title: Site-specific dual labeling of proteins by using small orthogonal tags at neutral pH.
abstract: To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FcεRI, which is a key event in allergic disease.
authors.affiliation: Genomics Institute of the Novartis Research Foundation, 10675 John-Jay-Hopkins Drive, San Diego, CA 92121 (USA).
authors.forename: Jan
authors.initials: J
authors.lastname: Grünewald
authors.forename: David H
authors.initials: DH
authors.lastname: Jones
authors.forename: Ansgar
authors.initials: A
authors.lastname: Brock
authors.forename: Hsien-Po
authors.initials: HP
authors.lastname: Chiu
authors.forename: Badry
authors.initials: B
authors.lastname: Bursulaya
authors.forename: Kenneth
authors.initials: K
authors.lastname: Ng
authors.forename: Todd
authors.initials: T
authors.lastname: Vo
authors.forename: Paula
authors.initials: P
authors.lastname: Patterson
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Uno
authors.forename: James
authors.initials: J
authors.lastname: Hunt
authors.forename: Glen
authors.initials: G
authors.lastname: Spraggon
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Geierstanger
mesh_terms: D015374:Biosensing Techniques; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000031:analogs & derivatives* / Q000737:chemistry; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
chemical_list: C561311:2-amino-6-(3,4-dihydro-2H-pyrrole-2-carboxamido)hexanoic acid; D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins; D008239:Lysine
keywords: FRET; IgE; biosensors; phosphopantetheinyl transferase; pyrroline-carboxy-lysine