Identification of protein-protein interactions using a magnetic modulation biosensing system

Roth, Shira; Zander, Itzhak; Michelson, Yehudit; Ben-David, Yossi; Banin, Ehud; Danielli, Amos

doi:10.1016/j.snb.2019.127228

Cited by 13 publications

(23 citation statements)

References 41 publications

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“…PfiA and other antitoxins of type II TA systems are proteins that inhibit their cognate toxin activity by binding to it and creating a protein–protein complex (Unterholzner et al ., 2013b). A magnetic modulation biosensing (MMB) system (Danielli et al ., 2010; Verbarg et al ., 2017; Roth et al ., 2020) was used to determine whether PfiT and PfiA bind to each other. His‐tagged PfiT was co‐overexpressed with Flag‐tagged PfiA.…”

Section: Resultsmentioning

confidence: 99%

“…Bacteria were then diluted 1:100 into M9 with l ‐(+)‐arabinose (33.3 μM) and grown to a turbidity of 0.5 at 595 nm. 1.5 OD (at 595 nm) of bacteria was taken from each strain and proteins were then extracted as previously described (Roth et al ., 2020).…”

Section: Methodsmentioning

confidence: 99%

“…5s). Identification of the protein–protein interaction using the MMB system was carried out as previously described (Roth et al ., 2020). All measurements were normalized to conjugated magnetic beads' signal.…”

Section: Methodsmentioning

confidence: 99%

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Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Zander

Shmidov

Roth

et al. 2020

Environmental Microbiology

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Toxin-antitoxin (TA) systems are small genetic modules usually consisting of two elements-a toxin and an antitoxin. The abundance of TA systems among various bacterial strains may indicate an important evolutionary role. Pseudomonas aeruginosa, which can be found in a variety of niches in nature, is an opportunistic pathogen for various hosts. While P. aeruginosa strains are very versatile and diverse, only a few TA systems were characterized in this species. Here, we describe a newly characterized TA system in P. aeruginosa that is encoded within the filamentous Pf4 prophage. This system, named PfiT/PfiA, is a homologue of the ParE/YefM TA system. It is a type II TA system, in which the antitoxin is a protein that binds the toxic protein and eliminates the toxic effect. PfiT/PfiA carries several typical type II characteristics. Specifically, it constitutes two small genes expressed in a single operon, PfiT inhibits growth and PfiA eliminates this effect, PfiA binds PfiT, and PfiT expression results in elongated cells. Finally, we assigned a novel function to this TA system, where an imbalance between PfiT and PfiA, favouring the toxin, resulted in cell elongation and an increase in virion production.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Zander

Shmidov

Roth

et al. 2020

Environmental Microbiology

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“…Then, the complexes were incubated for 20 min at room temperature with 100 µL of 1 µg/mL of streptavidin R-phycoerythrin (SA-PE, PJRS20-1, Agilent Technologies, Santa Clara, CA, USA). Subsequently, after a single buffer replacement to remove unbound fluorescent molecules, 100 µL of the final solution was loaded into a borosilicate glass cuvette and measured in the MMB system [ 25 ]. The LoD was calculated as three standard deviations over the blank measurement.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, we introduced a novel platform that uses magnetically modulated biosensors (MMB) to rapidly detect very low concentrations of antibodies [ 20 ], proteins [ 21 ], and specific nucleic acid sequences [ 22 , 23 , 24 ]. We have shown that the MMB can rapidly identify protein-protein interactions (PPI) in vitro with very high sensitivity [ 25 ]. Here, we demonstrate for the first time the applicability of the biosensors for rapid and highly sensitive screening of PPI inhibitors.…”

Section: Introductionmentioning

confidence: 99%

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

Roth

Danielli

2021

Sensors

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Inhibitor screening is an important tool for drug development, especially during the COVID-19 pandemic. The most used in vitro inhibitor screening tool is an enzyme-linked immunosorbent assay (ELISA). However, ELISA-based inhibitor screening is time consuming and has a limited dynamic range. Using fluorescently and magnetically modulated biosensors (MMB), we developed a rapid and sensitive inhibitor screening tool. This study demonstrates its performance by screening small molecules and neutralizing antibodies as potential inhibitors of the interaction between the spike protein 1 (S1) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the angiotensin-converting enzyme 2 (ACE2) receptor. The MMB-based assay is highly sensitive, has minimal non-specific binding, and is much faster than the commonly used ELISA (2 h vs. 7–24 h). We anticipate that our method will lead to a remarkable advance in screening for new drug candidates.

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Magnetic supercluster particles for highly sensitive magnetic biosensing of proteins

Kim

et al. 2022

Microchim Acta

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A strategy is reported to improve the detection limits of current giant magnetoresistance (GMR) biosensors by augmenting the effective magnetic moment that the magnetic tags on the biosensors can exert. Magnetic supercluster particles (MSPs), each of which consists of ~ 1000 superparamagnetic cores, are prepared by a wet-chemical technique and are utilized to improve the limit of detection of GMR biosensors down to 17.6 zmol for biotin as a target molecule. This value is more than four orders of magnitude lower than that of the conventional colorimetric assay performed using the same set of reagents except for the signal transducer. The applicability of MSPs in immunoassay is further demonstrated by simultaneously detecting vascular endothelial growth factor (VEGF) and C-reactive protein (CRP) in a duplex assay format. MSPs outperform commercially available magnetic nanoparticles in terms of signal intensity and detection limit.

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Identification of protein-protein interactions using a magnetic modulation biosensing system

Cited by 13 publications

References 41 publications

Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Characterization of PfiT/PfiA toxin–antitoxin system of Pseudomonas aeruginosa that affects cell elongation and prophage induction

Rapid and Sensitive Inhibitor Screening Using Magnetically Modulated Biosensors

Magnetic supercluster particles for highly sensitive magnetic biosensing of proteins

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