Total Chemical Synthesis of Correctly Folded Disulfide-Rich Proteins Using a Removable O-Linked β- <i>N</i> -Acetylglucosamine Strategy
Wei-Wei Shi, Chaowei Shi, Tong-Yue Wang, Yu-Lei Li, Yong-Kang Zhou, Xu-Han Zhang, Donald Bierer, Ji-Shen Zheng, Lei Liu
Abstract
Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked β- N -acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.