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 aci ds prescribed by the genetic code. Such an expansion is demonstrated here b y 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 cellu lar translation machinery. This is achieved during fermentation with tyrosi ne-auxotrophic E. coli host strain AT 2471 under the efficient selective pr essure. Since these halo-tyrosines show pharmaceutical activities, proteins substituted with these amino acid analogues might serve as shuttles for th eir specific delivery into target tissues. Such 'second' coding level in th e frame of the genetic code represents a novel form of protein engineering. (C) 2000 Elsevier Science Ltd. All rights reserved.