Genetic code expansion in mammalian cells: A plasmid system comparison

Zhou, Wenyuan; Wesalo, Joshua S.; Liu, Jihe; Deiters, Alexander

doi:10.1016/j.bmc.2020.115772

Cited by 17 publications

(31 citation statements)

References 70 publications

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“…These results revealed an inverse correlation between initial POI levels and degradation efficiency, presumably due to oversaturation of the proteasomal machinery. − This correlation is consistent with the increased level of protein degradation observed in translationally inhibited cells after 24 h, compared to a shorter (30 min) degradation period. POI levels suitable for rapid degradation via optoDeg should be readily achievable through simple concentration adjustments of HCK, which directly impacts protein expression levels …”

Section: Resultsmentioning

confidence: 99%

“…POI levels suitable for rapid degradation via optoDeg should be readily achievable through simple concentration adjustments of HCK, which directly impacts protein expression levels. 48 Cell signaling processes are rapid and highly dynamic and thus benefit from optical control and perturbation approaches. 49,50 We selected MKP3, a dual-specificity phosphatase with regulatory functions in cellular processes such as proliferation, differentiation, and development, as a target for our optoDeg methodology.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

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Targeted Protein Degradation through Fast Optogenetic Activation and Its Application to the Control of Cell Signaling

2021

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Development of methodologies for optically triggered protein degradation enables the study of dynamic protein functions, such as those involved in cell signaling, that are difficult to be probed with traditional genetic techniques. Here, we describe the design and implementation of a novel light-controlled peptide degron conferring N-end pathway degradation to its protein target. The degron comprises a photocaged N-terminal amino acid and a lysine-rich, 13-residue linker. By caging the N-terminal residue, we were able to optically control N-degron recognition by an E3 ligase, consequently controlling ubiquitination and proteasomal degradation of the target protein. We demonstrate broad applicability by applying this approach to a diverse set of target proteins, including EGFP, firefly luciferase, the kinase MEK1, and the phosphatase DUSP6 (also known as MKP3). The caged degron can be used with minimal protein engineering and provides virtually complete, light-triggered protein degradation on a second to minute time scale.

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Section: Resultsmentioning

confidence: 99%

Section: ■ Results and Discussionmentioning

confidence: 99%

Targeted Protein Degradation through Fast Optogenetic Activation and Its Application to the Control of Cell Signaling

2021

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“…Genetic code expansion connects the versatility of chemical synthesis to protein expression in living systems. − Protein modifications can be inserted site-specifically via natural translation machineries . By reprogramming the genetic code, non-canonical amino acids bearing a modification of choice are used in ribosomal polypeptide synthesis. − Non-canonical amino acids have many applications in protein engineering, as they can be equipped with isotopes for structural studies, − photoreactive groups and post-translational modifications for functional studies, − reactive groups for bio-orthogonal coupling, ,,− photocross-linkers, ,,− infrared-active probes to follow conformational dynamics, ,− fluorescent dyes for imaging, − biotin analogues for high-affinity interactions with streptavidin, and stable phosphotyrosine analogues for analysis of signal transduction. , Site-specific incorporation is achieved by suppressing a stop codon with an additional aminoacyl-tRNA-synthetase (aaRS)/tRNA pair. ,,, In bacterial or mammalian cells, the rarest amber codon (TAG) is most often used to minimize suppression throughout the proteome. ,,,, However, the underlying processes of the genetic code expansion are complex and hardly understood. , For efficient protein synthesis, an i...…”

