title: Multiple site-selective insertions of noncanonical amino acids into sequence-repetitive polypeptides.
abstract: A simple and efficient method is described for the introduction of noncanonical amino acids at multiple, defined sites within recombinant polypeptide sequences. Escherichia coli MRA30, a bacterial host strain with attenuated activity of release factor 1 (RF1), was assessed for its ability to support incorporation of a diverse range of noncanonical amino acids in response to multiple encoded amber (TAG) codons within genes derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNA(CUA) pair and the noncanonical amino acid. Elastin-mimetic protein polymers were prepared in which noncanonical amino acid derivatives were incorporated at up to 22 specific sites within the polypeptide sequence with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments from thermolysin digestion. The data suggest that this multisite suppression approach permits the preparation of protein-based materials in which novel chemical functionalities can be introduced at precisely defined positions within the polypeptide sequence.
authors.affiliation: Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.
authors.forename: I-Lin
authors.initials: IL
authors.lastname: Wu
authors.forename: Melissa A
authors.initials: MA
authors.lastname: Patterson
authors.forename: Holly E
authors.initials: HE
authors.lastname: Carpenter Desai
authors.forename: Ryan A
authors.initials: RA
authors.lastname: Mehl
authors.forename: Gianluca
authors.initials: G
authors.lastname: Giorgi
authors.forename: Vincent P
authors.initials: VP
authors.lastname: Conticello
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics*; D004549:Elastin / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000379:methods*; D010455:Peptides / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid
chemical_list: D000596:Amino Acids; D010455:Peptides; D011994:Recombinant Proteins; D004549:Elastin