Semisynthesis of homogeneous misfolded glycoprotein interleukin-8.

To uncover how cells distinguish between misfolded and correctly-folded glycoproteins, homogeneous misfolded glycoproteins are needed as a probe for analysis of their structure and chemical characteristic nature. In this study, we have synthesized misfolded glycosyl interleukin-8 (IL-8) by combining E. coli expression and chemical synthesis to improve the synthetic efficiency. In order to prepare N-terminal peptide-thioester segment (1-33), we prepared an E. coli expressed peptide and then activated the C-terminal Cys by using an intramolecular N-to-S acyl shift reaction, followed by trans-thioesterification of the Cys-thioester with an external bis(2-sulfanylethyl)amine (SEA). The glycopeptide segment (34-49) was prepared by solid phase peptide synthesis and the C-terminal peptide (50-72) was prepared in E. coli. These peptide and glycopeptide segments were successfully coupled by sequential native chemical ligation. To obtain homogeneous misfolded glycoproteins by shuffling the disulfide bond pattern, folding conditions were optimized to maximize the yield of individual homogeneous misfolded glycoproteins.