Dept. of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, U.K.
Biotechnol Prog. 2010 Jan-Feb;26(1):142-9. doi: 10.1002/btpr.305.
This study describes the use of a hexa-histidine tagged exopeptidase for the cleavage of hexa-histidine tags from recombinant maltose binding protein (MBP) when both tagged species are bound to an immobilized metal affinity chromatography (IMAC) matrix. On-column exopeptidase cleavage only occurred when the cleavage buffer contained an imidazole concentration of 50 mM or higher. Two strategies were tested for the on-column tag cleavage by dipeptidylaminopeptidase (DAPase): (i) a post-load wash was performed after sample loading using cleavage buffers containing varying imidazole concentrations and (ii) a post-load wash was omitted following sample loading. In the presence of 50 mM imidazole, 46% of the originally adsorbed hexa-histidine tagged MBP was cleaved, released from the column, and recovered in a sample containing 100% native (i.e., completely detagged) MBP. This strategy renders the subsequent purification steps unnecessary as any tagged contaminants remained bound to the column. At higher imidazole concentrations, binding of both hexa-histidine tagged MBP and DAPase to the column was minimized, leading to characteristics of cleavage more closely resembling that of a batch cleavage. An on-column cleavage yield of 93% was achieved in the presence of 300 mM imidazole, albeit with contamination of the detagged protein with tag fragments and partially tagged MBP. The success of the on-column exopeptidase cleavage makes the integration of the poly-histidine tag removal protocol within the IMAC protein capture step possible. The many benefits of using commercially available exopeptidases, such as DAPase, for poly-histidine tag removal can now be combined with the on-column tag cleavage operation.
本研究描述了一种六组氨酸标记外肽酶在重组麦芽糖结合蛋白（MBP）的六组氨酸标签与固定化金属亲和层析（IMAC）基质结合时从重组麦芽糖结合蛋白（MBP）中切割六组氨酸标签的用途。只有当切割缓冲液中咪唑浓度为 50mM 或更高时，柱上外肽酶切割才会发生。两种策略被用于二肽基氨肽酶（DAPase）的柱上标签切割：（i）在样品加载后，使用含有不同咪唑浓度的切割缓冲液进行负载后清洗，（ii）在样品加载后省略负载后清洗。在 50mM 咪唑存在下，46%的最初吸附的六组氨酸标记的 MBP 被切割，从柱上释放出来，并在含有 100%天然（即完全去标签）MBP 的样品中回收。该策略使随后的纯化步骤变得不必要，因为任何带标签的污染物都仍然与柱结合。在较高的咪唑浓度下，六组氨酸标记的 MBP 和 DAPase 与柱的结合都最小化，导致切割特征更类似于批处理切割。在 300mM 咪唑存在下，实现了 93%的柱上切割产率，尽管去标签蛋白与标签片段和部分标记的 MBP 污染。柱上外肽酶切割的成功使多组氨酸标签去除方案能够集成到 IMAC 蛋白质捕获步骤中。现在可以将使用商业可得的外肽酶（如 DAPase）去除多组氨酸标签的许多好处与柱上标签切割操作结合起来。
