An enhanced ascorbate peroxidase 2/antibody-binding domain fusion protein (APEX2–ABD) as a recombinant target-specific signal amplifier

Lee, Jisu; Song, Eun Kyung; Bae, Yoonji; Min, Ji-Young; Rhee, Hyun‐Woo; Park, Tae Joo; Kim, Moonil; Kang, Sebyung

doi:10.1039/c5cc02409a

Cited by 19 publications

(15 citation statements)

References 26 publications

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“…To complement this new application, we built a recombineering-ready plasmid series for C-terminal protein tagging. Coding regions for the auxin degron (AID 22 ), the fluorescent protein Venus 23 and the enzymes for biotin proximity labeling BirM 24 and APEX2 25 26 have been cloned into the same context in a plasmid series based on the R6K origin. The context includes on the 5′ side a flexible protein linker based on two copies of the Ty1 peptide, which serves two additional purposes.…”

Section: Resultsmentioning

confidence: 99%

RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

Baker

Gupta

Obst

et al. 2016

Sci Rep

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A fluent method for gene targeting to establish protein tagged and ligand inducible conditional loss-of-function alleles is described. We couple new recombineering applications for one-step cloning of gRNA oligonucleotides and rapid generation of short-arm (~1 kb) targeting constructs with the power of Cas9-assisted targeting to establish protein tagged alleles in embryonic stem cells at high efficiency. RAC (Recombineering And Cas9)-tagging with Venus, BirM, APEX2 and the auxin degron is facilitated by a recombineering-ready plasmid series that permits the reuse of gene-specific reagents to insert different tags. Here we focus on protein tagging with the auxin degron because it is a ligand-regulated loss-of-function strategy that is rapid and reversible. Furthermore it includes the additional challenge of biallelic targeting. Despite high frequencies of monoallelic RAC-targeting, we found that simultaneous biallelic targeting benefits from long-arm (>4 kb) targeting constructs. Consequently an updated recombineering pipeline for fluent generation of long arm targeting constructs is also presented.

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Section: Resultsmentioning

confidence: 99%

RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

Baker

Gupta

Obst

et al. 2016

Sci Rep

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“…APEX2 appears ideally suited as a reporter that is compatible with fusion to monomeric and dimeric sdAb targeted to the periplasm. While the cytosolic expression of APEX2 fused to the antibody binding domain of protein A was successful [30], being able to express high levels of a peroxidase in the E. coli periplasm immediately facilitates genetic fusion to any recombinant antibody of choice, not only specific for other filoviral proteins but any antigen of interest. In contrast, the cytosolic expression of recombinant antibody fragments, even in hosts engineered to have oxidizing cytosols to enable disulfide bond formation, can be fickle with success very much on a case-by-case basis even within the highly soluble single-domain family of antibodies [31].…”

Section: Discussionmentioning

confidence: 99%

Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among Marburgvirus and Dianlovirus

Sherwood

Hayhurst

2019

Viruses

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We explore evolved soybean ascorbate peroxidase (APEX2) as a reporter when fused to the C-termini of llama nanobodies (single-domain antibodies, sdAb; variable domains of heavy chain-only antibodies, VHH) targeted to the E. coli periplasm. Periplasmic expression preserves authentic antibody N-termini, intra-domain disulphide bond(s), and capitalizes on efficient haem loading through the porous E. coli outer membrane. Using monomeric and dimeric anti-nucleoprotein (NP) sdAb cross-reactive within the Marburgvirus genus and cross-reactive within the Ebolavirus genus, we show that periplasmic sdAb–APEX2 fusion proteins are easily purified at multi-mg amounts. The fusions were used in Western blotting, ELISA, and microscopy to visualize NPs using colorimetric and fluorescent imaging. Dimeric sdAb–APEX2 fusions were superior at binding NPs from viruses that were evolutionarily distant to that originally used to select the sdAb. Partial conservation of the anti-Marburgvirus sdAb epitope enabled the recognition of a novel NP encoded by the recently discovered Mĕnglà virus genome. Antibody–antigen interactions were rationalized using monovalent nanoluciferase titrations and contact mapping analysis of existing crystal structures, while molecular modelling was used to reveal the potential landscape of the Mĕnglà NP C-terminal domain. The sdAb–APEX2 fusions also enabled live Marburgvirus and Ebolavirus detection 24 h post-infection of Vero E6 cells within a BSL-4 laboratory setting. The simple and inexpensive mining of large amounts of periplasmic sdAb–APEX2 fusion proteins should help advance studies of past, contemporary, and perhaps Filovirus species yet to be discovered.

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“…Since the genetically encoded mAPEX2 was expressed in the tissue and compatible with microwave-enhanced chemical fixation containing glutaraldehyde, it replaced the use of pre-embedding antibody labeling to identify rAAV-targeted axons. mAPEX2 accommodates tyramide-conjugated fluorescent dyes, which are then deposited in the targeted cells by the TSA reaction [55]. The combination of mAPEX2-driven TSA reaction and post-embedding immunogold labeling allowed reliable identification of the targeted cells throughout the 50 μm vibraslices, at depths not accessible by pre-embedding antibody methods (< 10 μm).…”

Section: Mapex2 Labeled Axons Are Compatible With Ultrastructural Anamentioning

confidence: 99%

Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation

et al. 2020

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Analysis of neuronal compartments has revealed many state-dependent changes in geometry but establishing synapse-specific mechanisms at the nanoscale has proven elusive. We co-expressed channelrhodopsin2-GFP and mAPEX2 in a subset of hippocampal CA3 neurons and used trains of light to induce late-phase long-term potentiation (L-LTP) in area CA1. L-LTP was shown to be specific to the labeled axons by severing CA3 inputs, which prevented back-propagating recruitment of unlabeled axons. Membrane-associated mAPEX2 tolerated microwave-enhanced chemical fixation and drove tyramide signal amplification to deposit Alexa Fluor dyes in the light-activated axons. Subsequent post-embedding immunogold labeling resulted in outstanding ultrastructure and clear distinctions between labeled (activated), and unlabeled axons without obscuring subcellular organelles. The gold-labeled axons in potentiated slices were reconstructed through serial section electron microscopy; presynaptic vesicles and other constituents could be quantified unambiguously. The genetic specification, reliable physiology, and compatibility with established methods for ultrastructural preservation make this an ideal approach to link synapse ultrastructure and function in intact circuits.

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An enhanced ascorbate peroxidase 2/antibody-binding domain fusion protein (APEX2–ABD) as a recombinant target-specific signal amplifier

Cited by 19 publications

References 26 publications

RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses

Periplasmic Nanobody-APEX2 Fusions Enable Facile Visualization of Ebola, Marburg, and Mĕnglà virus Nucleoproteins, Alluding to Similar Antigenic Landscapes among Marburgvirus and Dianlovirus

Ultrastructure of light-activated axons following optogenetic stimulation to produce late-phase long-term potentiation

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