Expression of ‘Tailor-Made’ Proteins via Incorporation of Synthetic Amino Acids by Using Cell-Free Protein Synthesis
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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
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