利用双His₆-MBP标签拯救大肠杆菌中易于聚集的蛋白质。
Rescuing aggregation-prone proteins in Escherichia coli with a dual His₆-MBP tag.
作者信息
Needle Danielle, Waugh David S
机构信息
Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, B, Frederick, MD, 21702-1201, USA.
出版信息
Methods Mol Biol. 2014;1177:81-94. doi: 10.1007/978-1-4939-1034-2_7.
Insolubility of recombinant proteins in Escherichia coli is a major impediment to their production for structural and functional studies. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely recognized as a premier solubilizing agent. In this chapter, we describe how to construct dual His6-MBP-tagged fusion proteins by Gateway(®) recombinational cloning and how to predict their yield and solubility. We also describe a simple and rapid procedure to test the ability of a His6-MBP fusion protein to bind to Ni-NTA resin and to be digested by tobacco etch virus (TEV) protease, along with a method to assess the solubility of the target protein after it has been separated from His6-MBP.
重组蛋白在大肠杆菌中的不溶性是其用于结构和功能研究生产的主要障碍。解决这个问题的一种方法是将易于聚集的蛋白与高度可溶的伴侣蛋白融合。大肠杆菌麦芽糖结合蛋白（MBP）被广泛认为是一种首要的增溶剂。在本章中，我们描述了如何通过Gateway（®）重组克隆构建双His6-MBP标签的融合蛋白，以及如何预测它们的产量和溶解性。我们还描述了一种简单快速的程序，用于测试His6-MBP融合蛋白与Ni-NTA树脂结合以及被烟草蚀纹病毒（TEV）蛋白酶消化的能力，以及一种在目标蛋白与His6-MBP分离后评估其溶解性的方法。
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