Department of Molecular and Translational Medicine, University of Brescia, Viale Europa 11, 25123 Brescia, Italy.
Department of Biotechnology, Chemistry and Pharmacy, University of Siena, 53100 Siena, Italy.
Int J Mol Sci. 2022 Jan 21;23(3):1151. doi: 10.3390/ijms23031151.
Gremlin-1 is a secreted cystine-knot protein that acts as an antagonist of bone morphogenetic proteins (BMPs), and as a ligand of heparin and the vascular endothelial growth factor receptor 2 (VEGFR2), thus regulating several physiological and pathological processes, including embryonic development, tissue fibrosis and cancer. Gremlin-1 exerts all these biological activities only in its homodimeric form. Here, we propose a multi-step approach for the expression and purification of homodimeric, fully active, histidine-tagged recombinant gremlin-1, using mammalian HEK293T cells. Ion metal affinity chromatography (IMAC) of crude supernatant followed by heparin-affinity chromatography enables obtaining a highly pure recombinant dimeric gremlin-1 protein, exhibiting both BMP antagonist and potent VEGFR2 agonist activities.
Gremlin-1 是一种分泌的半胱氨酸结蛋白，作为骨形态发生蛋白 (BMPs) 的拮抗剂，以及肝素和血管内皮生长因子受体 2 (VEGFR2) 的配体，从而调节包括胚胎发育、组织纤维化和癌症在内的几种生理和病理过程。Gremlin-1 仅以同源二聚体的形式发挥所有这些生物学活性。在这里，我们提出了一种使用哺乳动物 HEK293T 细胞表达和纯化同源二聚体、完全活性、组氨酸标记的重组 Gremlin-1 的多步方法。粗上清液的离子金属亲和层析 (IMAC) 后接着进行肝素亲和层析，可获得高度纯化的重组二聚体 Gremlin-1 蛋白，具有 BMP 拮抗剂和强效 VEGFR2 激动剂活性。
