TY - CHAP
T1 - Expression and purification of Src-family kinases for solution NMR studies
AU - Piserchio, Andrea
AU - Cowburn, David
AU - Ghose, Ranajeet
PY - 2012
Y1 - 2012
N2 - 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.
AB - 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.
KW - Escherichia coli expression systems
KW - Protein tyrosine kinases
KW - Src-family kinases
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U2 - 10.1007/978-1-61779-480-3_7
DO - 10.1007/978-1-61779-480-3_7
M3 - Chapter
C2 - 22167671
AN - SCOPUS:84855870177
SN - 9781617794797
T3 - Methods in Molecular Biology
SP - 111
EP - 131
BT - Protein NMR Techniques
ER -