Synthesis of O-GlcNAcylated small heat shock proteins.

A protein's structure and function often depend not only on its primary sequence, but also the presence or absence of any number of non-coded posttranslational modifications. Complicating their study is the fact that the physiological consequences of these modifications are context-, protein-, and site-dependent, and there exist no purely biological techniques to unambiguously study their effects. To this end, protein semisynthesis has become an invaluable chemical biology tool to specifically install non-coded or non-native moieties onto proteins in vitro using synthetic and/or recombinant polypeptides. Here, we describe two facets of protein semisynthesis (solid-phase peptide synthesis and expressed protein ligation) and their use in generating site-specifically glycosylated small heat shock proteins for functional studies. The procedures herein require limited specialized equipment, employ mild reaction conditions, and can be extended to myriad other proteins, modifications, and contexts.