RESUMO
We recently developed a phage-based system for the evolution of proteins in bacteria with expanded amino acid genetic codes. Here we demonstrate that the unnatural amino acid p-boronophenylalanine (BF) confers a selective advantage in the evolution of glycan-binding proteins. We show that an unbiased library of naive antibodies with NNK-randomized V(H) CDR3 loops converges upon mutants containing BF when placed under selection for binding to a model acyclic amino sugar. This work represents a first step in the evolution of carbohydrate-binding proteins that use a reactive unnatural amino acid "warhead" and demonstrates that a "synthetic" genetic code can confer a selective advantage by increasing the number of functional groups available to evolution.
Assuntos
Compostos de Boro/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Evolução Molecular , Fenilalanina/análogos & derivados , Engenharia de Proteínas/métodos , Anticorpos/imunologia , Compostos de Boro/química , Códon sem Sentido , Escherichia coli , Proteínas de Escherichia coli/química , Humanos , Mutação , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Polissacarídeos/imunologia , Polissacarídeos/metabolismo , Seleção GenéticaRESUMO
We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V(H) CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded "synthetic" genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.
