Nonsense and Sense Suppression Abilities of Original and Derivative Methanosarcina mazei Pyrrolysyl-tRNA Synthetase-tRNA(Pyl) Pairs in the Escherichia coli BL21(DE3) Cell Strain

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. [Image: see text]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[Image: see text], [Image: see text]is also orthogonal in E. coli and can be coupled with PylRS to genetically incorporate BocK at an ochre mutation site. Although [Image: see text]is expected to recognize a UAG codon based on the wobble hypothesis, the PylRS-[Image: see text] 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-[Image: see text] pair can be used to encode BocK at an opal codon, the pair fails to suppress the incorporation of Trp at the same site. [Image: see text] fails to deliver BocK at an AGG codon when co-expressed with PylRS in E. coli.