Meng-Lin Tsao

Protein evolution with an expanded genetic code

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 VH 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.

Selective Staudinger modification of proteins containing p-azidophenylalanine.

Various methods have been developed to selectively modify proteins with synthetic agents and probes, and to covalently attach proteins to surfaces. These include semisynthesis, the use of electrophilic reagents that selectively label cysteine and lysine residues, and the selective introduction of amino acids with reactive side chains into proteins by in vitro biosynthesis with chemically aminoacylated tRNAs. Recently, we showed that one can genetically encode unnatural amino acids with reactive side chains directly in prokaryotic and eukaryotic organisms efficiently and with high fidelity. Amino acids with side chains containing keto, acetylenic, and azido groups have been incorporated into proteins in response to unique three-base (nonsense) and four-base (frameshift) codons. These amino acids were subsequently modified selectively with exogenous agents by oxime formation or [2+3] cycloaddition reactions. We now report that this approach can be extended to the modification of proteins with selectively incorporated aryl azide amino acids by using phosphine-derived probes and the Staudinger ligation reaction. The Staudinger ligation has been used previously to modify cell surface carbohydrates in both cellular and in vivo systems. The reaction proceeds with excellent yields under aqueous conditions and is highly selective for azides. The Staudinger ligation has also been used to selectively modify proteins that contain azidohomoalanine substituted for methionine residues. The selectivity of this approach, however, is intrinsically limited since all methionine residues throughout the proteins and proteome are substituted with azidohomoalanine, often in competition with the native amino acid. To overcome these limitations, we have taken advantage of a genetically encoded azide-containing amino acid that is incorporated at defined sites in a protein in response to a nonsense codon. The azide is subsequently modified by a Staudinger ligation to selectively incorporate exogenous reagents into the protein. Previously we demonstrated that the unnatural amino acid, p-azidophenylalanine (pAzPhe), which is essentially unreactive toward biomolecules, can be selectively incorporated into proteins in E. coli by using a heterologous suppressor tRNA–aminoacyl tRNA synthetase pair with altered specificity. Here we show that the Staudinger ligation can be used to modify this amino acid efficiently and selectively with spectroscopic probes in either proteins or phage-displayed peptides. A phage-display system was used in which the streptavidinbinding peptide (SBP) AGXTLLAHPQ was displayed pentavalently as a fusion to the pIII protein of M13 filamentous phage. The N-terminal AG sequence facilitates cleavage of the signal peptide; the third residue, X, encoded by the amber nonsense codon TAG, designates the unnatural amino acid to be incorporated. The phage Ph-Az (encoding SBP with pAzPhe at residue X) was prepared in E. coli strain TTS/RS in the presence of 2 mM pAzPhe with good efficiency and high fidelity. E. coli TTS/RS contains a plasmid that constitutively expresses a Methanococcus jannaschii mutant amber suppressor tRNA CUA (mutRNA CUA) and a mutant M. jannaschii tyrosyl-tRNA synthetase (MjTyrRS) which specifically charges mutRNA CUA with pAzPhe. As a negative control, another SBP-displayed phage (Ph-Q) was prepared in E. coli XL1-Blue, a natural amber suppression strain that incorporates glutamine at residue X. The fluorescein-derived phosphines 1 and 2 (Scheme 1) were used for the Staudinger ligation reaction since they can