RESUMO
Detailed kinetic analyses of inverse electron-demand DielsAlder cycloaddition and nitrilimine-alkene/alkyne 1,3-diploar cycloaddition reactions were conducted and the reactions were applied for rapid protein bioconjugation. When reacted with a tetrazine or a diaryl nitrilimine, strained alkene/alkyne entities including norbornene, trans-cyclooctene, and cyclooctyne displayed rapid kinetics. To apply these "click" reactions for site-specific protein labeling, five tyrosine derivatives that contain a norbornene, trans-cyclooctene, or cyclooctyne entity were genetically encoded into proteins in Escherichia coli using an engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pair. Proteins bearing these noncanonical amino acids were successively labeled with a fluorescein tetrazine dye and a diaryl nitrilimine both in vitro and in living cells.
Assuntos
Alcenos/química , Alcinos/química , Química Click , Proteínas/química , Coloração e Rotulagem/métodos , Catálise , Ciclo-Octanos/química , Proteínas de Escherichia coli/química , Cinética , Modelos Moleculares , Conformação Proteica , Tirosina/químicaRESUMO
Systematic studies of nonsense and sense suppression of the original and three derivative Methanosarcina mazei PylRS-tRNA(Pyl) pairs and cross recognition between nonsense codons and various tRNA(Pyl) anticodons in the Escherichia coli BL21(DE3) cell strain are reported. tRNA(CUA)(Pyl) is orthogonal in E. coli and able to induce strong amber suppression when it is co-expressed with pyrrolysyl-tRNA synthetase (PylRS) and charged with a PylRS substrate, N(ε)-tert-butoxycarbonyl-L-lysine (BocK). Similar to tRNA(CUA)(Pyl), tRNA(UUA)(Pyl) is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although tRNA(UUA)(Pyl) is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-tRNA(UUA)(Pyl) pair does not give rise to amber suppression that surpasses the basal amber suppression level in E. coli. E. coli itself displays a relatively high opal suppression level and tryptophan (Trp) is incorporated at an opal mutation site. Although the PylRS-tRNA(UCA)(Pyl) pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. tRNA(CCU)(Pyl) fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.