Therapeutic applications of genetic code expansion

Huang, Yujia; Wang, Yong; Hu, Liming; Qin, Xue-Wen; Liu, Tao

doi:10.1360/n032018-00071

Cited by 7 publications

(7 citation statements)

References 75 publications

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“…More than 300 ncAAs have been genetically incorporated into proteins, providing powerful tools for investigating protein structures and functions. [1][2][3]7,[77][78][79][80][81][82][83][84][85] To date, utilizing these ncAAs in the context of Genetic Code Expansion has required both exogenous feeding and good membrane permeability of chemically-synthesized ncAAs. Cell membranes are poorly permeable to ncAAs with charged, highly hydrophobic, or hydrophilic structures.…”

Section: Discussionmentioning

confidence: 99%

Unleashing the Potential of Noncanonical Amino Acid Biosynthesis for Creation of Cells with Site-Specific Tyrosine Sulfation

Chen

Jin

Zhang

et al. 2022

Preprint

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Incorporation of noncanonical amino acids (ncAAs) into proteins holds great promise for modulating the structure and function of those proteins and for influencing evolutionary dynamics in organisms. Despite significant progress in improving the efficiency of translational machinery needed for incorporating ncAAs, exogenous feeding of high concentrations of chemically-synthesized ncAAs, especially in the case of polar ncAAs, is required to ensure adequate intracellular ncAA levels. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. We discovered the first enzyme catalyzing tyrosine sulfation, sulfotransferase 1C1 from Nipponia nippon (NnSULT1C1), using a sequence similarity network (SSN). The unique specificity of NnSULT1C1 for tyrosine has been systematically explored using both bioinformatics and computational methods. This NnSULT1C1 was introduced into both bacterial and mammalian cells so as to yield organisms capable of biosynthesizing high levels of intracellular sTyr. These engineered cells produced site-specifically sulfated proteins at a higher yield than cells fed exogenously even with the highest level of sTyr reported in literature. We have used these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

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Section: Discussionmentioning

confidence: 99%

Unleashing the Potential of Noncanonical Amino Acid Biosynthesis for Creation of Cells with Site-Specific Tyrosine Sulfation

Chen

Jin

Zhang

et al. 2022

Preprint

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“…Incorporation of the multiple bromo/chlorotyrosine residues into the polypeptide chains of redox enzymes has been found to increase the enzyme's thermostability due to better side-chain packing (Ohtake et al 2015). Protein functionalization can also be improved by the incorporation of ncAA, and specifically for the production of antibody-drug conjugates or bispecific antibodies, and to attach polyethylene glycol to the chain to improve protein stability (Huang and Liu 2018). A single-point mutation of the Phe264 residue to form p-isothiocyanate phenylalanine in the enzyme MetA has been found to increase the enzyme's thermal stability compared to the wild-type protein (Li et al 2019).…”

Section: Protein Engineering Using Ncaa As Building Blocksmentioning

confidence: 99%

Genome recoding strategies to improve cellular properties: mechanisms and advances

et al. 2020

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The genetic code, once believed to be universal and immutable, is now known to contain many variations and is not quite universal. The basis for genome recoding strategy is genetic code variation that can be harnessed to improve cellular properties. Thus, genome recoding is a promising strategy for the enhancement of genome flexibility, allowing for novel functions that are not commonly documented in the organism in its natural environment. Here, the basic concept of genetic code and associated mechanisms for the generation of genetic codon variants, including biased codon usage, codon reassignment, and ambiguous decoding, are extensively discussed. Knowledge of the concept of natural genetic code expansion is also detailed. The generation of recoded organisms and associated mechanisms with basic targeting components, including aminoacyl-tRNA synthetase-tRNA pairs, elongation factor EF-Tu and ribosomes, are highlighted for a comprehensive understanding of this concept. The research associated with the generation of diverse recoded organisms is also discussed. The success of genome recoding in diverse multicellular organisms offers a platform for expanding protein chemistry at the biochemical level with non-canonical amino acids, genetically isolating the synthetic organisms from the natural ones, and fighting viruses, including SARS-CoV2, through the creation of attenuated viruses. In conclusion, genome recoding can offer diverse applications for improving cellular properties in the genome-recoded organisms.

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“…As the field transitions to developing therapeutic protein-protein conjugates that replace genetic fusions with flexible organic polymer linkers, it is advantageous to develop modular, site-specific connections for efficient assembly of protein architectures. Site-specific functionalization with noncanonical amino acids (ncAA) using genetic code expansion (GCE) provides freedom of attachment and has been used successfully to link molecules intended for therapeutic usage, including drug-antibody conjugates, PEGylated proteins, and conjugative protein dimerization for bispecific antibody formation (13)(14)(15). Unfortunately, these ligation reactions can require multiple conjugation steps, toxic catalysts, nonphysiological pHs or long incubation times (on the order of days), and high label concentrations to achieve sufficient protein coupling yields (16).…”

Section: Introductionmentioning

confidence: 99%

Nanobody assemblies with fully flexible topology enabled by genetically encoded tetrazine amino acids

et al. 2022

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Assembling nanobodies (Nbs) into polyvalent multimers is a powerful strategy for improving the effectiveness of Nb-based therapeutics and biotechnological tools. However, generally effective approaches to Nb assembly are currently restricted to the amino or carboxyl termini, greatly limiting the diversity of Nb multimer topologies that can be produced. Here, we show that reactive tetrazine groups—site-specifically inserted by genetic code expansion at Nb surface sites—are compatible with Nb folding and function, enabling Nb assembly at any desired point. Using two anti–SARS-CoV-2 Nbs with viral neutralization ability, we created Nb homo- and heterodimers with improved properties compared with conventionally linked Nb homodimers, which, in the case of our tetrazine-conjugated trimer, translated into enhanced viral neutralization. Thus, this tetrazine-based approach is a generally applicable strategy that greatly increases the accessible range of Nb assembly topologies, and thereby adds the optimization of topology as an effective avenue to generate Nb assemblies with improved efficacy.

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Therapeutic applications of genetic code expansion

Cited by 7 publications

References 75 publications

Unleashing the Potential of Noncanonical Amino Acid Biosynthesis for Creation of Cells with Site-Specific Tyrosine Sulfation

Unleashing the Potential of Noncanonical Amino Acid Biosynthesis for Creation of Cells with Site-Specific Tyrosine Sulfation

Genome recoding strategies to improve cellular properties: mechanisms and advances

Nanobody assemblies with fully flexible topology enabled by genetically encoded tetrazine amino acids

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