title: Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies.
abstract: Progression of a host of human cancers is associated with elevated levels of expression and catalytic activity of the Src family of tyrosine kinases (SFKs), making them key therapeutic targets. Even with the availability of multiple crystal structures of active and inactive forms of the SFK catalytic domain (CD), a complete understanding of its catalytic regulation is unavailable. Also unavailable are atomic or near-atomic resolution information about their interactions, often weak or transient, with regulating phosphatases and downstream targets. Solution NMR, the biophysical method best suited to tackle this problem, was previously hindered by difficulties in bacterial expression and purification of sufficient quantities of soluble, properly folded protein for economically viable labeling with NMR-active isotopes. Through a choice of optimal constructs, co-expression with chaperones and optimization of the purification protocol, we have achieved the ability to bacterially produce large quantities of the isotopically-labeled CD of c-Src, the prototypical SFK, and of its activating Tyr-phosphorylated form. All constructs produce excellent spectra allowing solution NMR studies of this family in an efficient manner.
authors.affiliation: New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027-7556, USA.
authors.forename: Andrea
authors.initials: A
authors.lastname: Piserchio
authors.forename: Ranajeet
authors.initials: R
authors.lastname: Ghose
authors.forename: David
authors.initials: D
authors.lastname: Cowburn
mesh_terms: D000818:Animals; D002645:Chickens; D004926:Escherichia coli / Q000235:genetics; D019906:Nuclear Magnetic Resonance, Biomolecular; D010766:Phosphorylation; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018909:src Homology Domains / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D016392:Proto-Oncogene Proteins pp60(c-src)