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, Arianna O.; Zheng, Yunan; Roy, Soumya Jyoti Singha; Biris, Nikolaos; Hussain, Myer; Loynd, Conor; Jewel, Delilah; Italia, James S.; Chatterjee, Abhishek

doi:10.1002/anie.202219269

Cited by 25 publications

(13 citation statements)

References 43 publications

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“…Our study highlights opportunities and limitations of dual suppression systems, requiring two selective, efficient and mutual orthogonal incorporation machineries. The relatively lower efficiency of ochre and opal stop codon suppression limits overall yield of dual suppressed protein 4,5,8,9 . We chose AzF for encoding the CuAAC handle because of its efficient incorporation (Figure 2A, S2B), but this likely limited the fluorescent labeling efficiency on live cells.…”

Section: Discussionmentioning

confidence: 99%

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Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Meineke

Caridha

Block

et al. 2023

Preprint

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Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Suppressor tRNAs are aminoacylated with noncanonical amino acids (ncAAs) by dedicated aminoacyl-tRNA synthetases (aaRS) and direct ncAA incorporation site-specifically during translation. These pairs of tRNA/aaRS must be orthogonal to the host's tRNAs, aaRS and natural amino acids. Pyrrolysyl-tRNA (PylT)/PylRS pairs from methanogenic archaea, as well as engineered tRNA/aaRS pairs derived from bacteria, are used for genetic code expansion in mammalian cells. Amber suppression is routinely achieved by transient introduction of the components leading to short-term and heterogeneous expression. Here, we demonstrate that stable integration of tRNA/aaRS genes allows for efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We extend a general plasmid design and PiggyBac (PB) integration strategy developed for the Methanosarcina mazei PylT/PylRS pair to genomic integration of two tRNA/aaRS pairs of bacterial origin. We further explore suppression of ochre and opal stop codons and parallel incorporation of two distinct ncAAs, both accessible for click chemistry, by dual suppression in stable cell lines. Clonal selection allows for isolation of cells with high dual suppression efficiency and dual site-specific fluorescent labeling of a cell surface receptor using bioorthogonal click chemistries on live mammalian cells.

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Section: Discussionmentioning

confidence: 99%

“…The amber codon is the least abundant and most efficiently suppressed stop codon in mammals. The other two stop codons TAA (ochre) and TGA (opal), and synthetic quadruplets, have also been employed for ncAA incorporation in mammalian cells and enable dual ncAA incorporation via dual suppression 3–9 .…”

Section: Introductionmentioning

confidence: 99%

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Meineke

Caridha

Block

et al. 2023

Preprint

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“…Using a similar strategy, acetyllysine and selenocysteine have been genetically encoded into proteins simultaneously. , Recently, several approaches to site-specifically incorporating three distinct ncAAs into a single protein have been reported. For example, OTSs based on Ec TyrRS, Ec TrpRS, and Mb PylRS are mutually orthogonal to each other and are able to decode UAG, UGA, and UAA codons as three distinct ncAAs in mammalian cells respectively, while OTSs derived from Ec LeuRS, Mm PylRS and Ec TyrRS are also mutually orthogonal to each other and can read UAG, UGA, and UAA codons as three different ncAAs in mammalian cells separately . However, suppressing all three stop codons could lead to issues of cell growth, so sense codons or quadruplet codons have been selected for multiple ncAA encoding.…”

Section: Summary and Perspectivesmentioning

confidence: 99%

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

Gan,

Fan

2024

Chem. Rev.

