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

Abstract

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.

Annexin V Drives Stabilization of Damaged Asymmetric Phospholipid Bilayers

“…This is also consistent with the fluorescence images shown in Figure F that also indicated extensive AnV binding consistent with a network of AnV forming across the bilayer at the AnV and Ca 2+ concentration used. ,,, Notably, the AnV ACFs, Figure , fit well to single component models. This suggests that the extent of oligomerization was fairly uniform across the membrane at this AnV concentration.…”

“…In the presence of AnV, DOPS flip-flop was confirmed to have occurred as phototreatment of the bilayer led to AnV binding at the membrane reflected in fluorescence imaging. From EIS this was evident as a gradual recovery of resistance which then stabilized for over 15 h, indicating AnV bound to the negatively charged lipids appearing on the exterior of the membrane after 1 h incubation. − The slow recovery of the bilayer after photodamage is attributed to the low concentration of DOPS that translocates to the distal leaflet, mimicking early stages of apoptosis. The increased resistance of the bilayer for an extended period of time when AnV is present suggests that AnV seals and stabilizes the membrane.…”

Microvesicles in bronchoalveolar lavage as a potential biomarker of COPD

“…Of interest CD62E þ , which is promptly expressed after activation by inflammatory stimuli (21)(22)(23)(24), might have been induced by the irritation produced by the bronchoscopy itself, which would indicate that CD62E þ MVs are likely derived from pulmonary endothelium. MVs were also marked for Annexin V, which is commonly used for the detection of MVs derived from apoptotic cells (11)(12)(13)(14)(15). The expression of Annexin V was higher in macrophage-derived MVs from smokers with COPD compared with nonsmokers.…”

“…For the characterization and analysis of the MVs, 20 μL (ml) of MV suspension were incubated in the dark for 30 min at room temperature with 2 ml of fluorescent-conjugated monoclonal antibodies against cell-type specific antigens and 2 ml of Annexin V-FITC (Bender MedSystems), which binds exposed phosphatidylserine on MV surface and can signal apoptotic but also immune-stimulated cells/MVs (11)(12)(13)(14)(15).…”

Rosiglitazone Shows Sequence-Specific Synergy With Chemotherapeutic Drugs in Inhibition of McF-7 Breast Cancer Cell Line

“…At 72-h post-treatment, the cells were detached using trypsin-EDTA and washed twice with PBS, then resuspended in serumfree medium. For the Apoptosis/Necrosis assay, cells were stained using Annexin-V-GFP 14 (Atomic Energy Commission of Syria (AECS), Syria) and propidium iodide (PI) and analyzed using BD FACSCalibur Flow Cytometer (BD Biosciences, San Jose, CA). For cell cycle analysis, the DNA content of PIstained cells was analyzed by flow cytometer as described by Darzynkiewicz et al 15 .…”