Epitope Tagging

Jarvik, Jonathan W.; Telmer, Cheryl A.

doi:10.1146/annurev.genet.32.1.601

Cited by 162 publications

(115 citation statements)

References 118 publications

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“…1B): one for the expression of full-length ORFs with their native stop codon (untagged clones, stop clones), and a second for ORFs that are fused to a C-terminal 3xHA (hemagglutinin) tag after the shuttling event (tagged clones, HA or 3xHA clones). Three tandem copies of the HA epitope tag were used to increase the sensitivity and signal-to-noise ratio in biochemical and histochemical assays (Jarvik and Telmer, 1998). These vectors are equipped with a partially randomised oligonucleotide of 36 bp for vector identification.…”

Section: Vector Design Barcoding and Attp Site Selectionmentioning

confidence: 99%

“…A tagged library holds major advantages over an untagged library: (1) a single antibody can be used to detect any ORF; (2) crossreaction with related proteins can be avoided, as an antibody specific to the tag can be used; (3) the tagged protein can be distinguished from the endogenous, untagged protein; and (4) immunochemistry becomes possible for even poorly immunogenic proteins or proteins that lack a specific antibody (Jarvik and Telmer, 1998). However, because epitope tagging can also interfere with protein function, we examined the potential of the C-terminal 3xHA tag to alter protein function in overexpression experiments.…”

Section: Functional Comparison Of Untagged and 3xha-tagged Transgenesmentioning

confidence: 99%

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A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

et al. 2013

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SUMMARYOverexpression screens are used to explore gene functions in Drosophila, but this strategy suffers from the lack of comprehensive and systematic fly strain collections and efficient methods for generating such collections. Here, we present a strategy that could be used efficiently to generate large numbers of transgenic Drosophila strains, and a collection of 1149 UAS-ORF fly lines that were created with the site-specific ΦC31 integrase method. For this collection, we used a set of 655 genes that were cloned as two variants, either as an open reading frame (ORF) with a native stop codon or with a C-terminal 3xHA tag. To streamline the procedure for transgenic fly generation, we demonstrate the utility of injecting pools of plasmids into embryos, each plasmid containing a randomised sequence (barcode) that serves as a unique identifier for plasmids and, subsequently, fly strains. We also developed a swapping technique that facilitates the rapid exchange of promoters and epitope tags in vivo, expanding the versatility of the ORF collection. The work described here serves as the basis of a systematic library of Gal4/UAS-regulated transgenes.

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Section: Vector Design Barcoding and Attp Site Selectionmentioning

confidence: 99%

Section: Functional Comparison Of Untagged and 3xha-tagged Transgenesmentioning

confidence: 99%

A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

et al. 2013

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“…A crucial aspect of this effort is the utilization of simple and efficient methodologies that are amenable to highthroughput approaches for the purification of proteins and protein complexes (1,2). As a result, a number of generic affinity-based methodologies have been developed for these purposes, based primarily on the use of specific antibodies or affinity tags that are fused to the protein of interest (3,4).…”

mentioning

confidence: 99%

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

Boer

Rodriguez²,

Bonte³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

400

438

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Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin͞streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

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“…bionanotechnology | synthetic biology | metastasis | antibody | nanobiotechnology P eptide tags are powerful tools for analyzing protein function, but have major limitations for controlling protein function (1,2). The flexibility and small surface area of peptides mean that peptide interactions are typically weak and reversible (3), and generally depend on binding of large protein partners (4-6).…”

mentioning

confidence: 99%

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

Fierer

Veggiani

Howarth

2014

Proc. Natl. Acad. Sci. U.S.A.

155

162

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Individual proteins can now often be modified with atomic precision, but there are still major obstacles to connecting proteins into larger assemblies. To direct protein assembly, ideally, peptide tags would be used, providing the minimal perturbation to protein function. However, binding to peptides is generally weak, so assemblies are unstable over time and disassemble with force or harsh conditions. We have recently developed an irreversible protein-peptide interaction (SpyTag/SpyCatcher), based on a protein domain from Streptococcus pyogenes, that locks itself together via spontaneous isopeptide bond formation. Here we develop irreversible peptide-peptide interaction, through redesign of this domain and genetic dissection into three parts: a protein domain termed SpyLigase, which now ligates two peptide tags to each other. All components expressed efficiently in Escherichia coli and peptide tags were reactive at the N terminus, at the C terminus, or at internal sites. Peptide-peptide ligation enabled covalent and site-specific polymerization of affibodies or antibodies against the tumor markers epidermal growth factor receptor (EGFR) and HER2. Magnetic capture of circulating tumor cells (CTCs) is one of the most promising approaches to improve cancer prognosis and management, but CTC capture is limited by inefficient recovery of cells expressing low levels of tumor antigen. SpyLigase-assembled protein polymers made possible the isolation of cancerous cells expressing lower levels of tumor antigen and should have general application in enhancing molecular capture.bionanotechnology | synthetic biology | metastasis | antibody | nanobiotechnology

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Epitope Tagging

Cited by 162 publications

References 118 publications

A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila

Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture

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