| Literature DB >> 20566322 |
Sherwin J Abraham1, Ismaeel Muhamed, Ryan Nolet, Fung Yeung, Vadim Gaponenko.
Abstract
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. Copyright 2010 Elsevier Inc. All rights reserved.Entities:
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Year: 2010 PMID： 20566322 DOI： 10.1016/j.pep.2010.05.015
Source DB: PubMed Journal: Protein Expr Purif ISSN： 1046-5928 Impact factor: 1.650