Abraham Sherwin J, Muhamed Ismaeel, Nolet Ryan, Yeung Fung, Gaponenko Vadim
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, United States.
Protein Expr Purif. 2010 Oct;73(2):125-31. doi: 10.1016/j.pep.2010.05.015. Epub 2010 Jun 8.
A p21 GTPase K-Ras4B plays an important role in human cancer and represents an excellent target for cancer therapeutics. Currently, there are no drugs directly targeting K-Ras4B. In part, this is due to the lack of structural information describing unique features of K-Ras4B. Here we describe a methodology allowing production of soluble, well-folded K-Ras4B for structural analysis. The key points in K-Ras4B preparation are low temperature expression and extraction of K-Ras4B from the insoluble fraction using a nucleotide loading procedure in the presence of Mg(2+) and citrate, a low affinity chelator. Additionally, a significant amount of K-Ras4B could be extracted from the soluble fraction. We show that recombinant K-Ras4B is monomeric in solution. Excellent NMR signal dispersion suggests that the protein is well-folded and is amenable to solution structure determination. In addition, using phospholipid bilayer nanodiscs we show that recombinant K-Ras4B interacts with lipids and that this interaction is mediated by the C-terminal hypervariable region.
一种p21 GTP酶K-Ras4B在人类癌症中起重要作用，是癌症治疗的一个理想靶点。目前，尚无直接靶向K-Ras4B的药物。部分原因是缺乏描述K-Ras4B独特特征的结构信息。在此，我们描述了一种用于生产可溶、折叠良好的K-Ras4B以进行结构分析的方法。制备K-Ras4B的关键点是低温表达以及在存在低亲和力螯合剂Mg(2+)和柠檬酸盐的情况下，通过核苷酸加载程序从不溶性部分中提取K-Ras4B。此外，还可从可溶性部分中提取大量的K-Ras4B。我们证明重组K-Ras4B在溶液中为单体。出色的核磁共振信号分散表明该蛋白折叠良好，适合进行溶液结构测定。此外，使用磷脂双层纳米盘，我们证明重组K-Ras4B与脂质相互作用，且这种相互作用由C端高变区介导。
