Quantitative analysis of annexin V–membrane interaction by flow cytometry

Wang, Jie; He, Liangqiang; Chen, Dianhua; Pi, Yazhou; Zhou, Wenping; Xiong, Xingkui; Ren, Yongzhe; Lai, Yueyang; Zhang, Hua

doi:10.1007/s00249-015-1026-9

Cited by 8 publications

(13 citation statements)

References 45 publications

(89 reference statements)

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“…Even though annexin V was previously expressed in a fusion form with GFP (Ernst et al, 1998; Wang et al, 2015), this work describes the first report of a functional annexin V fusion with the superfolder derivative of GFP. The most significant addition to the GFP palette in the past several years is the superfolder GFP, which possesses the two characteristic amino acid mutations of EGFP, and has unique six extra mutations at positions; S30R, Y39N, N105T, Y145F, I171V, and A206V (Pedelacq et al, 2006).…”

Section: Discussionmentioning

confidence: 91%

“…The plasmid pRSET-a was used in this work to establish in E. coli a high-level protein expression, via the T7 promoter, of cytoplasmic and N-terminal 6× His tagged sf GFP-annexin V fusion, and thus could be efficiently purified using nickel-charged resin. The same system for annexin V purification was previously tested, where a C-terminal 6× His tag was added and successfully used to purify the protein (Wang et al, 2015). Interestingly in their model, EGFP was used and cloned downstream the annexin V gene just before the 6× His tag, and the expressed fusion protein was successfully used for the detection of apoptosis (Wang et al, 2015).…”

Section: Discussionmentioning

confidence: 99%

“…The same system for annexin V purification was previously tested, where a C-terminal 6× His tag was added and successfully used to purify the protein (Wang et al, 2015). Interestingly in their model, EGFP was used and cloned downstream the annexin V gene just before the 6× His tag, and the expressed fusion protein was successfully used for the detection of apoptosis (Wang et al, 2015). Previously, Broaddus and coworkers conceived that GFP fusion preferably should be at the N-terminal side of annexin V; otherwise it might lose its capacity for PS binding (Ernst et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…Previously, Broaddus and coworkers conceived that GFP fusion preferably should be at the N-terminal side of annexin V; otherwise it might lose its capacity for PS binding (Ernst et al, 1998). However, in EGFP model a short linker was used to separate annexin V from the C-terminal fluorescent moiety (Wang et al, 2015). …”

Section: Discussionmentioning

confidence: 99%

“…Ever since, annexin V became a universal marker of apoptosis and a versatile tool to inspect the changes at the level of cell membranes (Lizarbe et al, 2013; Wang et al, 2015). Because of its implication in Ca 2+ signaling, expression of annexin V was found to be in strict correlations with several diseases, including cancer, autoimmune disorders, and diabetes (Fatimathas and Moss, 2010), and it may be considered as a specific marker for Alzheimer's disease (Sohma et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

et al. 2017

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Programed cell death is a critical and unavoidable part of life. One of the most widely used markers for dying cells, by apoptosis or pyroptosis, is the redistribution of phosphatidylserine (PS) from the inner to the outer plasma membrane leaflet. Annexin V protein is a sensitive and specific probe to mark this event because of its high affinity to the exposed PS. Beyond that, annexin V can bind to any PS-containing phospholipid bilayer of almost all tiny forms of membranous vesicles like blood platelets, exosomes, or even nanostructured liposomes. In this work, recombinant human annexin V was produced as a fusion with a highly fluorescent superfolder derivative of the green fluorescent protein (sfGFP) in Escherichia coli. The fusion protein(sfGFP-ANXV, 64 kDa), annexin V (ANXV, 40 kDa), and sfGFP (27 kDa) were separately produced after cloning their encoding genes in pRSET plasmid, and all proteins were expressed in a soluble form, then purified in high yields because of their N-terminal 6× His tag (~150 mg of pure protein per 1 L culture). Superiority of this fluorescent fusion protein over fluorescein-conjugated annexin V was demonstrated in binding to phospholipids (and their liposomes), prepared from natural sources (soya bean and egg yolk) that have different content of PS, by using different methods including ELISA, dot-blotting, surface plasmon resonance, and flow cytometry. We also applied fluorescent annexin V in the detection of apoptotic cells by flow cytometry and fluorescent microscopy. Interestingly, sfGFP-ANXV fusion was more sensitive to early apoptotic stressed HeLa cells than fluorescein-conjugated-ANXV. This highly expressed and functional sfGFP-ANXV fusion protein provides a promising ready-to-use molecular tool for quantifying liposomes (or similarly exosomes) and detecting apoptosis in cells.

