Institute of Molecular Biology "Roumen Tsanev", Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.
Institute for Interdisciplinary Research and Technologies, 1421 Sofia, Bulgaria.
Biomed Res Int. 2017;2017:3018608. doi: 10.1155/2017/3018608. Epub 2017 Jun 8.
In order to obtain glycosylated human interferon-gamma (hIFN) and its highly prone to aggregation mutant K88Q, a secretory expression in insect cells was employed. To facilitate recombinant proteins purification, detection, and stability the baculovirus expression vectors were constructed to bear N-terminal His-FLAG tag. Although the obtained proteins were glycosylated, we found that their biological activity was 100 times lower than expected. Our attempts to recover the biological properties of both proteins by tag removal failed due to enterokinase resistance of the tag. Surprisingly, the tag was easily cleaved when the proteins were expressed in cells and the tag-free proteins showed fully restored activity. To shed light on this phenomenon we performed molecular dynamics simulations. The latter showed that the tags interact with the receptor binding domains and the flexible C-termini of the fusion proteins thus suppressing their complex formation with the hIFN receptor. We hypothesize that in the case of glycosylated proteins the tag/C-terminal interaction positions the FLAG peptide in close proximity to the glycans thus sterically impeding the enterokinase access to its recognition site.
为了获得糖基化的人干扰素-γ（hIFN）及其高度聚集的突变体 K88Q，我们采用了昆虫细胞中的分泌表达。为了便于重组蛋白的纯化、检测和稳定性，我们构建了带有 N 端 His-FLAG 标签的杆状病毒表达载体。尽管获得的蛋白是糖基化的，但我们发现它们的生物活性比预期低 100 倍。我们试图通过去除标签来恢复这两种蛋白的生物活性，但由于标签对肠激酶的抗性而失败。令人惊讶的是，当蛋白在 细胞中表达时，标签很容易被切割，并且无标签的蛋白显示出完全恢复的活性。为了阐明这一现象，我们进行了分子动力学模拟。模拟结果表明，标签与受体结合域和融合蛋白的柔性 C 端相互作用，从而抑制它们与 hIFN 受体的复合物形成。我们假设，在糖基化蛋白的情况下，标签/C 端相互作用将 FLAG 肽定位在接近聚糖的位置，从而阻碍肠激酶进入其识别位点。
