title: Semisynthesis of Membrane-Attached Proteins Using Split Inteins.
abstract: The site-selective installation of lipid modifications on proteins is critically important in our understanding of how membrane association influences the biophysical properties of proteins as well as to study certain proteins in their native environment. Here, we describe the use of split inteins for the C-terminal attachment of lipid-modified peptides to virtually any protein of interest (POI) via protein trans-splicing (PTS). To achieve this, the protein of interest is expressed in fusion with the N-terminal split intein segment and the C-terminal split intein segment is prepared by solid phase peptide synthesis. A synthetic peptide carrying two lipid chains is also made chemically to serve as a membrane anchor and subsequently linked to the C-terminal split intein by native chemical ligation. Proteins of interest for our work are the prion protein as well as small GTPases; however, extensions to other POIs are possible. Detailed information for the C-terminal introduction of a lipidated membrane anchor (MA) peptide using split intein systems from Synechocystis spp. and Nostoc punctiforme for the Prion protein (PrP, as a challenging protein of interest) and the enhanced green-fluorescent protein (eGFP, as an easily trackable target protein) are provided here.
authors.affiliation: Department of Chemistry, Institute of Biological Chemistry, University of Vienna, Waehringer Str. 38, 1090, Vienna, Austria.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Hackl
authors.affiliation: Department of Chemistry, Institute of Biological Chemistry, University of Vienna, Waehringer Str. 38, 1090, Vienna, Austria.
authors.forename: Alanca
authors.initials: A
authors.lastname: Schmid
authors.affiliation: Department of Chemistry, Institute of Biological Chemistry, University of Vienna, Waehringer Str. 38, 1090, Vienna, Austria. christian.becker@univie.ac.at.
authors.forename: Christian F W
authors.initials: CF
authors.lastname: Becker
mesh_terms: D047668:Inteins; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D046937:Nostoc / Q000737:chemistry / Q000235:genetics; D046939:Synechocystis / Q000737:chemistry / Q000235:genetics
chemical_list: D008565:Membrane Proteins
keywords: Green fluorescent protein; Lipid-coated particles; Liposomes; Membrane-associated proteins; Prion protein; Protein semisynthesis; Protein trans-splicing; Split inteins; Synthetic membrane anchor