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

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

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 Escheri...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS Vol. 108; no. 26; pp. 10437 - 10442
Main Authors: Ou, Weijia, Uno, Tetsuo, Chiu, Hsien-Po, Grünewald, Jan, Cellitti, Susan E, Crossgrove, Tiffany, Hao, Xueshi, Fan, Qian, Quinn, Lisa L, Patterson, Paula, Okach, Linda, Jones, David H, Lesley, Scott A, Brock, Ansgar, Geierstanger, Bernhard H
Format: Journal Article
Language:English
Published: United States National Academy of Sciences 28-06-2011
National Acad Sciences
Subjects:
Adducts
Amino acids
Biochemistry
Biological Sciences
Biosynthesis
biotin
Codons
Culture Media
E coli
enzymes
Escherichia coli
Escherichia coli - genetics
ethylene glycol
fluorescence
Gene expression
Genetic code
Lysine - chemistry
mammals
Nuclear Magnetic Resonance, Biomolecular
oligonucleotides
oligosaccharides
peptides
Pharmaceutical chemistry
pharmacology
polyethylene glycol
protein synthesis
Proteins
Proteins - chemistry
Pyrroles - chemistry
Reagents
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Summary: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.
Bibliography:http://dx.doi.org/10.1073/pnas.1105197108
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1W.O. and T.U. contributed equally to this work.
Author contributions: W.O., T.U., S.E.C., S.A.L., A.B., and B.H.G. designed research; W.O., T.U., H.-P.C., J.G., S.E.C., T.C., X.H., Q.F., L.L.Q., P.P., L.O., D.H.J., A.B., and B.H.G. performed research; W.O., T.U., H.-P.C., J.G., S.E.C., X.H., and A.B. contributed new reagents/analytic tools; W.O., T.U., H.-P.C., J.G., S.E.C., T.C., D.H.J., S.A.L., A.B., and B.H.G. analyzed data; and W.O., T.U., H.-P.C., J.G., S.E.C., T.C., D.H.J., A.B., and B.H.G. wrote the paper.
Edited* by Peter G. Schultz, The Scripps Research Institute, La Jolla, CA, and approved May 11, 2011 (received for review April 4, 2011)
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.1105197108