Division of Molecular Biomedicine, Department of Molecular Biology and Biotechnology, Atomic Energy Commission of Syria (AECS), P. O. Box 6091, Damascus, Syria.
Department of Animal Biology, Faculty of Sciences, Damascus University, Damascus, Syria.
J Membr Biol. 2021 Apr;254(2):175-187. doi: 10.1007/s00232-021-00169-y. Epub 2021 Feb 19.
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.
膜联蛋白 V（ANXV）的主要特征是能够以依赖于钙的方式与生物膜相互作用。ANXV 主要与磷脂酰丝氨酸（PS）相互作用，因此荧光 ANXV 被广泛生产并用作标记凋亡细胞或任何含有 PS 的双层膜的敏感和特异性探针。许多报告描述了重组人 ANXV 的原核表达。为了克服大肠杆菌表达的一些限制，我们旨在本工作中研究哺乳动物细胞中的非常规替代表达系统，以融合超折叠绿色荧光蛋白（sfGFP）产生分泌型人 ANXV。使用聚乙烯亚胺（PEI）和 pcDNA-sfGFP-ANXV 质粒转染 HEK239T 细胞。转染后 48 小时，直接荧光测量、免疫印迹和 ELISA 证实细胞上清液中存在分泌的 sfGFP-ANXV。用亲和层析纯化后，估计分泌的 6×His 标记的 sfGFP-ANXV 的产量约为每 1ml 细胞上清液 2µg。该分泌系统适合产生足够数量的功能齐全的 sfGFP-ANXV 融合蛋白，以识别和结合含有 PS 的表面或脂质体。此外，流式细胞术和荧光显微镜等生物测定证实，分泌的 sfGFP-ANXV 能够检测凋亡细胞上 PS 的暴露。总之，我们提出哺乳动物表达是一种快速、经济且无内毒素的系统，可生产 sfGFP-ANXV 融合蛋白。真核系统中分泌的 sfGFP-ANXV 是一种很有前途的生物技术工具，它为在体外和体内测定中检测具有 PS 的表面和凋亡开辟了新的应用前景。
