An efficient system for incorporation of unnatural amino acids in response to the four-base codon AGGA in Escherichia coli

Lee, Byeong Sung; Kim, Su Yeon; Ko, Byoung Joon; Yoo, Tae Hyeon

doi:10.1016/j.bbagen.2017.02.017

“…Three plasmids, pET21a-PE24-TAG, pEVOL [58] containing the tRNA/azidophenylalanyl-tRNA synthetase (AzF-RS) pair originated from Methanococcus jannaschii [41], and pBbS2K containing E. coli prolyl-tRNA synthetase (ProRS) [59, 60] were co-transformed in E. coli BL21(DE3). The cells were cultured in 400 ml 2 × YT medium at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A

Lee

¹

,

Lee

²

,

Park

³

et al. 2019

Self Cite

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Background Immunotoxins consisting of a toxin from bacteria or plants and a targeting module have been developed as potent anti-cancer therapeutics. The majority of them, especially those in preclinical or clinical testing stages, are fusion proteins of a toxin and antibody fragment. Immunotoxins based on full-length antibodies are less studied, even though the fragment crystallizable (Fc) domain plays an important role in regulating the concentration of immunoglobulin G (IgG) in the serum and in antibody-mediated immune responses against pathogens. Results We devised a method to site-specifically conjugate IgG and another protein using a cysteine residue introduced into the IgG and a bio-orthogonally reactive unnatural amino acid incorporated into the other protein. The human epidermal growth factor receptor 2 (Her2)-targeting IgG, trastuzumab, was engineered to have an unpaired cysteine in the heavy chain, and an unnatural amino acid with the azido group was incorporated into an engineered Pseudomonas exotoxin A (PE24). The two protein molecules were conjugated site-specifically using a bifunctional linker having dibenzocyclooctyne and maleimide groups. Binding to Her2 and interaction with various Fc receptors of trastuzumab were not affected by the conjugation with PE24. The trastuzumab-PE24 conjugate was cytotoxic to Her2-overexpressing cell lines, which involved the inhibition of cellular protein synthesis due to the modification of elongation factor-2. Conclusions We constructed the site-specifically conjugated immunotoxin based on IgG and PE24, which induced target-specific cytotoxicity. To evaluate the molecule as a cancer therapeutic, animal studies are planned to assess tumor regression, half-life in blood, and in vivo immunogenicity. In addition, we expect that the site-specific conjugation method can be used to develop other antibody-protein conjugates for applications in therapeutics and diagnostics. Electronic supplementary material The online version of this article (10.1186/s13036-019-0188-x) contains supplementary material, which is available to authorized users.

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“…One reason for this is the poor recoding efficiency of the +1-frameshifting aminoacyl (aa)-tRNA, and the second is the failure of the +1-frameshifting aa-tRNA to compete with canonical aa-tRNAs that read the first three nucleotides of the quadruplet codon at the ribosomal aa-tRNA binding (A) site during the aa-tRNA selection step of the translation elongation cycle. While directed evolution by synthetic biologists has yielded +1frameshifting tRNAs, efficient recoding requires cell lines that have been engineered to deplete potential competitor tRNAs [3][4][5][6][7][8] . These problems emphasize the need to better understand the mechanism through which quadruplet codons are translated by +1-frameshifting tRNAs.…”

Section: Introductionmentioning

confidence: 99%

Insights into Genome Recoding from the Mechanism of a Classic +1-Frameshifting tRNA

Gamper

¹

,

Li

²

,

Masuda

³

et al. 2021

Preprint

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While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

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“…One reason for this is the poor recoding efficiency of the +1-frameshifting aminoacyl (aa)-tRNA, and the second is the failure of the +1-frameshifting aa-tRNA to compete with canonical aa-tRNAs that read the first three nucleotides of the quadruplet codon at the ribosomal aa-tRNA binding (A) site during the aa-tRNA selection step of the translation elongation cycle. While directed evolution by synthetic biologists has yielded +1-frameshifting tRNAs, efficient recoding requires cell lines that have been engineered to deplete potential competitor tRNAs [3][4][5][6][7][8] . These problems emphasize the need to better understand the mechanism through which quadruplet codons are translated by +1-frameshifting tRNAs.…”

mentioning

confidence: 99%

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

Gamper

¹

,

Li

²

,

Masuda

³

et al. 2021

View full text Add to dashboard Cite

While genome recoding using quadruplet codons to incorporate non-proteinogenic amino acids is attractive for biotechnology and bioengineering purposes, the mechanism through which such codons are translated is poorly understood. Here we investigate translation of quadruplet codons by a +1-frameshifting tRNA, SufB2, that contains an extra nucleotide in its anticodon loop. Natural post-transcriptional modification of SufB2 in cells prevents it from frameshifting using a quadruplet-pairing mechanism such that it preferentially employs a triplet-slippage mechanism. We show that SufB2 uses triplet anticodon-codon pairing in the 0-frame to initially decode the quadruplet codon, but subsequently shifts to the +1-frame during tRNA-mRNA translocation. SufB2 frameshifting involves perturbation of an essential ribosome conformational change that facilitates tRNA-mRNA movements at a late stage of the translocation reaction. Our results provide a molecular mechanism for SufB2-induced +1 frameshifting and suggest that engineering of a specific ribosome conformational change can improve the efficiency of genome recoding.

show abstract

An efficient system for incorporation of unnatural amino acids in response to the four-base codon AGGA in Escherichia coli

Cited by 18 publications

References 50 publications

Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A

Construction of an immunotoxin via site-specific conjugation of anti-Her2 IgG and engineered Pseudomonas exotoxin A

Insights into Genome Recoding from the Mechanism of a Classic +1-Frameshifting tRNA

Insights into genome recoding from the mechanism of a classic +1-frameshifting tRNA

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