title: Protein-liposome conjugates using cysteine-lipids and native chemical ligation.
abstract: Liposomes have become popular drug delivery vehicles and have more recently also been applied as contrast agents for molecular imaging. Most current methods for functionalization of liposomes with targeting proteins rely on reactions of amine or thiol groups at the protein exterior, which generally result in nonspecific conjugation at multiple sites on the protein. In this study, we present native chemical ligation (NCL) as a general method to covalently couple recombinant proteins in a highly specific and chemoselective way to liposomes containing cysteine-functionalized phospholipids. A cysteine-functionalized phospholipid (Cys-PEG-DSPE) was prepared and shown to readily react with the MESNA thioester of EYFP, which was used as a model protein. Characterization of the EYFP-liposomes using fluorescence spectroscopy showed full retention of the fluorescent properties of conjugated EYFP and provides a lower limit of 120 proteins per liposome. The general applicability of NCL was further tested using CNA35, a collagen-binding protein recently applied in fluorescent imaging of collagen. NCL of CNA35 thioester yielded liposomes containing approximately 100 copies of CNA35 per liposome. The CNA35-liposomes were shown to be fully functional and bind collagen with a 150-fold higher affinity compared to CNA35. Our results show that NCL is an attractive addition to existing conjugation methods that allows direct, covalent, and highly specific coupling of recombinant proteins to liposomes and other lipid-based assemblies.
authors.affiliation: Laboratory of Macromolecular and Organic Chemistry, and Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, and SyMO-Chem BV, P.O. Box 513, 5600 MB Eindhoven, The Netherlands.
authors.forename: Sanne W A
authors.initials: SW
authors.lastname: Reulen
authors.forename: Wilco W T
authors.initials: WW
authors.lastname: Brusselaars
authors.forename: Sander
authors.initials: S
authors.lastname: Langereis
authors.forename: Willem J M
authors.initials: WJ
authors.lastname: Mulder
authors.forename: Monica
authors.initials: M
authors.lastname: Breurken
authors.forename: Maarten
authors.initials: M
authors.lastname: Merkx
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003094:Collagen / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D004337:Drug Carriers; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes; D008164:Luminescent Proteins / Q000737:chemistry / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001426:Bacterial Proteins; D004337:Drug Carriers; D008051:Lipid Bilayers; D008081:Liposomes; D008164:Luminescent Proteins; D010714:Phosphatidylethanolamines; D010743:Phospholipids; C107570:polyethylene glycol-distearoylphosphatidylethanolamine; C054562:yellow fluorescent protein, Bacteria; D011092:Polyethylene Glycols; D003094:Collagen; D003545:Cysteine