A Facile Platform to Engineer <i>Escherichia coli</i> Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic

Grasso, Katherine T.; Roy, Soumya Jyoti Singha; Osgood, Arianna O.; Yeo, Megan Jin Rae; Soni, Chintan; Hillenbrand, Christen M.; Ficaretta, Elise D.; Chatterjee, Abhishek

doi:10.1021/acscentsci.1c01465

Cited by 24 publications

(32 citation statements)

References 54 publications

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“…To this end, we attempted the expression of full-length Trastuzumab harboring a TAG and a TGA codon at positions 121 of the heavy chain (HC) and 169 of the light chain (LC), respectively, using the EcTrp+EcTyr system (Figure 5A), which showed the highest dual suppression efficiency. The antibody expression cassettes were incorporated into a vector containing a recently developed EcTyrRS (pAAFRS), 39 capable of incorporating an azide-containing amino acid pAAF ( 8 , Figure 1), as well as eight copies of tRNA CUA EcTyr . The pAAF incorporation system was selected due to its high efficiency, as well as the post-translational stability of this azide-ncAA within the reducing cellular environment.…”

Section: Resultsmentioning

confidence: 99%

“…The pAAF incorporation system was selected due to its high efficiency, as well as the post-translational stability of this azide-ncAA within the reducing cellular environment. 39 Expi293 suspension cultures were co-transfected with the resulting plasmid along with another plasmid encoding the EcTrpRS/tRNA UCA EcTrp pair. A wild-type antibody, lacking any nonsense codons was also expressed in parallel.…”

Section: Resultsmentioning

confidence: 99%

“…Although the yield of the triply-labeled protein was low, the efficiency of this first-generation technology can be improved through expression plasmid optimization, as well as enhancing the efficiency of the aaRS/tRNA pairs. 39,40 The work described here significantly expands the scope of multi-ncAA mutagenesis in mammalian systems and would facilitate numerous applications.…”

Section: Site-specific Incorporation Of Three Distinct Ncaas Into One...mentioning

confidence: 98%

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An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells

Osgood

Zheng

Roy

et al. 2022

Preprint

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The site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is an emergent technology with much potential. For each different ncAA to be incorporated, this technology requires a distinct orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that recognizes a distinct nonsense codon. The aaRS/tRNA pairs currently available for ncAA mutagenesis in eukaryotes are all traditionally used to decode the TAG nonsense codon. Unfortunately, these pairs suppress the other two nonsense codons, TGA or TAA, at a significantly lower level, compromising the scope of multi-ncAA mutagenesis. Here we report that the bacteria-derived tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells. Additionally, we show that this pair does not cross-react with any of the three previously established aaRS/tRNA pairs. Consequently, the TGA-suppressing EcTrp pair can be combined with TAG-suppressing pyrrolysyl (archaeal), tyrosyl (bacterial), or leucyl (bacterial) pairs to develop three new routes for dual-ncAA incorporation in mammalian cells. We show that all three platforms enable site-specific incorporation of two distinct ncAAs into proteins – including a full-length humanized antibody – with excellent fidelity and good efficiency. Finally, we combined the EcTrp pair with the bacterial Tyr pair and the archaeal pyrrolysyl pair to site-specifically incorporate different combinations of three distinct ncAAs into a reporter protein in mammalian cells.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Site-specific Incorporation Of Three Distinct Ncaas Into One...mentioning

confidence: 98%

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An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells

Osgood

Zheng

Roy

et al. 2022

Preprint

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“…These machineries were identified from an extensive repository of OTSs available in these organisms that facilitate specific and efficient decoding of multiple orthogonal codons with structurally and functionally diverse ncAAs. (Beránek et al, 2018;Biddle et al, 2022;Cervettini et al, 2020;Gan et al, 2017;Grasso et al, 2022Grasso et al, , 2022Italia et al, 2017;Kelemen et al, 2022;Lang et al, 2012;Wang et al, 2014;Zheng et al, 2018a).…”

