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Author:
Date:
2014-10-20
[Abstract] Here, we present the full-length protocol for purifying the recombinant MOS1 transposase from insect cells used in our recent publication (Pflieger et al., 2014), which involved a N-terminal MBP-tag and maltose-affinity chromatography.
Due to their overall basic properties, transposases are often difficult to purify, especially because they tend to aggregate. Since the 90s, we chose a method of purification without a denaturation step. Our first priority was to preserve the 3D structure of the protein in order to maintain its biochemical activities with the highest ...
[摘要] 在这里，我们提出了用于从我们最近出版物（Pflieger等，2014）中使用的昆虫细胞中纯化重组MOS1转座酶的全长方案，其涉及N-末端MBP标签和麦芽糖亲和层析。
由于其总体基本性质，转座酶通常难以纯化，特别是因为它们倾向于聚集。自九十年代以来，我们选择了一种纯化方法，而没有变性步骤。我们的首要任务是保留蛋白质的3D结构，以保持其具有最高比活度的生化活性。然而，我们由细菌制成的生产/纯化定期含有截短产物（或降解产物），并且其水平随着纯化转座酶的浓度而增加。相比之下，由真核细胞制成的生产/纯化不含有这样的降解产物。因此，我们开发了涉及pVL1392杆状病毒转移载体和BaculoGoldTM杆状病毒表达系统的方案，从杆状病毒感染的Sf21细胞中表达重组MOS1。
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