A simple and efficient method is described for introduction of non-canonical amino acids at multiple, structurally defined sites within recombinant polypeptide sequences. E. coli MRA30, a bacterial host strain with attenuated activity for release factor 1 (RF1), is assessed for its ability to support the incorporation of a diverse range of non-canonical amino acids in response to multiple encoded amber (TAG) codons within genetic templates derived from superfolder GFP and an elastin-mimetic protein polymer. Suppression efficiency and isolated protein yield were observed to depend on the identity of the orthogonal aminoacyl-tRNA synthetase/tRNACUA pair and the non-canonical amino acid substrate. This approach afforded elastin-mimetic protein polymers containing non-canonical amino acid derivatives at up to twenty-two positions within the repeat sequence with high levels of substitution. The identity and position of the variant residues was confirmed by mass spectrometric analysis of the full-length polypeptides and proteolytic cleavage fragments resulting from thermolysin digestion. The accumulated data suggest that this multi-site suppression approach permits the preparation of protein-based materials in which novel chemical functionality can be introduced at precisely defined positions within the polypeptide sequence.

Choose one: A A = Approved for public release; distribution is unlimited B = US Gov't agencies only --must have an approved reason for using this distribution An intriguing discovery reported in Science Magazine, in 2004 by Crookes and colleagues, outlined the first look at an unusual family of natural protein-based polymers called reflectins. Reflectins are characterized by unique spectral and optical properties. Our interest in elucidation of a functional unit that drives self-assembly of reflectin-based materials led us to develop an approach for systematic investigation of a library of reflectin-based protein sequences. Cassette-based DNA cloning and concatemerization techniques were used to synthesize genes encoding tandem repeats of reflectin-based amino acid sequences. Reflectin constructs of interest included a reflectin 1a domain 3 (D3) monomer, a domain 3 dimer, subdomain peptides, recombinant reflectin 1b, an elastin-reflectin diblock copolymer, and an elastin-reflectin-GFP fusion protein. After construction of the sequences of interest at the DNA level, protein expression was carried out in a bacterial host. Approximately 10-20 mg protein/L cell culture yields were obtained for each construct. Preliminary structural characterization was performed. The unique spectral properties associated with recombinant reflectin protein materials make elastin-reflectin chimeric protein polymers attractive potential targets for biomaterial engineering applications.