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Published online before print July 14, 2008
Protein Science, DOI: 10.1110/ps.035659.108
 Copyright © 2008 The Protein Society
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Towards the Semisynthesis of Multi-domain Trans-membrane Receptors: Modification of Eph Tyrosine Kinases

Nikhil Singla1, Juha Pekka Himanen1, Tom W Muir2, and Dimitar B Nikolov1,3

1 Memorial Sloan Kettering Cancer Center;
2 The Rockefeller University

(RECEIVED April 6, 2008; ACCEPTED July 11, 2008)

Expressed protein ligation (EPL) is a protein engineering approach that allows the modification or assembly of a target protein from multiple recombinant and synthetic polypeptides. EPL has been previously used to modify intra-cellular proteins and small integral membrane proteins for structural and functional studies. Here we describe the semisynthetic site-specific modification of the complete, multi-domain extracellular regions of both A- and B- classes of Eph receptor tyrosine kinases. We show that the ectodomains of these receptors can be ligated to different peptides under carefully established experimental conditions, while their biological activity is retained. This work extends the boundaries of the EPL technique for semisynthesis of multi-domain, extracellular, disulfide-bonded and glycosylated proteins and highlights its potential application for reconstituting entire single-pass transmembrane proteins.

Keywords: Structure/function studies; Membrane Proteins; Methods of protein and peptide synthesis; Synthesis of Peptides and Proteins; Expressed Protein Ligation; Protein Modification; Receptor Tyrosine Kinases

3 E-mail: nikolovd{at}mskcc.org


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