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Post-translational modifications (PTMs) endow proteins with new properties to respond to environmental changes or growth needs. With the development of advanced proteomics techniques, hundreds of distinct types of PTMs have been observed in a wide range of proteins from bacteria, archaea, and eukarya. To identify the roles of these PTMs, scientists have applied various approaches. However, high dynamics, low stoichiometry, and crosstalk between PTMs make it almost impossible to obtain homogeneously modified proteins for characterization of the site-specific effect of individual PTM on target proteins. To solve this problem, the genetic code expansion (GCE) strategy has been introduced into the field of PTM studies. Instead of modifying proteins after translation, GCE incorporates modified amino acids into proteins during translation, thus generating site-specifically modified proteins at target positions. In this review, we summarize the development of GCE systems for orthogonal translation for site-specific installation of PTMs.

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“…The first two sites were chosen because they were previously found to be incompatible with the THIOMAB technology due to incomplete reoxidation, 25 and the last was chosen as a known site for successful stop codon suppression and conjugation. 47 Plasmids encoding one of these mutants of trastuzumab were cotransfected into Expi293 suspension cells with another plasmid containing the EcLeuRS/tRNA machinery, and Nvoc-cysteine was supplied in the media at the time of transfection at a final concentration of 1 mM. The expression was allowed to continue for 7 days, and afterward the mutant antibodies were purified using protein-Gaffinity chromatography.…”

Section: ■ Introductionmentioning

confidence: 99%

“…The ncAA-containing antibody can be fully decaged after just 10 min of irradiation, followed by direct conjugation to a cytotoxic payload to create a functional ADC. Although the GCE technology has been used to site-specifically engineer other bioorthogonal reactive handles within the antibody structure for use with other bioconjugation techniques such as SPAAC, − IEDDA, , oxime, , CRACR, − and others, , the reagents needed for these methods are often less accessible than their thiol-reactive counterparts. , By contrast, the approach reported here benefits from the expansive thiol conjugation toolbox as well as the popularity of thiol-targeted chemistries in the industry, where these processes have been optimized at scale, and the resulting conjugates have been validated in clinical settings. In addition, this strategy can be potentially combined with other bioorthogonal ncAA handles for site-specific antibody labeling at multiple distinct sites to create more complex next-generation ADCs with multiple small molecule attachments. ,,− …”

Section: Introductionmentioning

confidence: 99%

A Genetically Encoded Photocaged Cysteine for Facile Site-Specific Introduction of Conjugation-Ready Thiol Residues in Antibodies

Osgood,

Roy,

Koo

et al. 2024

Bioconjugate Chem.

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Antibody−drug conjugates (ADCs) have emerged as a powerful class of anticancer therapeutics that enable the selective delivery of toxic payloads into target cells. There is increasing appreciation for the importance of synthesizing such ADCs in a defined manner where the payload is attached at specific permissive sites on the antibody with a defined drug to antibody ratio. Additionally, the ability to systematically alter the site of attachment is important to fine-tune the therapeutic properties of the ADC. Engineered cysteine residues have been used to achieve such site-specific programmable attachment of drug molecules onto antibodies. However, engineered cysteine residues on antibodies often get "disulfide-capped" during secretion and require reductive regeneration prior to conjugation. This reductive step also reduces structurally important disulfide bonds in the antibody itself, which must be regenerated through oxidation. This multistep, cumbersome process reduces the efficiency of conjugation and presents logistical challenges. Additionally, certain engineered cysteine sites are resistant to reductive regeneration, limiting their utility and the overall scope of this conjugation strategy. In this work, we utilize a genetically encoded photocaged cysteine residue that can be site-specifically installed into the antibody. This photocaged amino acid can be efficiently decaged using light, revealing a free cysteine residue available for conjugation without disrupting the antibody structure. We show that this ncAA can be incorporated at several positions within fulllength recombinant trastuzumab and decaged efficiently. We further used this method to generate a functional ADC site-specifically modified with monomethyl auristatin F (MMAF).

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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**

Cited by 25 publications

References 43 publications

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Orthogonal Translation for Site-Specific Installation of Post-translational Modifications

A Genetically Encoded Photocaged Cysteine for Facile Site-Specific Introduction of Conjugation-Ready Thiol Residues in Antibodies

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