Abstract
The activity and function of proteins can be improved by incorporation of non-canonical amino acids (ncAAs). To avoid the tedious synthesis of a large number of chiral phenylalanine derivatives, we synthesized the corresponding phenylpyruvic acid precursors. Escherichia coli strain DH10B and strain C321.ΔA.expΔPBAD were selected as hosts for phenylpyruvic acid bioconversion and genetic code expansion using the MmPylRS/pyltRNACUA system. The concentrations of keto acids, PLP and amino donors were optimized in the process. Eight keto acids that can be biotransformed and their coupled genetic code expansions were identified. Finally, the genetic encoded ncAAs were tested for incorporation into fluorescent proteins with keto acids.
Highlights
Natural proteins are typically produced from twenty different amino acids as buildingNatural proteins are typically produced from twenty different amino acids as building blocks, sometimes in combination with pyrrolysine and selenocysteine
In the past two decades, this technology, known as genetic code expansion (GCE), was further developed [7,8]. This powerful technology can incorporate non-canonical amino acids (ncAAs) into proteins in a site-specific manner. By introducing both the orthogonal aminoacyl-tRNA synthetase and the nonsense suppressor tRNA into a cell, a ncAA is loaded onto the suppressor tRNA, whose anti-codon recognizes the amber nonsense codon [9] or even a quadruplet codon [10]
We successfully developed a simple method to genetically encode phenylalanine derivatives that could be incorporated into a protein by supplementation of their α-keto acid precursors
Summary
Natural proteins are typically produced from twenty different amino acids as buildingNatural proteins are typically produced from twenty different amino acids as building blocks, sometimes in combination with pyrrolysine and selenocysteine. In the past two decades, this technology, known as genetic code expansion (GCE), was further developed [7,8]. This powerful technology can incorporate ncAAs into proteins in a site-specific manner. By introducing both the orthogonal aminoacyl-tRNA synthetase (aaRS) and the nonsense suppressor tRNA into a cell, a ncAA is loaded onto the suppressor tRNA, whose anti-codon recognizes the amber nonsense codon [9] or even a quadruplet codon [10]. The cell’s translation machinery introduces the ncAAs into the stop codon or quadruplet codon of the target gene
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