Expression, purification, and characterization of soluble K-Ras4B for structural analysis.
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.
Abraham
Sherwin J
SJ
Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, IL 60607, United States.
Muhamed
Ismaeel
I
Nolet
Ryan
R
Yeung
Fung
F
Gaponenko
Vadim
V
D000595:Amino Acid Sequence; D006801:Humans; D010743:Phospholipids / Q000235:genetics; D017510:Protein Folding; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics; D012995:Solubility
C117307:KRAS protein, human; D010743:Phospholipids; D011994:Recombinant Proteins; D016283:Proto-Oncogene Proteins p21(ras)