A genetically encoded aza-Michael acceptor for covalent cross-linking of protein-receptor complexes.
Selective covalent bond formation at a protein-protein interface potentially can be achieved by genetically introducing into a protein an appropriately "tuned" electrophilic unnatural amino acid that reacts with a native nucleophilic residue in its cognate receptor upon complex formation. We have evolved orthogonal aminoacyl-tRNA synthetase/tRNACUA pairs that genetically encode three aza-Michael acceptor amino acids, N(ε)-acryloyl-(S)-lysine (AcrK, 1), p-acrylamido-(S)-phenylalanine (AcrF, 2), and p-vinylsulfonamido-(S)-phenylalanine (VSF, 3), in response to the amber stop codon in Escherichia coli. Using an αErbB2 Fab-ErbB2 antibody-receptor pair as an example, we demonstrate covalent bond formation between an αErbB2-VSF mutant and a specific surface lysine ε-amino group of ErbB2, leading to near quantitative cross-linking to either purified ErbB2 in vitro or to native cellular ErbB2 at physiological pH. This efficient biocompatible reaction may be useful for creating novel cell biological probes, diagnostics, or therapeutics that selectively and irreversibly bind a target protein in vitro or in living cells.
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Jennifer L
JL
Furman
Mingchao
M
Kang
Seihyun
S
Choi
Yu
Y
Cao
Erik D
ED
Wold
Sophie B
SB
Sun
Vaughn V
VV
Smider
Peter G
PG
Schultz
Chan Hyuk
CH
Kim
D020106:Acrylamide / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000235:genetics; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry / Q000235:genetics; D013449:Sulfonamides / Q000737:chemistry; D000068878:Trastuzumab
D020106:Acrylamide; D000596:Amino Acids; D000604:Amino Acyl-tRNA Synthetases; D061067:Antibodies, Monoclonal, Humanized; D003432:Cross-Linking Reagents; D007140:Immunoglobulin Fab Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; D013449:Sulfonamides; D000068878:Trastuzumab; C440389:vinyl sulfonamide