Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies

Andrea Piserchio, Ranajeet Ghose, David Cowburn

Research output: Contribution to journalArticle

8 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)87-93
Number of pages7
JournalJournal of Biomolecular NMR
Volume44
Issue number2
DOIs
StatePublished - 2009

Fingerprint

src-Family Kinases
Purification
Catalytic Domain
Nuclear magnetic resonance
Phosphoric Monoester Hydrolases
Isotopes
Labeling
Catalyst activity
Crystal structure
Availability
Neoplasms
Proteins
Therapeutics

Keywords

  • E. coli expression and purification
  • Non-receptor protein tyrosine kinases
  • Solution NMR methods
  • Src-family kinases

ASJC Scopus subject areas

  • Spectroscopy
  • Biochemistry

Cite this

Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies. / Piserchio, Andrea; Ghose, Ranajeet; Cowburn, David.

In: Journal of Biomolecular NMR, Vol. 44, No. 2, 2009, p. 87-93.

Research output: Contribution to journalArticle

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