天青蛋白是在诸如假单胞菌和产碱杆菌的细菌菌株中发现的小的周质蓝铜蛋白,其促进反硝化。天青苷因其介导电子转移过程的能力而被广泛研究,但它也引起了制药界作为潜在的抗微生物或抗癌剂的兴趣。在这里,我们提供了一种在大肠杆菌中表达和单步纯化天青蛋白的新方法,具有高产量和最佳金属化作用。使用N-末端GST标签的融合标签策略用于获得纯蛋白质,而不需要任何额外的纯化步骤。用HRV3C蛋白酶进行柱上切割后,收集天青素,并与硫酸铜一起孵育以确保足够的金属化。UV-VIS吸收,质谱,和圆二色性分析都验证了天青素的有效生产,适当的蛋白质折叠和具有相关辅因子的活性位点的发育。MD模拟验证了N末端GPLGS片段的掺入不影响天青素结构。此外,在HeLa细胞中测试了天青素的生物学活性。
Azurin is a small periplasmic blue copper protein found in bacterial strains such as Pseudomonas and Alcaligenes where it facilitates denitrification.
Azurin is extensively studied for its ability to mediate electron-transfer processes, but it has also sparked interest of the pharmaceutical community as a potential antimicrobial or anticancer agent. Here we offer a novel approach for expression and single-step purification of
azurin in Escherichia coli with high yields and optimal metalation. A fusion tag strategy using an N-terminal GST tag was employed to obtain pure protein without requiring any additional purification steps. After the on-column cleavage by HRV 3C Protease,
azurin is collected and additionally incubated with copper sulphate to ensure sufficient metalation. UV-VIS absorption, mass spectroscopy, and circular dichroism analysis all validated the effective production of azurin, appropriate protein folding and the development of an active site with an associated cofactor. MD simulations verified that incorporation of the N-terminal GPLGS segment does not affect azurin structure. In addition, the biological activity of
azurin was tested in HeLa cells.