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Abstract:
The non-canonical amino acid (ncAA) analogue of methionine (Met), b-cyclopropylalanine (Cpa),
was successfully incorporated into recombinant proteins expressed in Escherichia coli in a
residue-specific manner. Proteins substituted in this way are congeners because they derive from
the same gene sequence as the parent protein but contain a fraction of ncAAs. We have expressed
congeners using parent and mutant gene sequences of various proteins (lipase, annexin A5,
enhanced green fluorescent protein, and barstar) and found that Cpa incorporation is highly
dependent on the protein sequence composition. These results indicate that the global amino acid
composition of proteins might be a crucial parameter that influences the outcome of unnatural
translation. In addition, we could also demonstrate that the chemical nature of the second residue
could be essential for successful ncAA incorporation.