Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system.
Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E. coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2-anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.
Doi
Yoshio
Y
Department of Bioscience and Biotechnology, Okayama University, Okayama, Japan.
Ohtsuki
Takashi
T
Shimizu
Yoshihiro
Y
Ueda
Takuya
T
Sisido
Masahiko
M
D000409:Alanine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis / Q000235:genetics / Q000502:physiology; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism
D000596:Amino Acids; D012346:RNA, Transfer, Amino Acyl; D010444:Peptide Elongation Factor Tu; D000409:Alanine