Abstract
Here we describe how a sequential version of the protein semi-synthesis technique, Expressed Protein Ligation (EPL), can be used to assemble multiple (i.e. 3 or more) recombinantly-derived polypeptides segments into a target protein. Sequential EPL was successfully used to assembly the 304 amino acid eukaryotic adaptor protein, Crk-II, from three recombinant polypeptide segments in good yield. Moreover, the resulting multi-component ligation product was found to possess the expected biological activity in a series of ligand binding studies. By allowing the controlled assembly of 3 or more recombinant polypeptide segments, sequential EPL opens the door to the segmental isotopic labeling of internal regions of large proteins with NMR probe-nuclei.
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