Toward the semisynthesis of multidomain transmembrane receptors: modification of Eph tyrosine kinases.
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 intracellular proteins and small integral membrane proteins for structural and functional studies. Here we describe the semisynthetic site-specific modification of the complete, multidomain 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 multidomain, extracellular, disulfide-bonded, and glycosylated proteins and highlights its potential application for reconstituting entire single-pass transmembrane proteins.
Singla
Nikhil
N
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Himanen
Juha Pekka
JP
Muir
Tom W
TW
Nikolov
Dimitar B
DB
D000818:Animals; D003001:Cloning, Molecular; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis / Q000138:chemical synthesis / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D036081:Receptors, Eph Family / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D036081:Receptors, Eph Family