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Section: Discussionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

et al. 2017

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Secretion of Recombinant Human Annexin V in Fusion with the Super Folder GFP for Labelling Phosphatidylserine-Exposing Membranes

et al. 2021

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Advances in high-speed atomic force microscopy (HS-AFM) reveal dynamics of transmembrane channels and transporters

Heath

Scheuring

2019

Current Opinion in Structural Biology

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Dynamics are fundamental to the functions of biomolecules and can occur on a wide range of time-and length-scales. Here we develop and apply high-speed AFM height spectroscopy (HS-AFM-HS), a technique whereby we monitor the sensing of a HS-AFM tip at a fixed position to directly detect the motions of unlabeled molecules underneath. This gives Angstrom spatial and microsecond temporal resolutions. In conjunction with HS-AFM imaging modes to precisely locate areas of interest, HS-AFM-HS measures simultaneously surface concentrations, diffusion coefficients and oligomer sizes of annexin-V on model membranes to decipher key kinetics allowing us to describe the entire annexin-V membrane-association and self-assembly process in great detail and quantitatively. This work displays how HS-AFM-HS can assess the dynamics of unlabeled bio-molecules over several orders of magnitude and separate the various dynamic components spatiotemporally.Introduction: Developing a full picture of how biomolecules function requires an understanding of the intricate relationships between structure and dynamics. For molecules in isolation such as single proteins, these dynamics generally occur as conformational changes. For molecules that act in complexes, the dynamics are dependent on diffusion and partner interaction. These dynamic processes are of course not mutually exclusive, but occur in different spatiotemporal regimes. For membrane proteins these dynamics are crucial as they allow the cell to reorganize proteins in space and time, to form temporal functional units for a particular biochemical function or to regulate the function of the membrane protein itself. 1,2 Biomolecule dynamics occur over a range of length and timescales. Local flexibility, which generally concerns side chain rotations, bond vibrations and loop motions, happens over the femtosecond to nanosecond time range. Whereas collective motions of groups of atoms, loops and domains, typically occur on timescales of the microsecond or longer. Such collective motions are at the basis of most important biomolecular functions including conformational changes between functional states of proteins, the working of molecular machines, enzyme catalysis, protein folding and protein-protein interactions, though the later phenomena can extend into the millisecond to second time range depending on the process or the origin of the molecules under investigation. 3 Thus, developing techniques to directly access structural changes from the microsecond to second timescales is key to understanding the behavior of biomolecules. X-ray crystallography and electron microscopy (EM), are most powerful techniques to study biomolecular structures, 4,5 whilst able to provide unparalleled spatial resolution, the structures obtained from these methods are limited by ensemble averaging and static snapshots of fixed conformations. Consequently, dynamics must be inferred, missing vital information describing how the biomolecules truly function in native conditions, such as their fluctuations, rates, int...

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Quantitative analysis of annexin V–membrane interaction by flow cytometry

Cited by 8 publications

References 45 publications

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

Characterization of Annexin V Fusion with the Superfolder GFP in Liposomes Binding and Apoptosis Detection

Secretion of Recombinant Human Annexin V in Fusion with the Super Folder GFP for Labelling Phosphatidylserine-Exposing Membranes

Advances in high-speed atomic force microscopy (HS-AFM) reveal dynamics of transmembrane channels and transporters

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