Section: Introductionmentioning

confidence: 99%

“…Parallel efforts from our lab have led to the discovery of a broad range of E. coli tyrosyl-tRNA synthetase (EcTyrRS) and E. coli Leucyl-tRNA synthetase (EcLeuRS) variants with a variety of ncAA incorporation properties, including new-to-yeast ncAA incorporation and improved selectivity and activity for known ncAAs . In addition, work by Chatterjee and coworkers in engineered E. coli strains has led to the identification of additional EcaaRS variants derived from EcTyRS and EcTrpRS with unique substrate preferences for use in mammalian cells (Grasso et al, 2022;Osgood et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

Lahiri

Deventer

2022

Preprint

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Proteins containing noncanonical amino acids (ncAAs) provide opportunities for dissecting fundamental biological processes and engineering protein therapeutics with diverse chemistries. Incorporation of more than one ncAA into a single construct can endow a protein with multiple useful features, such as unique molecular recognition and covalent crosslinking. Herein, for the first time, we encode two chemically distinct ncAAs into proteins prepared in Saccharomyces cerevisiae. To complement ncAA incorporation in response to the amber (TAG) stop codon in yeast, we evaluated opal (TGA) stop codon suppression using three distinct orthogonal translation systems (OTSs): E. coli tyrosyl-tRNA synthetase/tRNATyr, E. coli leucyl-tRNA synthetase/tRNALeu, and M. alvus pyrrolysyl-tRNA synthetase/tRNAPyl. We observed selective TGA readthrough without detectable cross-reactivity from host translation components. Further, we found that multiple factors impacted TGA readthrough efficiency in a manner analogous to TAG readthrough, including the local nucleotide context surrounding stop codons and deletion of selected genes from the yeast genome. In contrast, the identity of the suppressor tRNA affected TGA readthrough efficiency quite differently than TAG readthrough efficiency. Altogether, these observations facilitated the systematic investigation of dual ncAA incorporation in response to TAG and TGA codons within both intracellular and yeast-displayed protein constructs. Employing a proficient combination of OTSs, pairs of ncAAs could be encoded within both protein formats with efficiencies up to 6% of wildtype protein controls. Moreover, exploration of dual ncAA incorporation in yeast display format aided in demonstrating important applications of doubly-substituted proteins. First, dual ncAA incorporation within a simple synthetic antibody construct resulted in retention of antigen binding functionality, indicating that the doubly-substituted proteins can retain their basic functions. Second, presentation of reactive alkyne and azide functionalities within yeast-displayed constructs enabled sequential installation of two distinct chemical probes using bioorthogonal reactions--strain-promoted- and copper-catalyzed azide-alkyne cycloadditions (SPAAC and CuAAC)--on the yeast surface. This confirmed the ability to chemoselectively modify yeast-displayed proteins at two distinct sites. Furthermore, by utilizing a soluble form of a doubly substituted construct, we validated the feasibility to selectively double label proteins in solution in a single pot reaction. Overall, our work facilitates the addition of a 22nd amino acid to the yeast genetic code and provides an important toolkit that expands the scope of applications of ncAAs for basic biological research and drug discovery in yeast.

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An Efficient Opal‐Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells**

et al. 2023

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Site‐specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl‐tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA‐suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual‐ncAA incorporation. Using these platforms, we site‐specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site‐specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

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A Facile Platform to Engineer Escherichia coli Tyrosyl-tRNA Synthetase Adds New Chemistries to the Eukaryotic Genetic Code, Including a Phosphotyrosine Mimic

Cited by 24 publications

References 54 publications

An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells

An efficient opal-suppressor tryptophanyl pair creates new routes for simultaneously incorporating up to three distinct noncanonical amino acids into proteins in mammalian cells

Dual Noncanonical Amino Acid Incorporation Enabling Chemoselective Protein Modification at Two Distinct Sites in Yeast

An Efficient Opal‐Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells**

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