Abstract
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.
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Abbreviations
- ANXV:
-
Annexin V
- ELISA:
-
Enzyme-linked immunosorbant assay
- sfGFP:
-
Super folder green fluorescent protein
- PI:
-
Probidium iodide
- PS:
-
Phosphatidylserine
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The authors would like to thank the Director General of the Atomic Energy Commission of Syria and the head of the Molecular Biology and Biotechnology department for their continuous support throughout this work.
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Twair, A., Kassem, I., Murad, H. et al. Secretion of Recombinant Human Annexin V in Fusion with the Super Folder GFP for Labelling Phosphatidylserine-Exposing Membranes. J Membrane Biol 254, 175–187 (2021). https://doi.org/10.1007/s00232-021-00169-y
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DOI: https://doi.org/10.1007/s00232-021-00169-y