Using Genetic Code Expansion for Protein Biochemical Studies

Chung, Christina Z.; Amikura, Kazuaki; Söll, Dieter

doi:10.3389/fbioe.2020.598577

Cited by 33 publications

(30 citation statements)

References 62 publications

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“…Genetic code expansion is a technique that allows the integration of such unnatural building blocks by hijacking the synthetic machinery of the target organism. [664][665][666][667][668][669] Also, genetic code expansion can be applied to introduce photoresponsive building blocks into a complex protein. [670][671][672] It is of pivotal importance that the photoswitchable substrate is sufficiently water-soluble, nontoxic, and successfully uptaken by the organism for efficient incorporation.…”

Section: Water-soluble Molecular Photoswitches As Biomolecular Building Blocksmentioning

confidence: 99%

Molecular photoswitches in aqueous environments

et al. 2021

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Section: Water-soluble Molecular Photoswitches As Biomolecular Building Blocksmentioning

confidence: 99%

Molecular photoswitches in aqueous environments

et al. 2021

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“…Furthermore, protein–protein interactions and protein topography (i.e., mapping a protein’s internal structure), topics that are crucial to understanding ECM assembly and architecture, can be studied by modifying BONCAT and using mutant tRNA to insert ncAAs onto pre-determined sites on a protein. The inserted ncAAs can then help identify or alter parts of the protein structure and its interaction with other proteins [ 114 , 115 , 143 , 144 ]. We direct the readers to the following reviews for more information on site-specific ncAA labeling and its applications [ 114 , 115 , 143 ].…”

Section: Introductionmentioning

confidence: 99%

Extracellular matrix dynamics: tracking in biological systems and their implications

Ling

Ren

2022

J Biol Eng

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The extracellular matrix (ECM) constitutes the main acellular microenvironment of cells in almost all tissues and organs. The ECM not only provides mechanical support, but also mediates numerous biochemical interactions to guide cell survival, proliferation, differentiation, and migration. Thus, better understanding the everchanging temporal and spatial shifts in ECM composition and structure – the ECM dynamics – will provide fundamental insight regarding extracellular regulation of tissue homeostasis and how tissue states transition from one to another during diverse pathophysiological processes. This review outlines the mechanisms mediating ECM-cell interactions and highlights how changes in the ECM modulate tissue development and disease progression, using the lung as the primary model organ. We then discuss existing methodologies for revealing ECM compositional dynamics, with a particular focus on tracking newly synthesized ECM proteins. Finally, we discuss the ramifications ECM dynamics have on tissue engineering and how to implement spatial and temporal specific extracellular microenvironments into bioengineered tissues. Overall, this review communicates the current capabilities for studying native ECM dynamics and delineates new research directions in discovering and implementing ECM dynamics to push the frontier forward.

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“…However, protein sequence motifs located at the N- or C- terminus must be incorporated into the protein of interest to ensure appropriate protein modification. In contrast, genetic code expansion strategies have the advantage that amino acid positions in proteins can be modified site-specifically ( Chung et al, 2020 ). This requires an orthogonal aminoacyl-tRNA synthetase (aaRS)-tRNA pair, which does not cross-react with endogenous aaRS, tRNAs and amino acids, while maintaining high specificity towards the non-canonical amino acids (ncaa).…”

Section: Introductionmentioning

confidence: 99%

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems

Schloßhauer

Cavak

Zemella

et al. 2022

Front. Mol. Biosci.

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The investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonal aminoacyl-tRNA synthetase.

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Using Genetic Code Expansion for Protein Biochemical Studies

Cited by 33 publications

References 62 publications

Molecular photoswitches in aqueous environments

Molecular photoswitches in aqueous environments

Extracellular matrix dynamics: tracking in biological systems and their implications

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems

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