Recognition of Non-α-amino Substrates by Pyrrolysyl-tRNA Synthetase

Pyrrolysine Analogs as Substrates for Bacterial Pyrrolysyl-tRNA Synthetasein Vitroandin Vivo

“…In this reaction, about half of the tRNA was aminoacylated and a band derived from the Lys(Cyc)-tRNA complex was observed above the nonacylated tRNA. Similarly to the case of Lys(Cyc) (1), Lys(Proc) (8), Lys(Aloc) (11), and Lys(Meoc) (15) were well recognized by DhPylRS and the aminoacylated tRNAs were observed as thick bands on the gel. Lys(Aloc) (11) gave the thickest band of the all substrates tested, and the band derived from nonacylated tRNA was detected as only a faint band.…”

“…14,15) By the use of this pair, various Lys derivatives were incorporated into recombinant proteins, and the pair was applied to the sitespecific modification of recombinant proteins in which a non-canonical amino acid was used as a target functional group. [5][6][7][8][9][10] On the other hand, although it has been found that DhPylRS functions in E. coli cells with D. hafnience tRNA Pyl as an orthogonal pair 12) and is presumed to recognize wide variety of Lys derivatives as substrates, similarly to the archaeal PylRS, no direct evidence as to which various Lys derivatives are incorporated into recombinant proteins by the E. coli expression system has been found. In the present study, we investigated the substrate specificity of bacterial DhPylRS in vitro and the orthogonality of the DhPylRStRNA…”

“…3). Bands derived from the GST mutant were observed when Lys(Cyc) (1), Lys(Proc) (8), or Lys(Aloc) (11) was added to the culture medium, indicating that adding these lysine derivatives suppressed the UAG amber codon with incorporation of them into the recombinant protein through the function of the DhPylRS/tRNA…”

“…4) This is consistent with previous reports that PylRS recognized not only Pyl but also various Lys derivatives carrying an acyl group attached to the sidechain amino group. [5][6][7][8][9][10] In particular, N " -cyclopentyloxycarbonyl-lysine [Lys(Cyc)] has been found to be a good substrate compatible with Pyl, and is widely used in the enzymology of PylRS.…”

Protein Engineering with Non-Natural Amino Acids

“…However, the Pyl-tRNA is not hardwired for amber codon suppression, and can be adapted to decode a number of other codons (Ambrogelly et al, 2007). In addition, the PylRS has been shown to readily accept a variety of side chain structures (Polycarpo et al, 2006;Yanagisawa et al, 2008;Neumann et al, 2008;Chen et al, 2009;Nguyen et al, 2009;Li et al, 2009;Hancock et al, 2010;Ou et al, 2011;Plass et al, 2011;Takimoto et al, 2011) as well as a set of non-alpha amino derivatives (Kobayashi et al, 2009). This feature makes the PylRS/tRNA pair an ideal candidate for site specific integration of NNAAs.…”