title: Multisite clickable modification of proteins using lipoic acid ligase.
abstract: Approaches that allow bioorthogonal and, in turn, site-specific chemical modification of proteins present considerable opportunities for modulating protein activity and stability. However, the development of such approaches that enable site-selective modification of proteins at multiple positions, including internal sites within a protein, has remained elusive. To overcome this void, we have developed an enzymatic approach for multisite clickable modification based on the incorporation of azide moieties in proteins using lipoic acid ligase (LplA). The ligation of azide moieties to the model protein, green fluorescent protein (GFP), at the N-terminus and two internal sites using lipoic acid ligase was shown to proceed efficiently with near-complete conversion. Modification of the ligated azide groups with poly(ethylene glycol) (PEG), α-d-mannopyranoside, and palmitic acid resulted in highly homogeneous populations of protein-polymer, protein-sugar, and protein-fatty acid conjugates. The homogeneity of the conjugates was confirmed by mass spectrometry (MALDI-TOF) and SDS-PAGE electrophoresis. In the case of PEG attachment, which involved the use of strain-promoted azide-alkyne click chemistry, the conjugation reaction resulted in highly homogeneous PEG-GFP conjugates in less than 30 min. As further demonstration of the utility of this approach, ligated GFP was also covalently immobilized on alkyne-terminated self-assembled monolayers. These results underscore the potential of this approach for, among other applications, site-specific multipoint protein PEGylation, glycosylation, fatty acid modification, and protein immobilization.
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, United States.
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Plaks
authors.affiliation: ‡Department of Chemical, Paper, and Biomedical Engineering, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Falatach
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, United States.
authors.forename: Mark
authors.initials: M
authors.lastname: Kastantin
authors.affiliation: ‡Department of Chemical, Paper, and Biomedical Engineering, Miami University, Oxford, Ohio 45056, United States.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Berberich
authors.affiliation: †Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309, United States.
authors.forename: Joel L
authors.initials: JL
authors.lastname: Kaar
mesh_terms: D001386:Azides / Q000737:chemistry* / Q000378:metabolism; D057930:Click Chemistry / Q000379:methods; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D008958:Models, Molecular; D011092:Polyethylene Glycols / Q000737:chemistry / Q000378:metabolism; D008063:Thioctic Acid / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001386:Azides; D005227:Fatty Acids; D055767:Immobilized Proteins; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D008063:Thioctic Acid; D008025:Ligases