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.
- SourceAvailable from: Hiroshi Murakami[Show abstract] [Hide abstract]
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
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ABSTRACT: Genetic code expansion, for the site-specific incorporation of unnatural amino acids into proteins, is currently limited to cultured cells and unicellular organisms. Here we expand the genetic code of a multicellular animal, the nematode Caenorhabditis elegans.Journal of the American Chemical Society 08/2011; 133(36):14196-9. DOI:10.1021/ja2054034 · 11.44 Impact Factor