BACKGROUND The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic β-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. RESULTS rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. CONCLUSION Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering.

中文翻译：

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在人HEK293细胞中稳定表达的重组人R-spondin1（RSPO1）蛋白的生产，纯化和表征。

背景技术R-Spondin蛋白包含分泌蛋白家族，其通过诱导Wnt途径在细胞增殖，分化和死亡中起重要作用而闻名。多项研究表明，RSPO在调节许多组织特异性过程中的重要性，这些过程包括：骨形成，骨骼肌组织发育，胰腺β细胞和肠干细胞的增殖，甚至癌症。由于其在干细胞中的促有丝分裂活性，RSPO1在其潜在的治疗用途中尤其在再生医学领域中在RSPOs分子中脱颖而出。在这里，我们使用HEK293细胞系生成了重组人RSPO1（rhRSPO1），获得了纯化，表征和具有生物活性的蛋白质产品，可用于细胞疗法。合成了hRSPO1编码序列，并将其亚克隆到哺乳动物细胞表达载体中。用含有hRSPO1编码序列和潮霉素抗性质粒的重组表达载体稳定地共转染HEK293细胞，选择用于潮气的并进行细胞克隆分离。结果在无血清的情况下，从转染的HEK293细胞的培养上清液中获得了rhRSPO1，并使用一种新颖的纯化策略进行纯化，涉及两个连续的色谱步骤，即：肝素亲和色谱，然后进行分子排阻色谱，旨在获得高分离度纯度的产品。纯化的蛋白通过蛋白质印迹，质谱和体外（C2C12细胞）和体内（BALB / c小鼠）生物活性测定进行了表征，证实该人类细胞表达系统的结构完整性和生物学功效。此外，如前所述，rhRSPO1糖基化分析使我们首次描述了该寡糖链的聚糖组成，证实了多肽链Asn137残基中存在N-糖基化。另外，该分析揭示了聚糖结构的存在，例如末端唾液酸，N-乙酰氨基葡糖胺和/或半乳糖。结论因此，产生了用于从HEK293细胞生产和纯化重组hRSPO1的稳定平台，从而导致了纯化，具有充分表征和生物活性的蛋白质产品的生产，可用于组织工程。如前所述，rhRSPO1糖基化分析使我们首次描述了该寡糖链的聚糖组成，从而证实了多肽链Asn137残基中存在N-糖基化。另外，该分析揭示了聚糖结构的存在，例如末端唾液酸，N-乙酰氨基葡糖胺和/或半乳糖。结论因此，产生了用于从HEK293细胞生产和纯化重组hRSPO1的稳定平台，从而导致了纯化，具有充分表征和生物活性的蛋白质产品的生产，可用于组织工程。如前所述，rhRSPO1糖基化分析使我们首次描述了该寡糖链的聚糖组成，从而证实了多肽链Asn137残基中存在N-糖基化。另外，该分析揭示了聚糖结构的存在，例如末端唾液酸，N-乙酰氨基葡糖胺和/或半乳糖。结论因此，产生了用于从HEK293细胞生产和纯化重组hRSPO1的稳定平台，从而导致了纯化，具有充分表征和生物活性的蛋白质产品的生产，可用于组织工程。如前所述。另外，该分析揭示了聚糖结构的存在，例如末端唾液酸，N-乙酰氨基葡糖胺和/或半乳糖。结论因此，产生了用于从HEK293细胞生产和纯化重组hRSPO1的稳定平台，从而导致了纯化，具有充分表征和生物活性的蛋白质产品的生产，可用于组织工程。如前所述。另外，该分析揭示了聚糖结构的存在，例如末端唾液酸，N-乙酰氨基葡糖胺和/或半乳糖。结论因此，产生了用于从HEK293细胞生产和纯化重组hRSPO1的稳定平台，从而导致了纯化，具有充分表征和生物活性的蛋白质产品的生产，可用于组织工程。