Premium
Multiple Site‐Selective Insertions of Noncanonical Amino Acids into Sequence‐Repetitive Polypeptides
Author(s) -
Wu ILin,
Patterson Melissa A.,
Carpenter Desai Holly E.,
Mehl Ryan A.,
Giorgi Gianluca,
Conticello Vincent P.
Publication year - 2013
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201300069
Subject(s) - amino acid , thermolysin , escherichia coli , peptide sequence , biochemistry , recombinant dna , transfer rna , cleavage (geology) , chemistry , sequence (biology) , gene , biology , trypsin , enzyme , rna , paleontology , fracture (geology)
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.