Section: Introductionmentioning

confidence: 99%

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

et al. 2022

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Genetic code expansion is a versatile method for in situ synthesis of modified proteins. During mRNA translation, amber stop codons are suppressed to site-specifically incorporate non-canonical amino acids. Thus, nanobodies can be equipped with photocaged amino acids to control target binding on demand. The efficiency of amber suppression and protein synthesis can vary with unpredictable background expression, and the reasons are hardly understood. Here, we identified a substantial limitation that prevented synthesis of nanobodies with N-terminal modifications for light control. After systematic analyses, we hypothesized that nanobody synthesis was severely affected by ribosomal inaccuracy during the early phases of translation. To circumvent a background-causing read-through of a premature stop codon, we designed a new suppression concept based on ribosomal skipping. As an example, we generated intrabodies with photoactivated target binding in mammalian cells. The findings provide valuable insights into the genetic code expansion and describe a versatile synthesis route for the generation of modified nanobodies that opens up new perspectives for efficient site-specific integration of chemical tools. In the area of photopharmacology, our flexible intrabody concept builds an ideal platform to modulate target protein function and interaction.

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“…We selected a dual-plasmid system for the expression of photocaged SNX3−enhanced green fluorescent protein (EGFP) in mammalian cells, since these plasmids offer excellent UAA incorporation efficiency in different cell lines. 47 This two-plasmid system, originally developed by the Chin lab, 48 is comprised of (1) the pE323 plasmid containing the pyrrolysyl tRNA-synthetase and four copies of the tRNA CUA expression cassette, and (2) the pE363 plasmid containing the gene of interest and four extra copies of the tRNA CUA (Figure 1C). To generate the plasmid bearing the gene of interest, the SNX3 and EGFP genes were separately cloned into the pE363 plasmid to generate pE363-SNX3− In order to quantify the extent of endosomal localization before and after optical release of the PX domain, we developed a CellProfiler (Broad Institute) 49 image processing pipeline for quantification of endosomal fluorescence.…”

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confidence: 99%

“…We selected a dual-plasmid system for the expression of photocaged SNX3–enhanced green fluorescent protein (EGFP) in mammalian cells, since these plasmids offer excellent UAA incorporation efficiency in different cell lines . This two-plasmid system, originally developed by the Chin lab, is comprised of (1) the pE323 plasmid containing the pyrrolysyl tRNA-synthetase and four copies of the tRNA CUA expression cassette, and (2) the pE363 plasmid containing the gene of interest and four extra copies of the tRNA CUA (Figure C).…”

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confidence: 99%

Optical Control of Phosphoinositide Binding: Rapid Activation of Subcellular Protein Translocation and Cell Signaling

2021

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Cells utilize protein translocation to specific compartments for spatial and temporal regulation of protein activity, in particular in the context of signaling processes. Protein recognition and binding to various subcellular membranes is mediated by a network of phosphatidylinositol phosphate (PIP) species bearing one or multiple phosphate moieties on the polar inositol head. Here, we report a new, highly efficient method for optical control of protein localization through the site-specific incorporation of a photocaged amino acid for steric and electrostatic disruption of inositol phosphate recognition and binding. We demonstrate general applicability of the approach by photocaging two unrelated proteins, sorting nexin 3 (SNX3) and the pleckstrin homology (PH) domain of phospholipase C delta 1 (PLCδ1), with two distinct PIP binding domains and distinct subcellular localizations. We have established the applicability of this methodology through its application to Son of Sevenless 2 (SOS2), a signaling protein involved in the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade. Upon fusing the photocaged plasma membrane-targeted construct PH–enhanced green fluorescent protein (EGFP), to the catalytic domain of SOS2, we demonstrated light-induced membrane localization of the construct resulting in fast and extensive activation of the ERK signaling pathway in NIH 3T3 cells. This approach can be readily extended to other proteins, with minimal protein engineering, and provides a method for acute optical control of protein translocation with rapid and complete activation.

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Genetic code expansion in mammalian cells: A plasmid system comparison

Cited by 17 publications

References 70 publications

Targeted Protein Degradation through Fast Optogenetic Activation and Its Application to the Control of Cell Signaling

Targeted Protein Degradation through Fast Optogenetic Activation and Its Application to the Control of Cell Signaling

Efficient Amber Suppression via Ribosomal Skipping for In Situ Synthesis of Photoconditional Nanobodies

Optical Control of Phosphoinositide Binding: Rapid Activation of Subcellular Protein Translocation and Cell Signaling

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