Site-specific protein modifications through pyrroline-carboxy-lysine residues.

Genomics Institute of Novartis Research Foundation, 10675 John-Jay-Hopkins Drive, San Diego, CA 92121-1125, USA.

Proceedings of the National Academy of Sciences of the United States of America 2011 Jun 28

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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.

Citation

Weijia Ou, Tetsuo Uno, Hsien-Po Chiu, Jan Grünewald, Susan E Cellitti, Tiffany Crossgrove, Xueshi Hao, Qian Fan, Lisa L Quinn, Paula Patterson, Linda Okach, David H Jones, Scott A Lesley, Ansgar Brock, Bernhard H Geierstanger. Site-specific protein modifications through pyrroline-carboxy-lysine residues. Proceedings of the National Academy of Sciences of the United States of America. 2011 Jun 28;108(26):10437-42