Expression, purification, and characterization of soluble K-Ras4B for structural analysis

Protein Expr Purif. 2010 Oct;73(2):125-31. doi: 10.1016/j.pep.2010.05.015. Epub 2010 Jun 8.

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.

MeSH terms

  • Amino Acid Sequence
  • Humans
  • Phospholipids / genetics
  • Protein Folding
  • Proto-Oncogene Proteins p21(ras) / chemistry*
  • Proto-Oncogene Proteins p21(ras) / genetics
  • Proto-Oncogene Proteins p21(ras) / isolation & purification*
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Signal Transduction / genetics
  • Solubility

Substances

  • Phospholipids
  • Recombinant Proteins
  • K-Ras4B protein, human
  • Proto-Oncogene Proteins p21(ras)