Therapeutic applications of genetic code expansion

Huang, Yujia; Liu, Tao

doi:10.1016/j.synbio.2018.09.003

Cited by 60 publications

(32 citation statements)

References 174 publications

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“…Several biotech companies are utilizing NSAAs or genetic code expansion to produce therapeutic proteins. Ambrx (La Jolla, CA, USA) is developing NSAA-containing ADCs, αHER2 ADC (ARX788) and PEG-FGF21 (ARX618), which are currently in phase 1 and phase 2 clinical trials, respectively (Huang and Liu 2018). To incorporate the NSAAs, Ambrx developed a mammalian cell-based production platform called EuCODE™ (Tian et al 2014).…”

Section: Industrial Applications Of Nsaasmentioning

confidence: 99%

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

Jin

Park

Hong

2019

Appl Microbiol Biotechnol

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The natural genetic code only allows for 20 standard amino acids in protein translation, but genetic code reprogramming enables the incorporation of non-standard amino acids (NSAAs). Proteins containing NSAAs provide enhanced or novel properties and open diverse applications. With increased attention to the recent advancements in synthetic biology, various improved and novel methods have been developed to incorporate single and multiple distinct NSAAs into proteins. However, various challenges remain in regard to NSAA incorporation, such as low yield and misincorporation. In this review, we summarize the recent efforts to improve NSAA incorporation by utilizing orthogonal translational system optimization, cell-free protein synthesis, genomically recoded organisms, artificial codon boxes, quadruplet codons, and orthogonal ribosomes, before closing with a discussion of the emerging applications of NSAA incorporation.

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Section: Industrial Applications Of Nsaasmentioning

confidence: 99%

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

Jin

Park

Hong

2019

Appl Microbiol Biotechnol

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“…Amino acid analogues are indispensable tools for the study of protein structure and function, facilitating protein characterization, detection, visualization, enrichment, and modification. [1][2][3][4][5][6] The development of new amino acid analogues and use of existing ones for new applications are key to accelerating advances in protein science and engineering. Our group is interested in amino acid analogues containing the element tellurium (Te) which, despite scarce natural presence in biological systems, has the potential to be used as a versatile handle in structural and chemical biology.…”

Section: Introductionmentioning

confidence: 99%

Incorporation of TePhe into Expressed Proteins is Minimally Perturbing

Nitz

2021

ChemBioChem

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Tellurium is a versatile heavy chalcogen with numerous applications in chemical biology, providing valuable probes in mass cytometry, fluorescence imaging and structural biology. L-Tellurienylalanine (TePhe) is an analogue of the proteinogenic amino acid L-phenylalanine (Phe) in which the phenyl side chain has been replaced by a 5-membered tellurophene moiety. High incorporation level of TePhe in expressed proteins at defined sites is expected to facilitate studies in proteomics, protein NMR spectroscopy, and structure elucidation. As a model we chose immunoglobulin-binding Protein G, B1 domain (GB1) to validate TePhe as a suitable structural analogue for Phe. We demonstrate that approximately 1 in 2 of all Phe sites within GB1 can be substituted with TePhe through expression in standard non-Phe-auxotrophic E. coli in Phe-deficient media containing glyphosate, an inhibitor of aromatic amino acid biosynthesis. The TePhe content of the GB1 sample can be further increased to 85 % through HPLC. Using NMR and CD spectroscopy, we confirm that the Phe-to-TePhe substitution has negligible impact on the global structure and stability of GB1.

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“…[33][34][35] For almost all genetic code expansion applications, yield and purity are paramount. 36 One factor that can limit the yield of a protein carrying one or more ncAAs is competition between the misacylated suppressor tRNA and release factor 1 (RF1), both of which recognizes the amber UAG stop codon. Recognition of the mis-acylated suppressor tRNA leads to incorporation of the ncAA, whereas recognition by RF1 triggers early translation termination.…”

Section: Introductionmentioning

confidence: 99%

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

Santiago¹,

Shah

et al. 2021

Preprint

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We report that the recently introduced commercial strain of V. natriegens (Vmax X2) supports robust unnatural amino acid mutagenesis, generating exceptional yields of soluble protein containing up to 5 non-canonical L-amino acids (ncAA). The isolated yields of ncAA-containing superfolder green fluorescent protein (sfGFP) expressed in Vmax X2 are up to 25-fold higher than those achieved using commercial expression strains (Top10 and BL21) and more than 10-fold higher than those achieved using two different genomically recoded E. coli strains that lack endogenous UAG stop codons and release factor 1 and have been optimized for improved fitness and preferred growth temperature (C321.ΔA.opt and C321.ΔA.exp). In addition to higher yields of soluble protein, Vmax X2 cells also generate proteins with significantly lower levels of mis-incorporated natural L-amino acids at the UAG-programmed position, especially in cases where the ncAA is an imperfect substrate for the chosen orthogonal aminoacyl tRNA synthetase (aaRS). This increase in fidelity implies that use of Vmax X2 cells as the expression host can obviate the need for time-consuming directed evolution experiments to improve specific activity of highly desirable but imperfect ncAA substrates.

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Therapeutic applications of genetic code expansion

Cited by 60 publications

References 174 publications

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

Incorporation of non-standard amino acids into proteins: challenges, recent achievements, and emerging applications

Incorporation of TePhe into Expressed Proteins is Minimally Perturbing

Genetic code expansion in the engineered organism Vmax X2: High yield and exceptional fidelity

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