Genetically Encoded 1,2-Aminothiols Facilitate Rapid and Site-Specific Protein Labeling via a Bio-orthogonal Cyanobenzothiazole Condensation
ABSTRACT We report evolved orthogonal pyrrolysyl-tRNA synthetase/tRNA(CUA) pairs that direct the efficient, site-specific incorporation of N(ε)-L-thiaprolyl-L-lysine, N(ε)-D-cysteinyl-L-lysine, and N(ε)-L-cysteinyl-L-lysine into recombinant proteins in Escherichia coli . We demonstrate that the unique 1,2-aminothiol introduced by our approach can be efficiently, rapidly, and specifically labeled via a cyanobenzothiazole condensation to quantitatively introduce biophysical probes into proteins. Moreover, we show that, in combination with cysteine labeling, this approach allows the dual labeling of proteins with distinct probes at two distinct, genetically defined sites.
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- "The latter two, namely p-azido-l-phenylalanine (1) and p-propargyloxy-l-phenylalanine (2, Figure 1), are particularly useful for bioconjugation, as proteins containing these nnAAs can be directly coupled using the copper (I)-catalysed azide–alkyne cycloaddition reaction. More recently, orthogonal pairs derived from the pyrrolysyl tRNA-synthetase pair of the archaea Methanosarcina barkeri and Methanosarcina mazei were used to incorporate a wide variety of pyrrolysine and phenylalanine analogues during in vivo expression in E. coli; some of these analogues also contain azide or alkyne moieties (19–24). In one study, a single pyrrolysyl tRNA-synthetase mutant was used to incorporate as many as 12 different phenylalanine analogues (25). "
ABSTRACT: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.Nucleic Acids Research 04/2013; 41(11). DOI:10.1093/nar/gkt226 · 9.11 Impact Factor
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- "This technology allows us to not only synthesize a protein carrying a nonproteinogenic amino acid at a specific position in various types of cells, but also obtain a protein containing a nonproteinogenic amino acid in a larger quantity than by in vitro genetic code expansion    . The use of various orthogonal tRNA/aaRS pairs has allowed for the synthesis of proteins carrying various artificial functional groups, such as a biochemical group (e.g., sulfate, acetate, or methylate)    , fluorescent probe     , photo-cross-linker [44– 49], photo-caged group       , and bioorthogonal reactive group       , for the study of protein structure and function. Similarly to in vitro genetic code expansion, in vivo amber suppression using orthogonal tRNA/aaRS pairs was extended to ochre and opal codon suppression [64– 66] and four-base codon suppression to incorporate two nonproteinogenic amino acids simultaneously into proteins in vivo   "
ABSTRACT: The presence of a nonproteinogenic moiety in a nonstandard peptide often improves the biological properties of the peptide. Non-standard peptide libraries are therefore used to obtain valuable molecules for biological, therapeutic, and diagnostic applications. Highly diverse non-standard peptide libraries can be generated by chemically or enzymatically modifying standard peptide libraries synthesized by the ribosomal machinery, using posttranslational modifications. Alternatively, strategies for encoding non-proteinogenic amino acids into the genetic code have been developed for the direct ribosomal synthesis of non-standard peptide libraries. In the strategies for genetic code expansion, non-proteinogenic amino acids are assigned to the nonsense codons or 4-base codons in order to add these amino acids to the universal genetic code. In contrast, in the strategies for genetic code reprogramming, some proteinogenic amino acids are erased from the genetic code and non-proteinogenic amino acids are reassigned to the blank codons. Here, we discuss the generation of genetically encoded non-standard peptide libraries using these strategies and also review recent applications of these libraries to the selection of functional non-standard peptides.Journal of nucleic acids 10/2012; 2012:713510. DOI:10.1155/2012/713510
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ABSTRACT: An Escherichia coli strain was engineered to synthesize 1-hexanol from glucose by extending the coenzyme A (CoA)-dependent 1-butanol synthesis reaction sequence catalyzed by exogenous enzymes. The C4-acyl-CoA intermediates were first synthesized via acetyl-CoA acetyltransferase (AtoB), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt), and trans-enoyl-CoA reductase (Ter) from various organisms. The butyryl-CoA synthesized was further extended to hexanoyl-CoA via β-ketothiolase (BktB), Hbd, Crt, and Ter. Finally, hexanoyl-CoA was reduced to yield 1-hexanol by aldehyde/alcohol dehydrogenase (AdhE2). Enzyme activities for the C6 intermediates were confirmed by assays using HPLC and GC. 1-Hexanol was secreted to the fermentation medium under anaerobic conditions. Furthermore, co-expressing formate dehydrogenase (Fdh) from Candida boidinii increased the 1-hexanol titer. This demonstration of 1-hexanol production by extending the 1-butanol pathway provides the possibility to produce other medium chain length alcohols using the same strategy.Journal of the American Chemical Society 06/2011; 133(30):11399-401. DOI:10.1021/ja203814d · 11.44 Impact Factor