Towards New Protein Engineering: In Vivo Building and Folding of Protein Shuttles for Drug Delivery and Targeting by the Selective Pressure Incorporation (SPI) Method

In vivo incorporation of non-canonical amino acids enables an expansion of the amino acid repertoire beyond the standard set of 20 canonical amino acids prescribed by the genetic code. Such an expansion is demonstrated here by reassignment of the tyrosine codons TAT or TAC to the non-canonical amino acids 3-fluoro-tyrosine and 3-chloro-tyrosine, without change of the cellular translation machinery. This is achieved during fermentation with tyrosine–auxotrophic E. coli host strain AT 2471 under the efficient selective pressure. Since these halo-tyrosines show pharmaceutical activities, proteins substituted with these amino acid analogues might serve as shuttles for their specific delivery into target tissues. Such ‘second’ coding level in the frame of the genetic code represents a novel form of protein engineering.