Expression and purification of Src-family kinases for solution NMR studies

Methods Mol Biol. 2012;831:111-31. doi: 10.1007/978-1-61779-480-3_7.

Abstract

NMR analyses of the structure, dynamics, and interactions of the Src family kinases (SFKs) have been hindered by the limited ability to obtain sufficient amounts of properly folded, soluble protein from bacterial expression systems, to allow these studies to be performed in an economically viable manner. In this chapter, we detail our attempts to overcome these difficulties using the catalytic domain (SrcCD) of c-Src, the prototypical SFK, as an illustrative example. We describe in detail two general methods to express and purify SrcCD from Escherichia coli expression systems in both fully active wild-type and kinase-deficient mutant forms, allowing the efficient and cost-effective labeling by NMR-active isotopes for solution NMR studies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bioreactors*
  • Catalytic Domain / genetics
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / metabolism*
  • Genetic Vectors / genetics
  • HSP90 Heat-Shock Proteins / metabolism
  • Isotope Labeling / methods
  • Models, Molecular*
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • src-Family Kinases / genetics
  • src-Family Kinases / isolation & purification*
  • src-Family Kinases / metabolism*

Substances

  • HSP90 Heat-Shock Proteins
  • src-Family Kinases