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Chem Biol. 2011 Mar 25;18(3):361-71. doi: 10.1016/j.chembiol.2011.01.011.

A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation.

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1
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.

Abstract

We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization.

PMID:
21439481
PMCID:
PMC3286648
DOI:
10.1016/j.chembiol.2011.01.011
[Indexed for MEDLINE]
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