Bacterial Two Hybrid: A Versatile One-Step Intracellular Selection Method

Pellis, Mireille; Vincke, Cécile

doi:10.1007/978-1-61779-968-6_9

Cited by 4 publications

(4 citation statements)

References 15 publications

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“…The “two-hybrid” system is a screening system used in protein–protein and protein–DNA interactions to activate a downstream gene by binding it to a transcription factor [ 49 , 50 ]. One of the advantages of this system lies in the capability to quantify the downstream reporter genes in vivo .…”

Section: Deep Mutational Scanningmentioning

confidence: 99%

Rational Protein Engineering Guided by Deep Mutational Scanning

Shin

Cho

2015

IJMS

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Sequence–function relationship in a protein is commonly determined by the three-dimensional protein structure followed by various biochemical experiments. However, with the explosive increase in the number of genome sequences, facilitated by recent advances in sequencing technology, the gap between protein sequences available and three-dimensional structures is rapidly widening. A recently developed method termed deep mutational scanning explores the functional phenotype of thousands of mutants via massive sequencing. Coupled with a highly efficient screening system, this approach assesses the phenotypic changes made by the substitution of each amino acid sequence that constitutes a protein. Such an informational resource provides the functional role of each amino acid sequence, thereby providing sufficient rationale for selecting target residues for protein engineering. Here, we discuss the current applications of deep mutational scanning and consider experimental design.

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Section: Deep Mutational Scanningmentioning

confidence: 99%

Rational Protein Engineering Guided by Deep Mutational Scanning

Shin

Cho

2015

IJMS

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“…For the development of functional intrabodies, it is crucial to establish their selection platform which works directly inside living cells. To date, two‐hybrid systems in bacterial, yeast, and mammalian hosts have been developed . Although these systems allow selection of intrabodies in the bacterial cytosol and eukaryotic nucleus, versatile methods for selecting intrabodies in the eukaryotic cytosol have yet to be reported.…”

Section: Introductionmentioning

confidence: 99%

“…To date, two-hybrid systems in bacterial, yeast, and mammalian hosts have been developed. [9][10][11][12][13][14][15] Although these systems allow selection of intrabodies in the bacterial cytosol and eukaryotic nucleus, versatile methods for selecting intrabodies in the eukaryotic cytosol have yet to be reported. Therefore, we aimed to develop a robust intrabody screening method in the cytosol of mammalian cells, which may be a potential platform for drug discovery directed toward therapeutics.…”

Section: Introductionmentioning

confidence: 99%

A Suicide Switch Directly Eliminates Intracellular scFv Oligomers in the Cytoplasm of Mammalian Cells

Nguyen

Nagamune

Kawahara

2018

Biotechnology Journal

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As intracellular antibodies (intrabodies) are highly promising tools for drug discovery, an innovative antibody screening platform in mammalian cells was previously developed by a single-chain Fv (scFv)-c-kit growth sensor, which successfully selected rabies nucleoprotein and phosphoprotein-specific intrabodies from a synthetic scFv library. Since the scFv-c-kit growth sensor releases a growth signal after forming oligomers due to binding to an oligomeric antigen, it is critical to use a library which does not contain self-oligomeric scFvs to avoid the off-target signal of the growth sensor. Here, a novel method to eliminate self-oligomeric scFvs directly in the cytoplasm of mammalian cells is presented. A suicide switch by fusing an scFv with a cell-death signaling domain to eliminate scFv oligomers is developed. It is found that among four cell-death signaling domains, a suicide switch by fusing scFv with Fas-associated death domain (FADD) can selectively reduce oligomeric scFvs. Furthermore, the library after eliminating scFv oligomers results in higher efficiency in the intrabody selection platform with a growth sensor. Collectively, the scFv-FADD suicide switch can be applied to eliminate oligomeric scFvs from a library, which can consequently improve the quality of intracellular scFv libraries and accelerate the discovery of intrabodies in the future.

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“…This feature necessitates additional laborious procedures for checking solubility [11,12] and antigen binding [2,13,14] in the cytoplasm to finally select intracellularly functional antibody fragments named "intrabodies". To select intrabodies from a library, bacterial [15,16], yeast [17][18][19][20] and mammalian [21][22][23] two hybrid systems were developed. These systems employ DNA binding and transactivator domains that are fused with a target antigen and antibody fragments, and antigen-antibody association induces a reporter gene expression.…”

Section: Introductionmentioning

confidence: 99%

Growth signalobody selects functional intrabodies in the mammalian cytoplasm

Lee

Kaku

Inoue

et al. 2016

Biotechnology Journal

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A versatile strategy to inhibit protein functions in the cytoplasmic environment is eagerly anticipated for drug discovery. In this study, we demonstrate a novel system to directly select functional intrabodies from a library in the mammalian cytoplasm. In this system, a target homo-oligomeric antigen is expressed together with a single-chain Fv (scFv) library that is linked to the cytoplasmic domain of a receptor tyrosine kinase (RTK) in the cytoplasm of murine interleukin-3 (IL-3)-dependent cells. As the tyrosine kinase is activated by dimerization, only scFv-RTK clones that can bind to the target antigen would be oligomerized and transduce a growth signal under the IL-3-deprived condition, which leads to selection of functional intrabodies. To demonstrate this system, we used rabies virus phosphoprotein (RV-P) that forms dimers in the cytoplasm as a target antigen. As a result, functional intrabodies were selected using our system from a naïve scFv library as well as from a pre-selected anti-RV-P library generated by phage display. This system may be applied for screening intrabodies that can prevent progression of various severe diseases.

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Bacterial Two Hybrid: A Versatile One-Step Intracellular Selection Method

Cited by 4 publications

References 15 publications

Rational Protein Engineering Guided by Deep Mutational Scanning

Rational Protein Engineering Guided by Deep Mutational Scanning

A Suicide Switch Directly Eliminates Intracellular scFv Oligomers in the Cytoplasm of Mammalian Cells

Growth signalobody selects functional intrabodies in the mammalian cytoplasm

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