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Expression, purification and characterization of murine Dkk1 protein

机译:鼠Dkk1蛋白的表达,纯化和表征

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摘要

Dickkopf-1 (Dkk1) protein is a secreted inhibitor of canonical Writ signaling and modulates that pathway during embryonic development. It is also implicated in several diseases and hence Dkk1 is a potential target for therapeutic intervention. In the present study 6His-tagged Dkk1 expression and secretion was assessed in five mammalian cell types. Only FreeStyle 293-F cells showed significant Dkk1 protein expression in culture medium. High and stable expression of the Dkk1 protein was obtained from a selected stable FreeStyle 293-F clone 3178, that grows in suspension in serum-free medium. The 3178 clone showed a high Dkk1 production level (10 mg/L) for up to 2 months of culture. A one step purification procedure resulting in large amounts of highly pure and active Dkk1 protein was developed. Purified Dkk1 binds its receptors LRP5 and LRP6, and is able to dose dependently inhibit canonical Writ signaling. Recombinant Dkk1 is glycosylated, but this modification is not essential for its biological activity. In summary, an abundant source of pure and functionally active Dkk1 protein is developed that will support the identification of inhibitors such as neutralizing antibodies that could find therapeutic use. (c) 2008 Elsevier Inc. All rights reserved.
机译:Dickkopf-1(Dkk1)蛋白是经典Writ信号的分泌抑制剂,可在胚胎发育过程中调节该途径。它也与多种疾病有关,因此Dkk1是治疗干预的潜在靶标。在本研究中,评估了5种哺乳动物细胞类型中6His标记的Dkk1的表达和分泌。只有FreeStyle 293-F细胞在培养基中显示出明显的Dkk1蛋白表达。 Dkk1蛋白的高稳定表达是从选定的稳定FreeStyle 293-F克隆3178中获得的,该克隆在无血清培养基的悬浮液中生长。 3178克隆在长达2个月的培养中均显示出高Dkk1产量水平(10 mg / L)。开发了一步纯化程序,可产生大量高纯度和高活性的Dkk1蛋白。纯化的Dkk1结合其受体LRP5和LRP6,并能够剂量依赖性地抑制规范的Writ信号传导。重组Dkk1被糖基化,但这种修饰对其生物学活性不是必需的。总之,已开发了丰富的纯净的,具有功能活性的Dkk1蛋白,这将支持鉴定抑制剂,例如可用于治疗的中和抗体。 (c)2008 Elsevier Inc.保留所有权利。

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