein and the lipid membrane. It was recently found that the site-specific 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 turn the cysteine in the consensus motif into a formylglycine, thus leading to the site-specific introduction of an aldehyde side chain for further chemical modification. We have engineered the consensus sequence to the C-terminus of an Enhanced Green Fluorescence Protein (EGFP) which was co-expressed with FGE in E. Coli. Lipids were chemically modified to bear a reactive aminooxy group and then conjugated with the aldehyde tagged EGFP. The resulting EGFP-lipid constructs were successfully incorporated into solid supported lipid bilayer as verified by fluorescence microscopy. Membrane integrity as well as protein and lipid motilities were investigated using both fluorescence recovery after photo-bleaching and fluorescence correlation spectroscopy. In order to determine integration efficiency, the surface concentration of EGFP-lipid constructs was monitored as a function of their solution concentration and incubation time. This site specific lipidation strategy promises to allow for the use of a variety of possible lipid anchors as well as to provide unprecedented freedom in the choice of the lipidation site on the protein.