Department of Medical Genetics, The Second Military Medical University, Xiang Yin Road 800, Shanghai, China.
Appl Microbiol Biotechnol. 2010 Jan;85(3):605-14. doi: 10.1007/s00253-009-2080-y. Epub 2009 Jul 24.
Annexin B1 is a novel Ca(2+)-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure-function relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them in the early stages of apoptosis.
Annexin B1 是猪带绦虫囊尾蚴中一种新型的 Ca(2+)-依赖性磷脂结合蛋白，具有许多潜在的生物医学应用。尽管 Annexin B1 已在大肠杆菌中成功表达，但在室温下纯化的蛋白质稳定性较差，这阻碍了我们进一步研究其结构-功能关系的尝试。为了提高蛋白质的稳定性，我们检查并改变了构建和纯化程序，希望能提高其效率。在这项研究中，我们描述了一种新的重组 Annexin B1，其 N 端融合了一个六组氨酸标签，通过固定化金属亲和层析和分子筛层析两步法可将其纯化至均一性。最终的产量约为每升细菌培养物 23 毫克。等电聚焦和质谱分析表明，用这种方法纯化的蛋白质在室温下非常稳定，甚至 3 天后仍保持稳定。一系列功能测试表明，重组蛋白具有高抗凝活性，荧光标记的 Annexin B1 可以与凋亡哺乳动物细胞的外膜结合，并在细胞凋亡的早期有效地检测到它们。
