Expression of ‘Tailor-Made’ Proteins via Incorporation of Synthetic Amino Acids by Using Cell-Free Protein Synthesis
- 158 Downloads
In the past decade, it became clear that the set of 20 canonical amino acids prescribed by the universal genetic code does not span all dimensions of chemical variability that could be potentially advantageous for functional diversification of proteins, especially in a user-defined environment. Recent examples of such artificial (i.e., tailor-made) proteins include functional design of novel classes of protein pH sensors, novel classes of green fluorescent proteins and fluorous proteins with enhanced stability and inertia [1–3]. To break through the limits of traditional protein engineering approaches that combine the standard set of canonical amino acids as basic building blocks in protein synthesis, different methods for the expansion of the amino acid repertoire have been described in the literature . From these experimental efforts two main research directions have emerged. First, canonical amino acid attached on cognate tRNA can be chemically or enzymatically modified or even loaded onto desired tRNA before entering the ribosome . Second, the amino acid selection process in living cells could be affected by experimentally imposed selective pressure-a selective pressure incorporation (SPI) approach  mainly employed by using metabolically engineered (e.g.,auxotrophic) host expression cells with modified or even “orthogonal” translational components .
KeywordsGreen Fluorescent Protein Genetic Code Active Amino Acid Synthetic Amino Acid Universal Genetic Code
Unable to display preview. Download preview PDF.
- 2.Bae J, Rubini M, Jung G,Wiegand G, Seifert MHJ, Azim MK, Kim JS, Zumbusch A, Holak TA, Moroder L, Huber R, Budisa N (2003) Expansion of the genetic code enables design of a novel “gold” class of green fluorescent proteins. J Mol Biol 328: 977–1202Google Scholar
- 4.Minks C, Alefelder S, Huber R, Moroder L, Budisa N (2000) Towards new protein engineering: in vivo building and folding of protein shuttles for drug delivery and targeting by the selective pressure incorporation ( SPI) method. Tetrahedron 56: 9431–9442Google Scholar
- 8.Lossau H, Kummer A, Heinecke R, Pollinger-Dammer F, Kompa C, Bieser G, Jonsson T, Silva CM, Yang MM, Youvan DC, Michel-Beyerle ME (1996) Time-resolved spectroscopy of wild-type and mutant green fluorescent proteins reveals excited state deprotonation consistent with fluorophore-protein interactions Chem Phys 213: 1–16CrossRefGoogle Scholar
- 9.Bae,JH, Alefelder S, Kaiser JT, Friedrich R, Moroder L, Huber R, Budisa N (2001) Incorporation of fl-selenolo[3,2-b]pyrrolyl-alanine into proteins for phase determination in protein X-ray crystallography. J Mol Biol 309: 925–936Google Scholar
- 11.Budisa N, Steipe B, Demange P, Eckerskorn C, Kellermann J, Huber R (1995) High-level biosynthetic substitution of methionine in proteins by its analogues 2-aminohexanoic acid, selenomethionine, telluromethionine and ethionine in Escherichia coli. Eur J Biochem 230: 788–796PubMedCrossRefGoogle Scholar
- 14.Budisa N, Minks C, Medrano FJ, Lutz J, Huber R, Moroder L (1998) Residue-specific bioincorporation of non-natural biologically active amino acids into proteins as possible drug carriers. Structure and stability of per-thiaproline mutant of annexin V. Proc Natl Acad Sci USA 95: 455–459Google Scholar