title: Site-specific protein modifications through pyrroline-carboxy-lysine residues.
abstract: Pyrroline-carboxy-lysine (Pcl) is a demethylated form of pyrrolysine that is generated by the pyrrolysine biosynthetic enzymes when the growth media is supplemented with D-ornithine. Pcl is readily incorporated by the unmodified pyrrolysyl-tRNA/tRNA synthetase pair into proteins expressed in Escherichia coli and in mammalian cells. Here, we describe a broadly applicable conjugation chemistry that is specific for Pcl and orthogonal to all other reactive groups on proteins. The reaction of Pcl with 2-amino-benzaldehyde or 2-amino-acetophenone reagents proceeds to near completion at neutral pH with high efficiency. We illustrate the versatility of the chemistry by conjugating Pcl proteins with poly(ethylene glycol)s, peptides, oligosaccharides, oligonucleotides, fluorescence, and biotin labels and other small molecules. Because Pcl is genetically encoded by TAG codons, this conjugation chemistry enables enhancements of the pharmacology and functionality of proteins through site-specific conjugation.
authors.affiliation: Genomics Institute of Novartis Research Foundation, 10675 John-Jay-Hopkins Drive, San Diego, CA 92121-1125, USA.
authors.forename: Weijia
authors.initials: W
authors.lastname: Ou
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Uno
authors.forename: Hsien-Po
authors.initials: HP
authors.lastname: Chiu
authors.forename: Jan
authors.initials: J
authors.lastname: Grünewald
authors.forename: Susan E
authors.initials: SE
authors.lastname: Cellitti
authors.forename: Tiffany
authors.initials: T
authors.lastname: Crossgrove
authors.forename: Xueshi
authors.initials: X
authors.lastname: Hao
authors.forename: Qian
authors.initials: Q
authors.lastname: Fan
authors.forename: Lisa L
authors.initials: LL
authors.lastname: Quinn
authors.forename: Paula
authors.initials: P
authors.lastname: Patterson
authors.forename: Linda
authors.initials: L
authors.lastname: Okach
authors.forename: David H
authors.initials: DH
authors.lastname: Jones
authors.forename: Scott A
authors.initials: SA
authors.lastname: Lesley
authors.forename: Ansgar
authors.initials: A
authors.lastname: Brock
authors.forename: Bernhard H
authors.initials: BH
authors.lastname: Geierstanger
mesh_terms: D003470:Culture Media; D004926:Escherichia coli / Q000235:genetics; D008239:Lysine / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011506:Proteins / Q000737:chemistry*; D011758:Pyrroles / Q000737:chemistry*
chemical_list: D003470:Culture Media; D011506:Proteins; D011758:Pyrroles; C013231:pyrroline; D008239:Lysine