"In House" assays for the quantification of Annexin V and its autoantibodies in patients with recurrent pregnancy loss and in vitro fertilisation failures.

Several studies have been shown that Annexin V (ANXV) autoantibodies concentrations are associated with both early recurrent pregnancy losses (RPLs) or in vitro fertilization failure (IVFf). We investigated the association between ANXV autoantibodies and ANVX levels in RPL, IVFf and normal group women. The study was conducted on 22 female patients with RPLs, 66 patients with IVFf, and 16 normal samples from women who had given birth. ANXV autoantibodies were measured using an ELISA test developed by fixing a homemade recombinant ANXV protein and examined with labeled human antibodies, while ANXV concentrations were measured by a competitive ELISA using a homemade anti ANXV polyclonal antibody. The results showed a clear relationship between the high levels of ANXV autoantibodies and the recurrent abortion. On the other hand, ANXV measurement in those patients showed decreased concentrations compared to normal samples. Negative correlation between ANXV and its autoantibodies levels was reported in almost all patients' samples. Our data supports the possibility that ANXV autoantibodies are a risk factor for reproductive failures associated with both RPLs and/or IVFf and the significant role for ANXV in the maintenance of pregnancy.

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

Annexin V (ANXV), mostly characterized by its ability to interact with biological membranes in a calcium-dependent manner. ANXV interacts mainly with phosphatidylserine (PS), for that fluorescent ANXV widely produced and used as a sensitive and specific probe to mark apoptotic cells or any PS-containing bilayers membranes. Many reports described the prokaryotic expression of recombinant human ANXV. To overcome some of E. coli expression limitations, we aimed in this work to investigate unconventional alternative expression system in mammalian cells for producing secreted human ANXV in fusion with the super folder green fluorescent protein (sfGFP). HEK239T cells were transfected using polyethylenimine (PEI) and pcDNA-sfGFP-ANXV plasmid. Forty-eight hours post transfection, direct fluorescence measurement, immunoblotting and ELISA confirmed the presence of secreted sfGFP-ANXV in cells supernatant. The yield of secreted 6 × His-tagged sfGFP-ANXV after affinity purification was estimated to be around 2 µg per 1 ml of cells supernatant. The secretion system was proper to produce a fully functional sfGFP-ANXV fusion protein in quantities enough to recognize and bind PS-containing surfaces or liposomes. Besides, biological assays such as flow cytometry and fluorescent microscopy confirmed the capacity of the secreted sfGFP-ANXV to detect PS exposure on apoptotic cells. Taken together, we present mammalian expression as a quick, affordable and endotoxin-free system to produce sfGFP-ANXV fusion protein. The secreted sfGFP-ANXV in eukaryotic system is a promising biotechnological tool, it opens up new horizons for additional applications in the detection of PS bearing surfaces and apoptosis in vitro and in vivo assays.

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

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.

Characterization of camel nanobodies specific for superfolder GFP fusion proteins.

Superfolder green fluorescent protein (sfGFP) is a fusion tag which plays a dual role in monitoring and purifying the recombinant fusion proteins using specific binders. Nanobodies are the smallest intact antigen binding fragments derived from heavy chain-only antibodies (HCAbs) occurring in camelids. They are produced as recombinant proteins in E. coli and have different biotechnological applications, including the detection and purification of their specific antigens. To produce anti-sfGFP specific nanobodies, an adult one-humped camel was successfully immunized and immune response was evaluated by ELISA, which showed an active participation of HCAbs in this response. A relatively large nanobody "immune" library of 5 × 10(8) individual transformants, with 87.5 % positivity, was prepared from the blood of the immunized camel. Phage display biopanning on this nanobody library resulted in the isolation of seven anti-sfGFP specific nanobodies, referred to as NbsfGFP01, 02, 03, 04, 06, 07 and 08. These nanobodies were able to recognize sfGFP tag as free or in fusion with growth hormone in ELISA and immuno-blotting. Furthermore, they showed important apparent affinities in the detection and capture of sfGFP by ELISA, and they targeted three different epitopes on the surface of their antigen. The interesting characteristics of these molecular binders make them valuable tools for more in-depth structural and functional studies related to sfGFP fusion proteins.