In certain methanogenic archaea a new amino acid, pyrrolysine (Pyl), is inserted at in-frame UAG codons in the mRNAs of some methyltransferases. Pyl is directly acylated onto a suppressor tRNA(Pyl) by pyrrolysyl-tRNA synthetase (PylRS). Due to the lack of a readily available Pyl source, we looked for structural analogues that could be aminoacylated by PylRS onto tRNA(Pyl). We report here the in vitro aminoacylation of tRNA(Pyl) by PylRS with two Pyl analogues: N-epsilon-d-prolyl-l-lysine (d-prolyl-lysine) and N-epsilon-cyclopentyloxycarbonyl-l-lysine (Cyc). Escherichia coli, transformed with the tRNA(Pyl) and PylRS genes, suppressed a lacZ amber mutant dependent on the presence of d-prolyl-lysine or Cyc in the medium, implying that the E. coli translation machinery is able to use Cyc-tRNA(Pyl) and d-prolyl-lysine-tRNA(Pyl) as substrates during protein synthesis. Furthermore, the formation of active beta-galactosidase shows that a specialized mRNA motif is not essential for stop-codon recoding, unlike for selenocysteine incorporation.
Download full-text PDF |
Source |
|---|---|
| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1817836 | PMC |
| http://dx.doi.org/10.1016/j.febslet.2006.11.028 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Genetic Code Expansion of the Silkworm Using a Pyrrolysyl-tRNA Synthetase/tRNA Pair.
ACS Synth Biol
December 2024
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization (NARO), 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
The domesticated silkworm , an essential industrial animal for silk production, has attracted attention as a host for protein production due to its remarkable protein synthesis capability. Here, we applied genetic code expansion (GCE) using a versatile pyrrolysyl-tRNA synthetase (PylRS)/tRNA pair from to ; GCE enables synthetic amino acid incorporation into proteins to give them non-natural functions. Transgenic lines expressing PylRS and its cognate tRNA were generated and cross-mated to obtain their F hybrid.
View Article and Find Full Text PDFGenetic Incorporation of a Thioxanthone-Containing Amino Acid for the Design of Artificial Photoenzymes.
Angew Chem Int Ed Engl
December 2024
State Key Laboratory of Chemical Oncogenomics, Shenzhen Key Laboratory of Chemical Genomics, AI for Science (AI4S) Preferred Program, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, University Town of Shenzhen, Nanshan District, 518055 Shenzhen, P. R. China.
Genetically encodable photosensitizers allow the design of artificial photoenzymes to expand the scope of abiological reactions. Herein, we report the genetic incorporation of a thioxanthone-containing amino acid into a protein scaffold via an engineered pyrrolysyl-tRNA/pyrrolysyl-tRNA synthetase pair. The designer enzyme was engineered to catalyze a dearomative [2+2] cycloaddition reaction in high yields (up to>99 % yield) with excellent enantioselectivity (up to 98 : 2 e.
View Article and Find Full Text PDFSite-specific incorporation of noncanonical amino acids (ncAAs) into proteins in eukaryotes has predominantly relied on the pyrrolysyl-tRNA synthetase/tRNA pair. However, access to additional easily engineered pairs is crucial for expanding the structural diversity of the ncAA toolbox in eukaryotes. The -derived leucyl-tRNA synthetase (EcLeuRS)/tRNA pair presents a particularly promising alternative.
View Article and Find Full Text PDFAligning fermentation conditions with non-canonical amino acid addition strategy is essential for Nε-((2-azidoethoxy)carbonyl)-L-lysine uptake and incorporation into the target protein.
Sci Rep
October 2024
Christian Doppler Laboratory for Production of Next-Level Biopharmaceuticals in E. coli, Department of Biotechnology, BOKU University, Institute of Bioprocess Science and Engineering, Vienna, Austria.
Protein engineering with non-canonical amino acids (ncAAs) holds great promises for diverse applications, however, there are still limitations in the implementation of this technology at manufacturing scale. The know-how to efficiently produce ncAA-incorporated proteins in a scalable manner is still very limited. In the present study, we incorporated the ncAA N-[(2-azidoethoxy)carbonyl]-L-lysine (Azk) into an antigen binding fragment (Fab) in Escherichia coli.
View Article and Find Full Text PDFDirected Evolution of Pyrrolysyl-tRNA Synthetase for the Genetic Incorporation of Two Different Noncanonical Amino Acids in One Protein.
ACS Bio Med Chem Au
October 2024
Texas A&M Drug Discovery Center and Department of Chemistry, College of Arts and Sciences, Texas A&M University, College Station, Texas 77843, United States.
The genetic code expansion technique is a powerful chemical biology tool to install noncanonical amino acids (ncAAs) in proteins. As a key enzyme for this technique, pyrrolysyl-tRNA synthetase (PylRS), coupled with its cognate amber suppressor tRNA, has been engineered for the genetic incorporation of more than 200 ncAAs. Using PylRS clones from different archaeal origins, two ncAAs have also been genetically encoded in one protein.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!