Phage selection for site-specific incorporation of unnatural amino acids into proteins in vivo.
The development of a method for the site-specific incorporation of unnatural amino acids into proteins in vivo would significantly facilitate studies of the cellular function of proteins, as well as make possible the synthesis of proteins with novel structures and activities. Our approach to this problem consists of the generation of amber suppressor tRNA/aminoacyl-tRNA synthetase pairs that are not catalytically competent with all the endogenous Escherichia coli tRNAs and aminoacyl-tRNA synthetases, followed by directed evolution of such orthogonal aminoacyl-tRNA synthetases to alter their amino acid specificities. To evolve the desired amino acid specificity, a direct selection for site-specific incorporation of unnatural amino acids into a reporter epitope displayed on the surface of M13 phage has been developed and characterized. Under simulated selection conditions, phage particles displaying aspartate were enriched over 300-fold from a pool of phage displaying asparagine using monoclonal antibodies raised against the aspartate-containing epitope. The direct phage selection offers high specificity for the amino acid of interest, eliminating the potential for contamination with synthetases active towards wild-type amino acids in multiple rounds of selection.
Pastrnak
M
M
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Schultz
P G
PG
D000596:Amino Acids / Q000276:immunology / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017104:Bacteriophage M13 / Q000276:immunology / Q000378:metabolism; D003090:Coliphages / Q000276:immunology / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D000939:Epitopes / Q000378:metabolism; D004926:Escherichia coli; D010455:Peptides / Q000276:immunology / Q000302:isolation & purification; D014176:Protein Biosynthesis; D011506:Proteins / Q000276:immunology; D012343:RNA, Transfer / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012680:Sensitivity and Specificity
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000939:Epitopes; D010455:Peptides; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl; D012343:RNA, Transfer